Comparative Study of Serum and Biliary Lipid Profile in Libyan Gallstone Patients

Cardiac Markers
Tuesday, July 21, 2:00 pm - 4:30 pm

L, p<0.01). In multivariate analysis, considering CK-MB as the dependent variable, it was found that age (P<0.001), family history (P=0.016), dyslipidemia (P=0.006) and diabetes (P=0.004), CRP (P=0.043), multiple stent (P=0.015), post-dilatation (P=0.02) are independent predictors for post-procedural CK-MB elevation after controlling for sex, smoking history, hypertension, maximum deployment pressure and predilatation. After removing the effect of the above all other variables, CRP remained an independent predictor of CK-MB elevation (P=0.043). Conclusions: The inflammatory activity, as represented by plasma CRP level, is associated with procedure-related microvascular injury as assessed by CK-MB elevation after coronary stenting. As distal microembolization is a determinant of short- and long-term morbidity and mortality after PCI, specific strategies may be developed to minimize myocardial injury in subjects with elevated CRP level.
Tuesday PM, July 21

Poster Session: 2:00 pm - 4:30 pm Cardiac Markers
B-1 Serum Gamma Glutamyl Transferase: A Novel Biomarker for Screening of Premature Coronary Artery Disease D. A. Khan, S. Shabbir, F. A. Khan. AM College, National University of Sciences and Technology, Rawalpindi, Pakistan
Background: Gamma Glutamyl transferase (GGT) is an enzyme responsible for extracellular catabolism of glutathione, associated with oxidative stress and cardiovascular disease. We aimed to elucidate the role of GGT activity in association with premature coronary artery disease (CAD) and screening of high risk young patients for angiography. Methods: In this case control study 218 young adults (age < 45 years) consisting of 111 angiographically determined CAD (>70% occlusion) patients and 107 healthy control were recruited from National Institute of Heart Disease/ AFIC Rawalpindi, Pakistan. Detailed history and clinical examination was carried out. The patients of hepatic disease and alcoholic were excluded. Atherosclerotic coronary artery lesions were angiographically diagnosed in these patients with at least 70% stenos in the one of the main coronary arteries. Serum GGT activity was analyzed at 37 C by using glutamyl-3-carboxy-4-nitroanilide as substrate by using Human kit (Germany) on Selectra E autoanalyzer (Netherlands). Serum cholesterol was measured by cholesterol oxidase method and serum triglyceride by GPO-POD colorimetric standard methods. The data was analyzed by using SPSS-16.0. Results: The patients were predominantly male (96%) with mean (SD) age of 38.7(4.3) years. The main risk factors identified in the CAD patients were hypertension 71%, dyslipidemia 69%, smoking 68% and diabetes 30%. The patients had significantly increased oxidative stress with median GGT 53 U/L (IQR 42-69) as compared to controls 28 IU/L (IQR 21-33; p=0.001). Serum GGT exhibited a significant positive correlation with blood pressure (r=0.53), cholesterol(r= 0.38), blood glucose (r= 0.41), smoking (r=0.19) and negative correlation with total antioxidant status (r=-0.48; p<0.01). Area under the receiver operative curve of GTT was 0.929 (95% CI: 0.896-0.962). The study revealed good diagnostic accuracy at cut off 35 U/L for CAD in high risk young patients with sensitivity 92%, specificity 81%, positive likelihood ratio 5, negative likelihood ratio 0.1 and diagnostic odd ratio of 48. Conclusion: The increase oxidative stress due to cardiovascular risk factors is associated with raised serum GGT activity in the non alcoholic patients of premature CAD. Estimation of GGT is an accurate and cost effective test for screening of the high risk CAD young patients before angiography in developing countries.
B-3 Persistent Elevated Troponin T in Patient with Thrombotic Thrombocytopenic Purpura Responsive to Treatment H. K. Lee1, M. Gautier2, F. A. Polito2, T. J. Brough2, Z. M. Szczepiorkowski1. 1Dartmouth Medical School & Dartmouth-Hitchcock Medical Center, Lebanon, NH, 2Dartmouth-Hitchcock Medical Center, Lebanon, NH
Background: A 39-year-old woman was transferred to our institution with profound thrombocytopenia, acute generalized abdominal pain, and intractable vomiting with volume depletion. Her laboratory results before transfer to our institution revealed a platelet count of 6,000, hemoglobin of 9.9 g/dL, presumptive positives in urine cocaine, THC, and opioids, and elevated Troponin I, CKMB, CPK, amylase, lipase, total bilirubin, and AST. Patient was diagnosed with Thrombotic Thrombocytopenic Purpura (TTP) and pancreatitis. Her ADAMTS-13 activity was found to be <5% (reference range: >67%) with inhibitor activity of 1.3 Inhibitor Units (reference range: < 0.4 Inhibitor Units). Six plasma exchanges were performed over 6 days and the patients platelet count rose from 6,000 to 352,000 (reference range: 160, 000450,000). While patient was responding to the plasma exchange treatment for TTP, her Troponin T (cTnT) rose from 0.78 to 1.32 ng/mL (reference range: 0-0.03 ng/mL) over 5 days. However, her CPK decreased from 415 to 88 U/L (reference range: 0-160 U/L) and no abnormality was revealed in 2 ECGs and an echocardiogram during the same time period. Objectives: We described a case of persistent elevated cTnT with normal ECG and echocardiogram in a patient with TTP. The elevated cTnT may signal the presence of microinfarcts in the patient, which was difficult to diagnose and could be easily missed. Methods: All cardiac marker measurements were performed on the Roche Modular System. Non-specific antibody interference to cTnT experiment was performed using Non-specific Antibody Blocking Tubes from Scantibodies Laboratory, Inc. Results: Non-specific antibody interference in the measurement of cTnT was first suspected in the patient as other cardiac markers were trending towards normal and no abnormality was found in radiologic studies. However, no interference was found. Aspirin 325 mg/day was then prescribed on day 5 of the plasma exchange and the patients cTnT decreased steadily over 3 days to <0.03 ng/mL. The patients cTnT did not increase over the next two months and she denied any cardiac symptoms. An in-vitro experiment was performed where the level of CPK and cTnT in platelet concentrate with and without freezing was measured. Intact platelets showed low levels of CPK while frozen-thawed platelets (i.e. damaged platelets) showed a significant release of CPK. It is speculated that the initial high level of CPK in the patient may be due to platelet destruction in early TTP. As she underwent plasma exchanges, which removes large molecules from the circulation, her CPK returned to normal levels. However, her cTnT levels remained elevated. Conclusions: Myocardial infarction (MI) in the presence of TTP is difficult to diagnose as the myocardial involvement in TTP frequently involves the conduction system and classic ECG manifestations of an acute MI may not be apparent. Monitoring serial levels of cardiac troponin may provide an early warning of subclinical myocardial damage in patients with TTP as elevated levels of CPK alone may be misleading. Cardiac rhythm monitoring is suggested for patients with elevated troponin and there may be a role for Aspirin in treating patients with MI in the presence of TTP.
B-2 Plasma C-Reactive Protein Level is a risk factor of Distal Microembolization During Percutaneous Coronary Intervention H. S. Chaudhury1, A. Rahman2, M. Ahmed2, S. Husnayen2, A. Mohibullah2. 1M Bhasani Medical College, Dhaka, Bangladesh, 2National Institute of Cardiovascular Diseases (NICVD), Dhaka, Bangladesh
Background: Elevated C-reactive protein (CRP) has been well known as a biomarker reflecting inflammatory process that might evoke the potential for microembolization of atheromatous plaque and imparts poor prognosis in patients with coronary artery disease. This study was designed to evaluate whether preprocedural CRP level is associated with procedure related myocardial injury after coronary stenting. Methods: Using a prospective analytical design we studied 377 subjects: 350 (92.8%) male and 27 (7.2%) female. Plasma CRP level was obtained from angina patient who underwent coronary stenting within 24 hours before procedure and divided patients into either normal (CRP < 6 mg/L) or elevated group (CRP > 6 mg/L). We evaluated procedure related myocardial damage by measuring CK-MB level before and after PCI. Results: A total of 377 patients (with 551 lesions) who were divided into two groups: Normal CRP group (n=171) and elevated CRP group (n=206). In normal CRP Group, there was no significant elevation of CK-MB level after stenting (preprocedural vs postprocedural: 17.8 + 5.4 vs 22.1 + 5.6 mg/L, p=ns). In elevated CRP Group, there was significant elevation of CK-MB level after stenting (18.1 + 6.7 vs 26.3 + 9.4 mg/
CLINICAL CHEMISTRY, Vol. 55, No. 6, Supplement, 2009
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B-4 A biomarker panel for identification of acute coronary syndrome patients at risk for subsequent heart failure/death P. Kavsak1, A. Newman2, D. Ko2, A. MacRae3, A. Jaffe4. 1McMaster University, Hamilton, ON, Canada, 2Institute for Clinical Evaluative Sciences, Toronto, ON, Canada, 3University of Manitoba, Winnipeg, MB, Canada, 4Mayo Clinic, Rochester, MN
Background: Elevated IL-6 and MCP-1 concentrations have previously been reported to be independent predictors of heart failure (HF)/death in an ACS population. We sought to assess if elevations in additional biomarkers involved in inflammation, cardiac fibrosis and function (IL-10, VEGF, EGF) would further facilitate identification of those at risk for HF/death. Methods: In an ACS cohort (n=216) where each subject had at least 2 EDTA specimens stored at -800C; the earliest available specimen and the next closest to 9 h post onset (2nd specimen) were thawed and measured for IL-6, MCP-1, IL-10, VEGF, EGF using the evidence investigator biochip array (Randox). For each subject, the concentrations of the cytokines in the 2nd specimen were used to score the subject positive or negative for the panel. Univariately any increase of VEGF, IL-6, IL-10, MCP-1 or decrease in EGF from the median concentration in the population was considered a positive result and 3 positive cytokines yielded a positive panel. NTproBNP (Elecsys, Roche) and cardiac troponin I (cTnI, AccuTnI, Beckman Coulter) had been measured in this cohort, and published cutoffs (NT-proBNP >183 ng/L and cTnI > 0.02 ug/L) were used in multivariate models. Health outcomes (HF/ death) were obtained via the Canadian Institutes for Health Information and Registered Persons databases. Both Kaplan Meier and Cox proportional hazard models were utilized for the outcome analysis. The study received research ethics approval. Results: Subjects with a positive panel test (i.e., at least 3 cytokines positive) had a higher probability for HF/death over 8 years (p<0.001, Figure). At 6 months, the hazard ratio (HR), after adjusting for age, sex, and history of HF was 3.8 (p=0.003). Additional adjustment for NT-proBNP and cTnI yielded a HR of 3.2 (p=0.009). (25th-75th):
Cardiac Markers
4 (2-6)). Outcomes (MI/death) within 1 year of presentation were obtained via databases (Canadian Institutes for Health Information and Registered Persons) and analyzed with Kaplan Meier and Cox proportional hazard analysis. The study received research ethics board approval. Results: There was no difference between the sexes in the prevalence of subjects meeting the change criteria (males (72%) and females (71%); p=0.77). Kaplan Meier analysis indicated that those subjects that met the hs-cTnI change criteria were at higher risk of MI/death (p=0.004, Figure). The hazard ratio (HR) at 6 months for MI/ death in those with hs-cTn change compared to those without a change was 4.0 (95%CI:1.4-11.3; p= 0.008) and 3.9 (95%CI:1.4-10.9; p=0.010) before and after adjusting for age and sex.
Conclusion: A novel hs-cTnI assay using change criteria facilitates risk stratification in patients presenting with ACS.
B-6 Long-term survival is predicted in patients presenting with chest pain by PAPP-A measurements P. Kavsak1, X. Wang2, M. Henderson1, D. Ko2, A. MacRae3, A. Jaffe4. 1McMaster University, Hamilton, ON, Canada, 2Institute for Clinical Evaluative Sciences, Toronto, ON, Canada, 3University of Manitoba, Winnipeg, MB, Canada, 4Mayo Clinic, Rochester, MN
Background: PAPP-A is increased in unstable plaques and circulating levels are detectable in patients with acute coronary syndromes. The present study sought to determine if measurement of PAPP-A could identify patients at high risk for death (up 10 years) after presenting with chest pain to an emergency department. Methods: Three hundred and twenty patients (60% male) with heparinized plasma specimens (frozen -800C, 2 thaws from an original chest pain population of 448 recruited in 1996) were included in this analysis. Specimens were assayed with a highly sensitive ELISA (DSL, Webster TX) measuring uncomplexed PAPP-A and the PAPP-A/proMBP complex in equimolar concentrations, but not proMBP. For survival analysis, Kaplan Meier and Cox proportional hazard methods were used. The PAPP-A concentration in the earliest specimen (median (25th-75th): 4 hours (2-9) from pain onset) was used to stratify the cohort into tertiles. Death date was obtained via the Registered Persons database. This study was approved by the research ethics board. Results: After 10 years 44% of the subjects had died. There were no differences in mortality between the sexes (p=0.58). Kaplan Meier analysis based on tertiles (tertile 1: PAPP-A<0.92 mIU/L; tertile 2: 0.92-1.62 mIU/L; tertile 3: >1.62 mIU/L) showed differences in long-term survival (p=0.05, Figure). The hazard ratio (HR) at 2 years for subjects with concentrations of PAPP-A in the upper third compared to the lowest third was 2.23 (95%CI:1.04-4.80; p=0.04) after adjusting for age and sex. Further adjustment for cardiac troponin (cTnI 0.02 ug/L at presentation) yielded a HR of 2.15 (95%CI: 1.00-4.63; p=0.05).
Conclusion: This panel test represents a new method for the identification of patients with ACS at risk for HF/death.
B-5 Impact of change criteria and a novel high sensitivity cardiac troponin I assay in predicting health outcomes P. Kavsak1, X. Wang2, D. Ko2, A. MacRae3, A. Jaffe4. 1McMaster University, Hamilton, ON, Canada, 2Institute for Clinical Evaluative Sciences, Toronto, ON, Canada, 3University of Manitoba, Winnipeg, MB, Canada, 4Mayo Clinic, Rochester, MN
Background: The 2007 Universal definition of myocardial infarction (MI) suggested the use of changes in cardiac troponin (cTn) to diagnose recurrent infarction. For patients with elevated cTn, a 20% increase (or >3SDs of the variance of the measure) would be suggestive of reinfarction. It has not been demonstrated whether these change criteria can be used for risk stratification in patients presenting to the emergency department with symptoms of acute coronary syndromes (ACS). A novel high sensitivity cTnI (hs-cTnI) assay was used. Methods: In an ACS cohort (n=257, median age 67 years, 58% male) recruited in 1996, a research hs-cTnI assay (reported units ng/L; Beckman Coulter) was measured in all available heparin specimens stored at -800C. A change was defined as an absolute difference of at least 3.34 ng/L for concentrations 100 ng/L and a 20% difference in higher concentrations, reflecting the >3SD/20% criteria between the lowest and highest concentrations for each subject (median # of specimens/subject
Conclusion: This PAPP-A assay may be useful for long-term risk stratification in patients presenting with chest pain.
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Cardiac Markers
B-7 Comparison of NT-proBNP concentrations measured with Elecsys proBNP and proBNP II Y. Gao. Shanghai Sixth People's Hospital, Shanghai Jiao Tong University, Shanghai, China
Objective: Replacing the polyclonal capture and detection antibodies with monoclonal ones, Roche Diagnostics upgrades Elecsys proBNP to proBNP II for determination of serum/plasma N-terminal pro B-type natriuretic peptide (NTproBNP). The aim of the study was to investigate the difference of clinical sample measurements with the two assays. Methods: Blood samples (60 serum and 50 plasma) from patients with cardiovascular diseases were collected and determined simultaneously with Elecsys proBNP and proBNP II on Elecsys 2010 immunoassay analyzer (ECLIA, Roche Diagnostics GmbH, Mannheim, Germany). Wilcoxon and McNemar test for possible differences in the two NT-proBNP assays were performed and their correlations were analyzed with SPSS 13.0 soft ware. Results: The median (P25~P75) values of NT-proBNP were 124.4 (25.5~606.4) pg/mL and 126.6 (17.6~643.7) pg/mL for proBNP assay and proBNP II assay, respectively. Though a slight difference in the measurements was seen statistically (Z=-2.663, P=0.008), a 100% agreement of sample classification was achieved (Kappa=1.000, P<0.001) with high correlation (Spearman r=0.997, P<0.001) between the two assays (Figure 1). Conclusion: The two Elecsys NT-proBNP assays, proBNP and proBNP II, though with a slight difference in sample measurements, have almost the same diagnostic performance for congestive cardiac failure.

6.62.3 vs. 6.42.2 M, p NS); (ii) vit B12 - median (range) - 349 (132-1196) vs. 299 (136-1177) pg/ml, p <0.001); (iii) folate - 15.44.4 vs. 15.05.0 ng/ml, p NS; (iv) CRP median (range) - 0.03 (0.01-1.57) vs. 0.16 (0.01-5.06) mg/dL, p < 0.001); (v) ICAM - 230.1 77.6 vs. 244.7 81.4 ng/ml, p = 0.040); (vi) VCAM - 1142.4 344.7 vs.1151.8 247.6 ng/ml, p NS. In correlation analysis, BMI had significant (p < 0.01) correlation with Vit. B12 (r -0.255), folate (r -0.18) and CRP (r 0.65). Homocysteine, as expected, was significantly correlated (p < 0.05) with vit B12 (r -0.30) and folate (r -0.42). ICAM-1 correlated (p < 0.05) with tHcys (r -0.19), vit B12 (r 0.10), folate (r 0.12) and CRP (r = 0.1). Conclusions: Circulating levels of inflammatory predictors of atherosclerosis (CRP and soluble adhesion molecules, but not homocysteine) are seen at increased levels in obese adolescents. Indeed, there were significant linear correlations between BMI and ICAM-1 and the other mediators, suggesting possible pathogenetic interconnectivity of these parameters. (This study was supported by Kuwait University Research Administration Grant # MC01/04)
B-9 Urocortin: a potential biomarker for the diagnosis of heart failure patients? D. Gruson, S. Ahn, J. Ketelslegers, M. Rousseau. Cliniques Universitaires St Luc, Bruxelles, Belgium
Objective: The available evidence suggests that Urocortin (UCN), a cardioprotective and vasoactive peptide derived from fish neuroendocrinology, may be involved in the physiopathology of cardiovascular diseases. The aim of this study was to determine UCN plasma concentrations in patients with congestive heart failure (CHF) and to evaluate its relationship with other established markers of CHF. Methods: Plasma concentrations of UCN were measured in 42 fully treated CHF patients (mean age: 6410 years; mean ejection fraction: 216%). UCN was determined using a specific ELISA assay (Phoenix pharmaceuticals, USA) after acidic extraction using Sep-Pak C18 columns. Circulating levels of BNP, Nt-proBNP, Nt-proANP and Big ET-1 were also determined. Reference values for biomarkers were obtained from 20 healthy ageand sex-matched subjects. Results: In comparison with controls, UCN plasma concentrations (geometric mean [95% CI]) were significantly increased in CHF patients (88 pmol/L [75-105] vs 46 [39-54], p<0.001). As expected, the other neurohormones were also significantly increased in CHF patients (BNP: 1443 pg/ml [1023-1862] vs 52 [36-67], Nt-proBNP: 3501 pg/ml [2356-5202] vs 35 [24-51], NtproANP: 5498 pg/ml [4336-6971] vs 324 [255-411] and Big ET-1: 15.8 pg/ml [13.618.4] vs 5.9 [5.2-6.8]; p<0.01 for all vs controls). Surprisingly, no significant correlation was observed between UCN and the other CHF biomarkers. Conclusions: Our results have demonstrated that plasma concentrations of UCN are significantly increased in patients with CHF. Further studies should be therefore performed to confirm the potential utility of UCN as a complementary biomarker for the diagnosis and prognosis of CHF.
B-8 Circulating soluble adhesion molecules and other inflammatory markers as atherosclerotic disease risk factors in adolescents influence of body mass A. O. Akanji, A. Al-Isa, L. Thalib. Kuwait University Faculty of Medicine, Kuwait, Kuwait
Introduction: Circulating levels of some markers of the inflammatory response (including CRP and soluble adhesion molecules) and oxidant activity (including total homocysteine (tHcys) and its determinants, vitamin B12 & folate) have been implicated as predictors of atherosclerosis susceptibility. There is a high prevalence of obesity in childhood in Kuwait with the inevitable consequence of increased risk for atherosclerotic vascular disease in adult life. This study explores the relationship between body mass and circulating levels of these biochemical risk markers - Hcys, vit B12, folate, CRP, and soluble adhesion molecules (ICAM & VCAM) - in Kuwaiti adolescents. Methods: We recruited 514 Kuwaiti adolescents aged 10-19 yr randomly from selected schools. 226 of them were of normal weight, and 288 overweight and obese (by age-standardized criteria). In all subjects, BMI was measured (wt/ ht), and a fasting blood sample was collected and analyzed for Hcys, vit B12, folate, CRP, ICAM and VCAM, using standard immunoassay techniques. Results: The results were compared for normal weight vs. overweight/obese, for each of the measured parameters as follows (meanSD, except where stated): (i)tHcys -
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B-10 The Effect of Sample Storage Conditions and Time to Assay Performance on the Measurement of Ischemia Modified Albumin. B. Seamonds. Mercy Health Laboratory, Darby, PA
Background: Ischemia Modified Albumin (IMA) is FDA cleared to assist in risk stratification of patients presenting to the ED with suspected ACS. Our studies have shown that IMA > 95U/mL, combined with troponin < 0.04 (Troponin T, Roche Diagnostics) and non-diagnostic ECG, may be used at presentation to identify a cohort of patients at low risk of adverse cardiac events resulting in an earlier ED discharge. Therefore, elevations of IMA, as a result of stability, will decrease the ability of IMA to identify a low risk cohort thereby diminishing its utility in clinical decision making. Specimen handling is an important consideration for reliable IMA assay results. Our purpose was to determine the affects of increasing the time from blood draw until IMA measurement and the effects of serum storage on re-assayed samples. Methods: Normal healthy volunteers each provided two tubes of blood which were immediately centrifuged after clotting. After serum isolation, samples were either: a) aliquoted and frozen at -20C and -70C for testing after 1 week and 4 weeks of storage; or b) collection tubes were left at room temperature and stoppered, except to measure IMA every 30 minutes over the next 4 hours. Frozen samples were thawed at room temperature, mixed by multiple inversions and then immediately tested for IMA. Room temperature samples were tested immediately for IMA after an aliquot was removed from the stoppered tube. IMA was analyzed using the Roche Integra 800 platform. Statistical analysis was by Students T test. Results: Blood was obtained from 14 subjects and 9 subjects for frozen and room temperature stability respectively. Mean baseline frozen and room temperature IMA was 85.9 + 4.6 and 86.9 2.3 U/mL respectively. Following 1 week of frozen storage, IMA rose to 94.4 + 4.9 (p<0.001) and 91.1 + 6.4 (p = 0.051) for -20C and -70C respectively. After 4 weeks, the mean value was 93.4 + 8.6 (p = 0.009) and 87.2 + 7.5 (p = 0.63) for -20C and -70C respectively.. Room temperature samples rose a mean of 2.3 U/mL in the first 30 minutes and continued to rise a mean of 1.3 0.7 U/mL at each 30 minute time interval (p<0.001). During the first 90 minutes, mean IMA rose a total of 4.5 0.7 U/mL. By 2 hours, 89% (8 of 9) of specimens had IMA values exceeding 90 U/mL. Mean IMA at 4 hours was 97.3 3.6 U/mL with a mean increase over 4 hours of 10.4 2.3 IU/mL. Conclusions: Once blood is obtained in a test tube, IMA rises significantly and rapidly while awaiting testing. The rise in initially negative IMA levels may reach the cutpoint with important clinical impact. Samples stored for future analysis or reanalysis of IMA should be stored at -70C to maintain sample stability and processed immediately aftering thawing. This study encompasses a 4 week time point and does indicate stability at longer frozen intervals.
Cardiac Markers
Discussion. By selectively choosing the zero-concentration calibrator matrixes for troponin-I, different concentration values were determined for samples from a normal population (both mean values and 99th percentiles). Because it is possible to have different values, it becomes critically important for studies assessing a normal population to define the zero-concentration calibrator. It may even be necessary for comparisons between assays to use a standard zero-concentration matrix. While the normal population values for the 99th percentile vary in this study with the experimental matrixes, they are all less than that expected for this assay, namely 0.04 ng/ml. The mean values are also all less than the expected analytical sensitivity of 0.02 ng/ml. Therefore, even with the variation in results, they all fall within the values reported for this assay.
B-12 Changes of BNP in Acute myocardial infarction compared with other related serum biomarkers D. Zhang, H. Jiang, Y. P. Tian. Chinese PLA General Hospital, BeiJing, China
Background: Acute myocardial infarction (AMI) is one of the most serious threats to human health. The diagnosis of AMI as early as possible is very important for clinical treatment and prognosis. However, many symptoms of AMI are found to be atypical in clinical practice. When patients feel chest-pain, there is no abnormal changes in ECG for the following 4 hours after chest-pain or typical myocardial infarction markers are rarely abnormal. Now there is also a lack of sensitive markers during the 4-hour window time between myocardial ischemia and significant rise of TnT. BNP could be mainly used as a heart failure marker in clinical diagnosis. But most of the researches focus BNP usage on risk stratification and prognosis. Objective: To evaluate potential value of BNP on diagnosis of AMI at the early stage by comparing BNP and typical myocardial infarction marker (TnT, CKMB mass). Materials and methods: By electrochemiluminescence immunoassay, Plasma TnT, BNP, CKMBmass level were measured in 35 patients with chest-pain and chest distress. All these case were diagnosed as initial AMI as well as without other diseases such as hypertension, diabetes, diseases of brain, lung liver, kidney and infection. Results: Whether TnT is positive or not, there were no significant difference in BNP and CKMB mass. There were no significant difference in BNP and TnT. But plasma BNP level of five patients, who all suffer from AMI with non ST segment elevation in the group of negative TnT, rise significantly. Conclusion: BNP, as an assistant indicator, can help to assess cardiac function and improve prognosis during AMI treatment. Discussion: As the specific protein of myocardial cell, TnT is released into blood when cardiac muscle is injured, but TnT remains normally if cardiac muscle is not yet injured. It is deemed that myocardial ischemia is another important cause of BNP rising in a recent report. Myocardial ischemia is precedent to cardiac muscle injury, so the rising of BNP may be observed before the rising of TnT. Thus, BNP has remarkable significant in identifying AMI with non ST segment elevating. Measurement of BNP helps to diagnose the suspicious patient with myocardial ischemia at early stage without ECG changes. With monitoring of CKMB mass and TnT, the clinic can confirm or exclude the diagnosis of AMI. BNP alone cant be used to identify myocardial infarction. But the combination of TnT and CKMB mass is still the golden standard of diagnosing AMI. BNP can be used as an secondary indicator to assess cardiac function and help to improve prognosis during AMI treatment.
B-11 Analyses of Samples from a Normal Population for Troponin-I Concentrations Using Four Different Matrixes For the ZeroConcentration Calibrator R. E. Hruska. Abbott Diagnostics Division, Abbott Park, IL
Objectives. This study used a troponin-I calibrator set substituted with four different experimental matrixes for the zero-concentration calibrator. For each calibration curve the troponin-I concentrations of samples from a normal population were measured. This is relevant as the reporting of troponin-I concentrations become closer to zero, the importance of the choice of the zero-concentration calibrator becomes critical. This study determined the impact of the zero-concentration calibrator matrix on the mean concentrations and the 99th percentiles of a normal population. Materials and Methods. Analyses used historical data generated with the Abbott AxSYM Troponin-I ADV assay. Responses of 1274 normal samples were analyzed using four different experimental matrixes for the zero-concentration calibrator as follows: a protein matrix, a pool of plasma samples from a normal population, a plasma pool that had been processed to remove clotting factors, and a plasma pool that was processed and then treated with a conjugate antibody to troponin-I. Results. A summary of the analyses is included in Table 1. Table 1. Analyses of Values (ng/ml) for Samples from a Normal Population. Zero-Concentration Calibrator Matrix (all other Mean 99th calibrators the same) Values Percentile Protein Matrix 0.0003 0.0088 Plasma Pool 0.0022 0.0172 Processed Plasma Pool 0.0070 0.0274 Processed and Treated Plasma Pool 0.0107 0.0321
B-13 C-REACTIVE PROTEIN IN PATIENTS WITH NSTEMI ACUTE CORONARY SYNDROME E. McNair1, M. Qureshi1, C. Wells2, R. Basran2, C. Pearce2, J. Devilliers2, K. Prasad3. 1Department of Pathology and Laboratory Medicine, Royal University Hospital, University of Saskatchewan, Saskatoon, SK, Canada, 2Department of Cardiology, Royal University Hospital, University of Saskatchewan, Saskatoon, SK, Canada, 3Department of Physiology, University of Saskatchewan, Saskatoon, SK, Canada
OBJECTIVE: C reactive protein (CRP) is an acute phase inflammatory mediator that has been implicated as a risk factor for cardiovascular disease (CAD). The clinical presentation of acute coronary syndrome (ACS) ranges from unstable angina (U/A) to non ST-segment elevation myocardial infarction (NSTEMI) and ST-segment elevation myocardial infarction (STEMI). Although, elevated levels of serum CRP have been reported in patients with CAD, the serum levels of CRP in patients with NSTEMI
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ACS is unknown. The purpose of this study was to determine whether: (1) the serum levels of CRP are higher in patients with NSTEMI ACS as compared to healthy controls; and (2) CRP levels are directly proportional to the number of diseased vessels in patients with NSTEMI ACS. METHODS: The study subjects consisted of two groups. Group 1: (controls) twentyeight age-matched healthy men and Group II: Forty-six NSTEMI ACS patients. Group II patients were subdivided into subjects with one vessel (1VD), two vessels (2VD) or three vessels disease (3VD). Fasting blood samples were collected and serum levels of CRP were measured by using highly sensitive near infrared particle immunoassay rate method technology. RESULTS: The serum levels of CRP in Group I and Group II were 3.1 0.26 and 10.23 2.2 mg/L, respectively and these values were significantly different from each other (p<0.05). Group II showed a 230% higher level of CRP as compared to Group I subjects. The serum levels of CRP in the patients with 1VD, 2VD, and 3VD were 11.4 3.9, 10.7 3.2, and 9.4 2.8 mg/L, respectively. The values of all subgroups were significantly different (p<0.05) from Group I. However, the values within the subgroup were not significantly different from each other. CONCLUSION: The results demonstrate that the serum levels of CRP are higher in patients with NSTEMI ACS as compared to controls. In addition, serum levels of CRP were higher in the subgroups of affected vessels as compared to control. However, there is no significant difference between the number of affected vessels and serum CRP levels. Serum CRP levels are elevated in patients with NSTEMI ACS and may contribute to the inflammation and thrombosis associated with acute coronary syndrome.

B-15 Evaluation of cardiac troponin T elevation in patients with chronic renal failure undergoing dialysis K. Hong, C. Jung, M. Lee, W. Chung. Ewha Womans University Mokdong Hospital, Seoul, Republic of Korea
Background : Cardiac troponin is widely used as the most sensitive and important biomarker of myocardial ischemia. However, it is frequently reported that cardiac troponin levels are elevated in patients with chronic renal failure (CRF) regardless of ischemic myocardial injury. Therefore, it is difficult to diagnosis ischemic heart diseases especially when other findings are obscure . We conducted a study to find causes of cardiac troponin T (cTnT) elevation and to evaluate relation between cTnT increase and cardiac disease in patients with CRF undergoing dialysis. Methods : We analyzed cTnT value of total 69 patients (male 30, female 39, mean age 58.713.2) measured at Ewha Womans University Mokdong Hospital between October and December, 2007. Reference value was 99th percentile of 20 healthy adults without cardiac diseases. We measured cTnT values by Elecsys 2010 (Roche diagnostics, Germany), the 4th generation assay and determined total imprecision. We investigated underlying disease, duration of dialysis, ischemic heart disease and measured CK, CK-MB, NT-proBNP, BUN, Creatinine, EGFR, LD, AST, glucose, triglyceride, cholesterol and albumin. Results : The 99th percentile of cTnT in healthy adults group was < 0.01 ng/mL and 10% CV of cTnT was 0.02 ng/mL. Ischemic heart disease (IHD) was 43.5% (30/69). cTnT, CK-MB, NT-proBNP and creatinine were significantly higher in patients with IHD than those without IHD. Mean cTnT values of myocardial infarction, IHD, cardiovascular disease (CVD), total population groups, and patients without CVD group were 0.81 ng/mL, 0.43 ng/mL, 0.34 ng/mL, 0.23 ng/mL, and 0.05 ng/mL, respectively. cTnT and CK-MB values were increased according to severity of cardiac disease. The cut-off values of cTnT in CVD and IHD groups by ROC curve analysis were both 0.07 ng/mL and that in myocardial infarction group was 0.11 ng/mL. Sensitivity, specificity, positive and negative predictive values, and efficiency in CVD and IHD groups were 76.7%, 71.8%, 67.6%, 80.0%, and 73.9%, respectively. And those in myocardial infarction group were 84.6%, 76.8%, 45.8%, 95.6%, and 78.3%, respectively. Conclusions : cTnT level was higher in CRF patients undergoing dialysis with cardiac disease than those without cardiac disease. The cut-off values of cTnT in CVD and IHD groups were both 0.07 ng/mL and that in myocardial infarction group was 0.11 ng/mL.
B-14 Harmony of Cardiac Troponin I Measurements Across Six Commercially Available Platforms W. E. Kelley1, C. deFilippi2, S. Duh1, M. Gantzer3, J. Todd4, R. H. Christenson2. 1Univ of Maryland Medical Center, Baltimore, MD, 2Univ of Maryland Sch of Med, Baltimore, MD, 3Siemens Healthcare Diagnostics, Newark, DE, 4Singulex, Inc., Alameda, CA
Objective: We compared the agreement (harmony) of cardiac troponin I (cTnI) measurements for six commercially available assays using samples from suspected acute coronary syndrome (ACS) patients with values near the 99th percentile of the normal reference population. Relevance: Cardiac troponin is the preferred cardiac marker for myocardial infarction diagnosis and risk stratification; the consensus cutpoint according to the joint ESC/ ACCF/AHA/WHF Task Force for the Redefinition of Myocardial Infarction is the 99th percentile of cTn concentration of a normal reference population as measured by each method. Harmonization of popular commercial assays in ACS samples at cTnI concentrations near the 99th percentile is uncertain. Methodology: Appropriate plasma samples from 60 ACS patients were analyzed on the Stratus CS (Siemens), Advia Centaur (Ultra assay, Siemens), Erenna (high sensitivity cTnI; Singulex), ACCESS II (AccuTnI; Beckman Coulter), Triage cTnI (Biosite) and i-Stat (Abbott) platforms. To determine harmonization, the results for each cTnI assay (y-axis) were compared by linear regression analysis to the median results of all platforms tested (x-axis; except Triage, see below). Results: For the Triage assay, 52 of the 60 measurements yielded results of <0.05 ng/ mL (non-numerical result); thus linear regression and harmony assessment was not possible. Triage results were excluded from calculation of the median. The results of the cTnI linear regression analysis for the other methods are displayed below. 25%tile 50%tile 75%tile slope Yintercept R Sy/x Access2 0.03 0.04 0.06 0.696 0.018 0.9588 0.033 Centaur 0.017 0.037 0.067 1.36 -0.003 0.9651 0.06 i-STAT 0 0.02 0.055 0.684 0.002 0.9647 0.026 Erenna 0.014 0.03 0.082 0.932 0.011 0.9352 0.056 Stratus CS 0.01 0.04 0.09 1.08 0.005 0.9685 0.045
B-16 The utility of Troponin T to the diagnosis of silent myocardial infarction in patients submitted to noncardiac surgery - preliminary results of the VISION Study G. S. Lima-Oliveira1, E. A. Suzumura2, V. F. Borges1, M. C. Dantas2, A. J. Duarte1, P. J. Devereaux3, O. Berwanger2. 1Clinical Lab - HCor, So Paulo - SP, Brazil, 2Research Institute - HCor, So Paulo - SP, Brazil, 3McMaster University, Hamilton, ON, Canada
Background:Several factors suggest that some perioperative myocardial infarctions (MI) may go undetected with no monitoring of cardiac biomarkers after surgery. The majority of perioperative MI occur when most patients receive analgesic medication that can blunt cardiac pain.Besides, some surgical patients remain intubated and sedated, limiting their ability to communicate symptoms. The vast majority of postoperative episodes of MI are silent. There is evidence that the troponin is more sensitive and specific than CK-MB for MI in acute coronary syndrome. Objective:The purpose of this study was to evaluate the Troponin T (TropT) as biomarker for silent MI diagnostic in patients undergoing to noncardiac surgery. Methods:Multicentre, international, prospective cohort study. Eligibility: Patients who are 45 years of age who have undergone noncardiac surgery, received general or regional anaesthesia, required at least an overnight hospital admission after surgery and consent prior to or within the first 24 hours after surgery. Monitoring: All patients had a TropT drawn between 6 to 12 hours postoperatively and on the 1st, 2nd and 3rd day after surgery. When elevated TropT was detected, an ECG and echocardiography were undertaken even if the patient had no ischemic symptoms (angina, nausea, dyspnea) or signs (hypotension, tachycardia) to fulfill the diagnostic criteria for MI. We compared the incidence of symptomatic and silent MI diagnostic according to the criteria above with the incidence found by Mangano et al. and Ashton et al.
Conclusion: Harmony is conformity of test results among analytical methods. In this analysis, harmony is reflected by the slopes, R values and agreement within the interquartile range. The slopes ranged from 0.70 to 1.4 and R values were all >0.93. The 50th and 75th percentile concentrations differed by less than or equal to 2-fold, as did 3 of the assays at the 25th percentile of data. The data in the table show a substantial degree of harmony among the methods.
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Results:The incidence of silent MI was four times higher in comparison with the previous studies. The results are summarized bellow. Incidence of MI in the VISION Study versus the studies conducted by Mangano and Ashton Study N Biomarker Symptomatic MI Silent MI p VISION 201 Troponin T 4(1.99%) 5(2.48%) Mangano 474 CK-MB 9(1.90%) 3(0.63) <0.001* Ashton 512 CK-MB 5(0.97%) 3(0.58) <0.001# Exact Fisher Test: *VISION versus Mangano; #VISION versus Ashton Conclusion:Our finds suggest that the TropT is a better biomarker than CK-MB to the diagnosis of silent MI in noncardiac surgery. In this sense, we are conducting the VISION Study with 40,000 patients in 10 countries to answer this question.
Cardiac Markers
precursor proBNP1-108. Although many studies have demonstrated elevated plasma measures of both of these cleavage products as important prognostic indicators of acutely decompensated and chronic heart failure, a subset of patients with moderate to severe heart failure who are at increased risk for disease progression and mortality have been noted to have low plasma BNP1-32 measures. We hypothesized that measurement of the precursor proBNP1-108 could further risk-stratify the group of patients chronic ambulatory heart failure and a low plasma BNP1-32. Methods: Baseline plasma measures of BNP (Biosite) and proBNP (Bio-Rad, in development) were analyzed in 694 participants from the African-American Heart Failure Trial (A-HeFT), a randomized study of fixed dose combination of isosorbide dinitrate / hydralazine in predominantly New York Heart Association Class III heart failure. Analysis of event free survival defined as time to death or hospitalization for heart failure was performed with Kaplan-Meier and Cox Proportional Hazards methods. We defined low BNP as a baseline measure below 200 pg/ml and also performed the analysis using baseline BNP=157 pg/ml as the median cutoff. Results: In the group of patients with a baseline BNP<200 (57.2% of cohort), a proBNP above median (198 pg/ml) was identified in 68 patients (11% of cohort) and was associated with an increased risk of death or hospitalization for heart failure (Risk ratio=1.51, p=0.0012). After adjustment for NYHA class, estimated glomerular filtration rate, body mass index, and qualifying left ventricular ejection fraction by echocardiographic measure, a proBNP above median remained highly significant as a baseline predictor of event free survival in multivariate analysis (Risk ratio=1.54, p=0.0018). Using the baseline median BNP as cutoff (BNP<157 pg/ml), an elevated proBNP remained significant as an independent risk factor of event free survival using the same multivariate adjustment (Risk ratio = 1.47, p=0.02). Conclusion: An elevated measure of precursor B-type natriuretic peptide (ProBNP) is a powerful predictor of event free survival in patients with ambulatory moderate to severe heart failure who were identified to have a low plasma BNP.
B-17 Comparison of effects of five biocompatible coating materials of cardiopulmonary bypass circuits on inflammatory response during coronary artery bypass grafting Q. H. Meng, N. Sohn, J. Marcoux, T. Mycyk, J. Krahn. University of Saskatchewan, Saskatoon, SK, Canada
Objectives: Cardiopulmonary bypass (CPB) applied to support circulation during coronary artery bypass grafting (CABG) can induce cardiac injury, inflammation, and oxidative stress. The aim of this study was to compare the impact of different biocompatible coating material used in CPB on inflammatory response and oxidative stress in patients undergoing CABG. Subjects and Methods: Eighty patients underwent elective CABG with CPB were recruited. All subjects were randomly divided into five groups based on the types of biocompatible materials used in CPB: Trillium, Phosphocholine, Bioline, PMEA (Polymethoxyethyl acrylate), and the control group (n=16 each group). Blood was drawn at three different time points: before CPB (T1), right-after CPB (T2), or 72 hours after CABG (T3). Serum high sensitivity CRP (hsCRP) levels were determined using a near infrared particle immunoassay on Beckman Synchron LX20. Serum levels of TNF-, IL-6, IL-10, oxidized nitric oxide (NOx), and myeloperoxidase (MPO) were assayed using commercially available ELISA kits. Results: Serum levels of TNF- in Trillium group were significantly increased at different time points (p < 0.05). Serum levels of TNF- in Bioline, Phosphocholine, and PMEA groups significantly increased only 72 hrs after CPB (p < 0.05). Serum levels of IL-6 significantly increased after initiation of CPB (T2) (P < 0.01) and remained elevated after termination of CPB by 72 hrs after cardiac surgery (T3) in all groups (P < 0.01). The changes of serum IL-10 levels were similar to serum IL-6. Serum hsCRP levels were steadily and significantly increased in all groups at T2 and T3 (p < 0.01). Serum levels of NOx in Trillium, Phosphocholine, and Bioline groups significantly decreased at T3 (P < 0.05). Morover, Phosphocholine group showed a significant decrease between T2 and T1 (p < 0.05). Both Phosphocholine and Bioline groups showed statistical decreases in the levels of serum oxidized nitric oxide compared with the control group at the corresponding T2 and T3 (p < 0.05). Serum MPO levels were significantly elevated at both T2 and T3 in all groups with levels at T3 to a higher degree (P < 0.05). Unless indicated, there were no statistical differences in all the markers determined among groups at the same time point. Conclusions: All coating biomaterials of CPB induce inflammatory response and oxidative stress to various extents. It appears that both Phosphocholine and Bioline coated-biocompatible materials induce less inflammatory responses and oxidative stress compared to the control.
B-19 Clinical Utility of NT-proBNP and BNP Ratio for the Accuracy Diagnosis of Heart Failure with and without Renal Dysfunction P. Srisawasdi1, S. Vanavanan1, C. Charoenpanichkit2, M. H. Kroll3. 1Department of Pathology, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok, Thailand, 2Department of Medicine, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok, Thailand, 3Department of Laboratory Medicine, Boston Medical Center, Boston, MA
Background: The use of BNP or NT-proBNP concentrations for the diagnosis of heart failure, especially for asymptomatic patients at risk for developing heart failure may be confused in patients with the concomitant renal dysfunction. Increased BNP and NT-proBNP were found from decreased renal function, chronic kidney disease (CKD), cardiac dysfunction (heart failure), or both. In the present study, we studied the use of the molar ratio of NT-proBNP to BNP to assess heart failure in the patient with renal dysfunction. Methods: Eligible patients had to be at least 30 years old with chest pain lasting more than 20 minutes suspected to be myocardial in origin and occurring within 3 to 72 hours of presentation. We excluded the patient who have angina with an established precipitating cause (e.g. Anemia or Tachydysrhythmia). Eighty-four Thai patients (54 males and 30 females) were included. We measured NT-proBNP (Roche Diagnostics, GmbH, D-68298 Mannheim, Germany) and BNP (Abbott Laboratories, Abbott Park IL) levels and regressed the geometric means of their concentrations and the ratio with NYHA functional classes (I to IV) and CKD stages (classified by estimated glomerular filtration rate). Results: Both peptides concentrations were progressively higher in patients with increasing heart failure class or CKD stage. Regression patterns of the geometric means of the peptides concentrations and the ratio with NYHA functional class or CKD stage demonstrated exponential curves. Exponential regression analysis demonstrated a strong correlation with functional class, with R2 of 0.996 (p = 0.002), 0.993 (p = 0.004) and 0.959 (p = 0.021) for BNP, NT-proBNP and their molar ratio, respectively, as well as with CKD stage, the ratio giving the highest correlation (R2 = 0.961, p = 0.003), followed by NT-proBNP (R2 = 0.909, p = 0.012) and BNP (R2 = 0.746, p = 0.059). Examination of the regression analyses of log geometric means of BNP, NT-proBNP against the CKD stage at different NYHA class reveals that only the molar ratio yields significant regressions for all cases (p<0.025), for each slope and essentially each intercept. The curves of the ratios for CKD1 through CKD5 have almost identical slopes, 0.250 (0.005) for class I, 0.218 (0.049) for class II, 0.241(0.017) for class III and 0.214 (0.025) for class IV. By ANOVA, there was no significant difference among the slopes for the molar ratios (p>0.95). For NT-proBNP
B-18 Elevated unprocessed brain natriuretic peptide (ProBNP1-108) is a powerful predictor of event free survival in stable moderate to severe African-American heart failure patients who have a low measured plasma brain natriuretic peptide (BNP1-32): Results from an A-HeFT substudy E. Rame1, A. H. Wu1, D. Dries2, T. Cappola3, W. Tam4, C. Larue5, I. Giuliani5, T. De Marco1, D. McGlothlin1, C. Hoopes1. 1University of California, San Francisco, San Francisco, CA, 2University of Pennsylvania, Philadelphia, PA, 3University of Pennsylvania, Philadephia, PA, 4NitroMed, Lexington, MA, 5Bio-Rad Laboratories, Marnes-la-Coquette, France
Background: Both BNP1-32 and the N-terminal fragment of the prohormone containing amino acid residues 1-76 (NT-proBNP1-76) are products of the cleaved
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Cardiac Markers
and BNP, not all the slopes were statistically different from zero. Conclusion: The molar ratio of NT-proBNP to BNP represents the most consistent description of the effect of decreased renal function by the NYHA class. Thus, examining the ratio may be more instructive than reference to either peptide alone. We recommended the use of the ratio together with their concentrations for further accurate interpretation in the diagnosis and management of combined heart and renal failure.

disease, hypertension, or heavy alcohol intake, receiving no cardiac medication; whose blood pressure was <160/90 mmHg as the mean of two readings; whose fasting blood glucose was <6 mmol/L, whose estimated creatinine clearance was >60 mL/ min/1.73 m2; and normal cardiac function (ejection fraction >50% by echocardiography or >55% by magnetic resonance imaging). The individual patient data from each study was then pooled and subdivided according to age cut-offs (<50, 50-60,50-75, <60,60-70, >70, 60-75, >75) and the 95% reference interval calculated for each dataset. Results: Data was obtained from 1086 individuals (521 males) from 3 centres in the UK, Denmark and the US. Median age was 47 years, range 29.6-86.7. There was a significant relationship between age and sex and NTproBNP levels. NTproBNP values where higher in women than men and increased with increasing age. Values converged and were not statistically significantly different between men and women over the age of 75. The reference intervals are summarised in table 1. Age Male n Median Lower Upper Female n Median Lower Upper <50 332 13.8 2.5 85.3 308 34.4 2.5 162.6 50-60 123 23.0 2.5 171.1 162 44.1 5.9 156.8 <60 455 17.0 2.5 110.7 470 39.7 2.5 158.3 50-75 181 25.0 2.5 172.1 248 49.5 7.0 177.9 60-75 58 43.0 5.1 212.4 86 69.3 7.2 200.2 60-70 49 33.9 3.9 216.8 76 61.6 7.0 181.3 >70 95 76.9 20.1 339.0 82 132.5 20.6 309.0 >75 All 84 113.9 20.0 357.0
B-20 The 99th centile reference limit for cardiac troponin I in a fully characterised reference population. P. O. Collinson1, O. Clifford-Mobley1, D. C. Gaze1, F. Boa1, R. Senior2. 1St George's Hospital, London, United Kingdom, 2Northwick Park Hospital, London, United Kingdom
Objective: To determine the assay performance and 99th centile of the Siemens Ultra assay using a well-defined subject population. Methods: 1392 general population subjects >45 years old were randomly selected from seven representative local community practices. Details were collected by questionnaires and subjects heart rate and blood pressure measurement (the average of two readings), spirometry, electrocardiography (ECG), echocardiography and they were venesected for fasting serum glucose, creatinine. Samples which were not analysed immediately were initially frozen to -20C then transferred for long-term storage to -70C and subsequently analysed for cardiac troponin I (cTnI). Total imprecision was using pools at 7 levels measured four times for five days. Five serum samples from post myocardial infarction patients were divided into three aliquots, stored 20 C, 4 C and -20 C and reanalysed after 1,2, 3, 6 and 13 days storage. All analyses were performed using the ADVIA centaur (Siemens Healthcare Diagnostics) using the manufacturers recommended methods and including manufacturer provided QC. The detection limit of the assay is 0.006 g/L with an upper limit of 50g/L. The claimed 10% CV is 0.03 g/L with a 99th centile of 0.04g/L. Troponin concentrations were calculated directly from the relative light units of the analyser by curve fitting from the calibration of the instrument. Normal subjects were defined as all attending general population subjects with no history of vascular disease, hypertension, or heavy alcohol intake, receiving no cardiac medication; whose blood pressure was <160/90 mmHg as the mean of two readings; whose fasting blood glucose was <6 mmol/L, whose estimated creatinine clearance was >60 mL/min/1.73 m2; and normal cardiac function. Precision was determined according to protocol EP 15A. Sample stability was assessed by one-way analysis of variance. All other analyses were performed by use of appropriate nonparametric statistical testing. All analyses were performed using the Analyse it add-in Excel (Analyse-it for Microsoft Excel (version 2.12), www.analyse-it.com). Results: 699/1392 ( 50.2%) of those invited attended for screening, (336 male) median age 58.0 years, interquartile range 53-71years. There were 309 normals (127 male) median age 53 years, interquartile range 49-62. Assay imprecision varied from 27.2% (0.0228 g/l) to 2.5% (0.79 g/l) with 10% imprecision at 0.045 g/l. Samples were stable for up to 6 days at 20oC, 3 days at 4oC and through 5 freeze thaw cycles. The 99th centile was 0.039 g/l (confidence interval 0.037-0.047) and was not influenced by age or gender. Conclusion: The 99th centile of the Siemens Ultra assay is in accordance with the manufacturers supplied data. Assay imprecision just meets current recommendations.
Conclusion: Reference intervals derived from this population were less than those recommended by the manufacturer and are comparable with rule out cut offs suggested for chronic heart failure.
B-22 Low levels of IgM antibodies cardiovascular disease in men. to phosphorylcholine predict
I. Dahlbom1, J. Su2, X. Hua2, A. Cederholm2, M. Halldin2, M. Hellenius2, M. Wikstrm2, H. Lettesj3, H. Grnlund2, J. Frostegrd2, U. de Faire2. 1Uppsala University, Uppsala, Sweden, 2Karolinska Institutet, Stockholm, Sweden, 3Athera Biotechnologies AB, Stockholm, Sweden
Objective. Immunogenic phosphorylcholine (PC) containing epitopes is expressed on apoptotic cells, oxidized LDL particles and several infectious agents The role of IgM antibodies against PC (anti-PC) in prediction of cardiovascular disease (CVD) and on macrophage uptake of OxLDL was investigated. Methods. From a screening of 4232 subjects at an age of 60 years, (2039 men and 2193 women), 211 incident cases of CVD (myocardial infarction, ischemic stroke, or hospitalized angina pectoris) and 633 age- and sex-matched controls were identified through a 5-7 year follow-up. Serum IgM anti-PC was determined by ELISA (CVDefine). The macrophage uptake of OxLDL before and after addition of affinity purified IgM anti-PC was studied by FACScan. Results: Relative risks (RR) with 95% confidence intervals (CI) of anti-PC levels adjusted for smoking, BMI, type II diabetes, hypercholesterolaemia, and high blood pressure yielded an increased risk for CVD only for subjects within the lowest quartile of anti-PC values with a RR of 1.37 ( CI 0.87 - 2.16 ). However, for men stronger associations were noted with successively increasing multivariately adjusted RRs from quartile 4 down to quartile 1. Men within quartile 1 (values below 29.7 U/ml) had a significantly increased RR of 1.96 (CI 1.09 - 3.55). Further adjustments for hsCRP gave essentially the same results. No enhanced risk was noted for women. Anti-PC extracted from a human IgM pool inhibited macrophage uptake of OxLDL. Conclusions. Our results indicates that low IgM anti-PC could be a novel risk marker for CVD among men, potentially by interfering with macrophage function in atherogenesis.
B-21 The REFerence INtEerval in the healthy (REFINE) study P. O. Collinson1, D. Gaze1, J. de Lemos2, P. Hildebrand3. 1St George's Hospital, London, United Kingdom, 2University of Texas Southwestern Medical Center, Dallas, TX, 3Copenhagen University Hospital Frederiksberg, Copenhagen, Denmark
Objective: To determine the reference interval for N-terminal pro B type natriuretic peptide (NTproBNP) measurement in a fully characterised healthy population. Method: In order to overcome the problems with existing published literature, we analysed a series of studies of populations where NTproBNP measurements had been performed and echocardiographic estimation of ejection fraction were available. All samples were analysed for NTproBNP using an Elecsys 2010 (Roche Diagnostics). The interassay %CV was 5.0 at 380 ng/L, 4.4 at 8700 ng/L, 5.0 at 13000 ng/L, with detection limit 20 ng/L and upper measuring limit 25000 ng/L. Normal subjects were defined as all attending general population subjects with no history of vascular
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B-23 Low levels of IgM antibodies against phosphorylcholine predict development of coronary artery disease in a population based cohort from Northern Sweden. I. Dahlbom1, H. Grnlund2, G. Hallmans3, J. H. Jansson3, K. Boman3, M. Wikstrm2, H. Lettesj4, U. de Faire2, J. Frostegrd2. 1Uppsala University, Uppsala, Sweden, 2Karolinska Institutet, Stockholm, Sweden, 3Ume University, Ume, Sweden, 4Athera Biotechnologies AB, Stockholm, Sweden
Objective: Phosphorylcholine (PC) is an epitope on oxidized low density lipoprotein (oxLDL) that may contribute to the atherogenicity of oxLDL. IgM antibodies against PC (anti-PC) are natural antibodies present ubiquitously in the population. The association between anti-PC and the incidence of myocardial infarction (MI) was investigated. Methods: Included were 462 incident cases of first MI events and 888 age- and sexmatched controls identified through 13 years of follow-up (1987-1999) of subjects included in the FIA-2 study from northern Sweden. Serum levels of anti-PC were measured by ELISA (CVDefine, Athera Biotechnologies, Sweden). Relative risks (RR) with 95% confidence intervals (CI) of incident MI with adjustments for age, gender, geographical region, hypertension, diabetes, BMI, smoking habits, scholesterol and hsCRP were determined. Results: Low anti-PC values were associated with increased risk of MI. Significant associations were found for values below 26.8 U/ml, corresponding to the lowest 25th percentile, and the highest association was seen below 16.9 U/ml, These results remained almost unaltered after adjustment for confounding factors (RR crude: 1.56, CI: 1.07-2.28 and RR adjusted, 1.69, CI: 1.09-2.54). Conclusion: Our results suggest that IgM anti-PC assessment adds independent information to the more traditional risk factors for MI. Low levels of natural IgM antibodies against PC could be an important risk marker for the development of MI development.
Cardiac Markerss
S100 concentrations were higher during general anaesthesia compared to local anaesthesia; however differences at each time point were not statistically significant. Conclusions: S100 concentrations rapidly increase in serum following cross clamping during CEA surgery, but return to baseline at 36 hours. S100 concentrations were higher but not statistically significant when general anaesthesia compared to local anaesthesia was used. Further studies are required using both methods of anaesthesia to fully investigate differences in S100 during CEA surgery. This meta-analysis of 4717 papers identified only 13 suitable studies for inclusion and consisted of 496 patients. There is a lack of published data supporting the kinetic release of S100 during CEA surgery. Furthermore, there is a lack of supporting evidence of the prognostic value of measuring S100 during CEA surgery.
B-25 Assessing the in vitro changes Ischemia Modified Albumin in relation to serum pH. D. C. Gaze, C. L. Seed, P. O. Collinson. St George's Hopsital & Medical School, London, United Kingdom
Objective: Ischemia modified albumin (IMA) is a Food and Drug Administration approved novel biomarker of cardiac ischemia. The in vitro stability of IMA is approximately 2-4hrs. Increases in IMA over time may be due to alteration in the sample pH. The in vitro changes to serum pH in relation to IMA were investigated. Clinical relevance: The clinical performance of IMA has been well characterised yet the assay suffers methodological and analytical pitfalls including sample stability, a factor paramount in the clinical interpretation of results in the emergency setting. Methods: Whole blood samples were collected in Vacutainer SSTTM tubes (Beckton Dickenson) containing silica cot activator gel. After clotting, samples were centrifuged at 3000rpm for 10 minutes (Spinchron, Beckman Coulter). Sample pH was measured by Ag/AgCl reference electrode (Mettler Toledo MP225 pH meter). Intra-assay %CV in pH was determined using pH solution standards (pH 7 and 10). IMA was determined using the albumin cobalt binding (ACB) spectrophotometric assay (Inverness medical). Analytical sensitivity was 14 kU/L, Inter-assay precision was 4.9-7.5% at 72.5-140.1kU/L, 95thcentile of 111 apparently healthy normals was 85 kU/L. Serum samples were obtained from apparently healthy normals (IMA <85kU/L) to serve as controls. Serum samples from known cardiac patients were also collected. Fresh samples were assayed for pH and IMA. The remaining serum was transferred to polypropylene screw-capped tubes in volumes of 1,2,4,5,8 and 10mL. Aliquots from each volume were assayed for IMA and pH at 30min intervals for 4hrs. Results: The intra-assay CV was 0.8 and 0.05% at pH 7 and 10 respectively. Samples in 1-4mL volumes had significantly higher pH compared to 10mL of serum. Both sample pH and IMA concentrations increased over time for each volume. IMA concentrations were significantly correlated to in vitro pH in fresh serum from both controls(r=0.98, (95%CI 0.93-0.99, p=<0.0001) and patient (r=0.98, 95%CI=0.960.99, p=<0.0001) samples. Conclusions: A kinetic increase in IMA occurs over time which is correlated to a change in sample pH. Increases in samples pH are greatest at smaller volumes probably due to increased air within the tube. IMA concentrations may be higher in sample tubes that are under filled giving rise to false positive results.
B-24 Meta-analytic calculation of the kinetic release of S100 protein in patients undergoing carotid endarteriectomy surgery. D. C. Gaze, A. Begum, P. O. Collinson. St George's Hospital & Medical School, London, United Kingdom
Objective: Measurement of S100 protein in serum has been considered to be a reliable and highly sensitive marker in the indication of ischaemic cerebral damage. The aim of this study was to analyse the kinetic release of S100 in patients undergoing carotid endarteriectomy (CEA) surgery by pooling studies in a metaanalysis. Clinical relevance: A small number of studies present conflicting information regarding the use of S100 during CEA surgery and data differs according to the use of either general or local anaesthesia. Methods: A Medline search (1996-2008) was used to source studies examining the release of S100 in CEA. Patient demographics, risk factors, type of anaesthesia and S100 concentrations were recorded on a standard proforma. Using double-entry techniques, S100 arterial blood concentrations pre-CEA, post cross clamping, and at various time intervals post-CEA up to 72 hours were recorded. Using a weighted mean method and generation of box-whisker plots, the serum S100 kinetic profile during CEA was constructed. Statistical differences in S100 concentrations at each time point from baseline were assessed as were differences in S100 kinetics when CEA was performed under general or local anaesthesia. Results: Of 4717 potentially relevant papers, 13 met the inclusion criteria and were abstracted. The analysis comprised 496 patients, 325(66%) male, median age 69 (95%CI=66-71) years. 72 (22%) patients had documented ischaemic heart disease. Only two studies reported a history of peripheral vascular disease (n=24). There were 120 (24%) episodes of previous myocardial infarction or angina and 36% of patients (n=181) had a history of previous cerebral vascular accident defined as stroke or transient ischaemic attack. 234 (47%) were hypertensive and 77 (16%) were either insulin dependent or non-insulin dependent diabetics. 99 (20%) subjects were known cigarette smokers. The weighted mean serum S100 prior to CEA surgery was 0.06 (95%CI = 0.04-0.11)g/L. The peak S100 was seen post-cross clamp 0.17, (95%CI=0.125-0.23)g/L. S100 concentrations decreased between 4-6hrs, were slightly elevated at 24hrs and returned to pre-CEA concentrations by 36hrs. Pre-CEA concentrations were significantly lower than those obtained post-cross clamp (p=0.0039), at 1hr post-surgery (p=0.04) and 24 hr post-CEA (p=0.0469). 389(78%) of patients received general anaesthesia and 107(22%) received local anaesthesia.
B-26 Effect of atorvastatin on CD40 ligand dependent induction of Eselectin expression in endothelial cells Z. Xia, Y. Li, M. Wang. Department of Clinical Laboratory, Renmin hospital of Wuhan University, Wuhan ,Hubei, China
Objective Beyond lipid lowering, statins have pleiotropic effects with favourable benefits against atherogenesis. The purpose of this study is to investigate the regulatory effects of atorvastatin on CD40 ligand (CD40L) induced E-selectin in human umbilical vein endothelial cells (HUVEC) and the association with signal pathway. Methods HUVEC were incubated with CD40L for 24 hours with or without pretreated by atorvastatin of the concentrations of 0.1, 1, or 10 mol/L. The protein and mRNA levels of E-selectin were detected by flow cytometry and reverse transcription polymerase chain reaction (RT-PCR). Extracellular signal regulated kinase (ERK1/2) activation was analyzed by western blotting. Results The expression of E-selectin mRNA was enhanced by CD40L in HUVEC with increased by 189.79% 21.56% (P<0.05) compared to control, while atorvastatin at 0.1~10 mol/L could decrease E-selectin mRNA in a concentration-dependent manner. The expression of E-selectin on HUVEC surface in CD40L stimulation group was significantly higher
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than that in control (P<0.05). Atorvastatin at 1 mol/L reduced E-selectin protein expression by 48.68% compared to CD40L stimulation group, with the maximal inhibition achieved as much as 70.25% at the concentration of 10 mol/L. At same time, atorvastatin inhibited CD40L induced phosphorylation of ERK1/2, the expression of phosphorylation of ERK1/2 decreased by 81.23%5.64%, 72.67%5.48% and 41.35%4.82% compared to CD40L stimulation group, respectively. Conclusion These results indicate that atorvastatin inhibited CD40L induced E-selectin expression in HUVEC through the inhibition of the activation of ERK1/2 pathway.

Data are expressed as median or n(%) Increased hsTnT (>0.018ng/ml) + (n=46) - (n=40) Age(year) 72 73 Ischemic etiology 65% 25% NYHA functional class IV 83% 68% Left ventricular ejection fraction (LVEF) 40% 46% Estimated glomerular filtration rate 50 63 (eGFR;mL/min/1.73m2) N-terminal pro-BNP (NT-proBNP;pg/ml) 4605 2175 Heart-type fatty acid-binding protein (H5.7 4.5 FABP;ng/ml) P value NS 0.0005 0.10 0.04 0.02 0.0003 0.01
B-27 Utility of Very Low Serum Concentrations of Cardiac Troponin T for Stratifying Risk of Cardiovascular Disease in General Population J. Ishii, F. Kitagawa, A. Kuno, A. Nakashima, H. Naruse, S. Matsui, T. Nakano, T. Ishikawa, I. Tanaka, Y. Ozaki. Fujita Health University, Toyoake, Japan
To investigate the utility of a new precommercial highly sensitive cardiac troponin T (hsTnT) assay (lower detection limit of 0.001ng/mL) for stratifying the risk of cardiovascular disease (CVD), cross-sectional study was conducted for 206 health checkup examinees without chronic kidney disease (estimated glomerular filtration rate [GFR] <60mL/min/1.73m2). Results: 1) Cardiac troponin T was detectable in all subjects with a new hsTnT assay compared with 0% with a traditional assay (lower detection limit of 0.01ng/mL). 2) Forty-eight patients had possible CVD defined as a history of CVD and/or abnormal electrocardiographic findings. 3) In 98 apparently healthy subjects who had no possible CVD, and no history of hypertension or diabetes, 95th upper limit for hsTnT was 0.010ng/mL. 4) Clinical characteristics between subjects with and without increased hsTnT (>0.010ng/mL) were shown in Table. Data are expressed as median or % Increased hsTnT + (n=32) - (n-174) P value (>0.010 ng/ml) Age(years) 75 70 NS Male 56% 49% NS Systolic blood pressure(mmHg) 145 138 0.005 79 74 NS Estimated GFR(mL/min/1.73m2) NT-proBNP(pg/ml) 111 66 0.0007 High-sensitive C-reactive protein(mg/L) 0.97 0.46 0.0005 10-year risk of developing coronary heart 16% 10% 0.002 disease using Framingham risk score Possible CVD 44% 20% 0.006 Conclusion: A new hsTnT assay may be useful for stratifying the cardiovascular risk in general population.
B-29 Clinical Utility of Very Low Concentrations of Cardiac Troponin T for after Additional Treatment in Patients with Chronic Heart Failure F. Kitagawa, J. Ishii, A. Kuno, A. Nakashima, H. Naruse, S. Matsui, T. Nakano, T. Ishikawa, I. Tanaka, Y. Ozaki. Fujita Health University, Toyoake, Japan
Circulating cardiac troponin T (TnT) predicts adverse outcome in patients with chronic heart failure but is detectable in only a small fraction of those with stable chronic heart failure. We prospectively evaluated the clinical utility of a new precommercial highly sensitive TnT (hsTnT) assay (lower detection limit of 0.001ng/ mL) on discharge in 77 chronic heart failure patients without detectable TnT with a traditional assay (lower detection limit of 0.01ng/mL). Results: 1) TnT was detectable in all patients with a new hsTnT assay. 2) During a mean follow-up period of 23 months, there were 25 (33%) cardiac events (3 cardiac deaths and 25 hospitalizations for worsening chronic heart failure. 3) Clinical and biochemical parameters between chronic heart failure patients with and without increased hsTnT (>median value of 0.015ng/mL) were shown in Table. Data are expressed as median or n(%) Increased hsTnT (>0.015ng/mL) + (n=40) - (n=37) Male 68% 49% Age(years) 77 67 Estimated glomerular filtration rate 45 61.4 (eGFR;mL/min/1.73m2) N-terminal pro-BNP 1498 541 (NT-proBNP;pg/ml) Heart-type fatty acid-binding protein (H5.5 4.2 FABP;ng/ml) Cardiac event 40% 24% Cardiac death 8% 0% P value NS 0.004 0.002 <0.0001 <0.0001 0.1 0.2
Conclusion: A new hsTnT assay may be useful for the risk stratification after additional treatment in chronic heart failure patients without undetectable TnT with a traditional assay.
B-28 B-30 Clinical Utility of Very Low Concentrations of Cardiac Troponin T on Admission in Patients Hospitalized for Worsening Chronic Heart Failure F. Kitagawa, J. Ishii, A. Kuno, A. Nakashima, H. Naruse, S. Matsui, T. Nakano, T. Ishikawa, I. Tanaka, Y. Ozaki. Fujita Health University, Toyoake, Japan
We prospectively evaluated the clinical utility of a new precommercial highly sensitive cardiac troponin T (hsTnT) assay (lower detection limit of 0.001ng/mL) on admission in 86 patients hospitalized for worsening chronic heart failure without detectable troponin T concentration with a traditional assay (lower detection limit of 0.01ng/mL). Also, troponin T concentrations were measured with both hsTnT and traditional assays in 98 normal volunteers. Results: 1) In normal volunteers, troponin T was detectable in 100% with a new hsTnT assay, but was in 0% with a traditional assay. 2) HsTnT concentration was significantly higher in chronic heart failure patients than normal volunteers (median value: 0.018 versus 0.005ng/mL, P <0.0001). 3) Clinical characteristics between chronic heart failure patients with and without increased hsTnT (>median value of 0.018ng/mL) were shown in Table. Conclusion: A new hsTnT assay may be useful for evaluating the severity of heart failure on admission in patients hospitalized for worsening chronic heart failure at previously undetectable troponin T concentration.
Utility of Very Low Serum Concentrations of Cardiac Troponin T for Early Risk Stratification in Patients with Acute Coronary Syndrome A. Kuno, J. Ishii, F. Kitagawa, A. Nakashima, H. Naruse, S. Matsui, T. Nakano, T. Ishikawa, I. Tanaka, Y. Ozaki. Fujita Health University, Toyoake, Japan
We prospectively evaluated the clinical utility of a new precommercial highly sensitive cardiac troponin T (hsTnT) assay (lower detection limit of 0.001ng/mL) for the early risk stratification in 82 patients with acute coronary syndrome without detectable TnT with a traditional assay (lower detection limit of 0.01ng/mL). Also, serum TnT concentrations were measured with both hsTnT and traditional assays in 98 normal volunteers. Results: 1) Acute myocardial infarction, defined as a peak value of cardiac troponin I more than the upper reference limit of 0.10ng/mL, was present in 49 patients. 2) In normal volunteers TnT was detectable in 100% with a new hsTnT assay, but in 0% with a traditional assay. 3) HsTnT concentration was significantly higher in patients with acute coronary syndrome than normal volunteers (median value: 0.018 versus 0.005ng/mL, P<0.0001). 4) Clinical characteristics between acute coronary syndrome patients with and without increased hsTnT (>median value of 0.018ng/mL) were shown in Table.
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Data are expressed as median or %; GFR = glomerular filtration rate; Increased hsTnT >0.018ng/mL - (n=42) + (n=40) P value Age (year) 67 66 NS Acute myocardial infarction 38% 70% 0.005 Emergent coronary intervention 17% 41% 0.02 Biochemical value on admission 78 70 0.06 Estimated GFR (mL/min/1.73m2) Increased H-FABP (>the upper reference limit 10% 53% <0.0001 of 6.2ng/mL) CK-MB (ng/mL) 1.4 1.6 NS Myoglobin (ng/mL) 27 40 0.004 NT-proBNP (pg/mL) 179 192 NS Conclusion: A new hsTnT assay on admission may be useful for the early risk stratification of patients with acute coronary syndrome at previously undetectable TnT concentrations.
Cardiac Markers
B-32 Polymorphisms in intercellular adhesion molecule-1 gene and their association with coronary heart disease in Han population in China M. Wang1, W. Li2, Y. Li1. 1Renmin Hospital of Wuhan University, Wuhan, China, 2Department of Hematology, Renmin Hospital of Wuhan University, Wuhan, China
Objectives: Epidemiologic studies and randomized clinical trials have provided compelling evidence that coronary heart disease (CHD) is largely an inflammatory disease. However, there is also reason to believe that there is a heritable component to the disease. Intercellular adhesion molecule-1 (ICAM-1) has been reported be involved in pathogenic mechanism of CHD. The present study aimed to explore the distribution of Gly241Arg and Lys469Glu polymorphisms of ICAM-1 gene, and to assess the association between ICAM-1 gene polymorphisms and CHD in Han population in China, as well as the interactions of the polymorphisms and the related risk factors of CHD. Methods: Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and PCR sequence-specific primers (PCR-SSP) were used for the detection of ICAM-1 genotypes in 211 CHD patients and 206 controls. Binary Logistic regression analysis was made to explore the risk factors for CHD. The serum sICAM-1 and high sensitivity C-reactive protein (hsCRP) levels were measured by ELISA and turbidimetric method, respectively. Results: The frequencies of Lys/Lys, Lys/Glu and Glu/Glu Genotypes of Lys469Glu were 55.5% and 44.7%, 38.9% and 46.1%, 5.7% and 9.2%, patients and controls respectively. The frequencye of Lys/Lys genotype in CHD patients was significantly higher than in controls (P<0.05), and the relative risk suffered from CHD of Lys/Lys genotype was 1.54 times of the Glu/Glu and Lys/Glu genotypes (OR=1.54,95%CI:1.05~2.27). In a multivariate Logistic regression analysis, hypertension, diabetes mellitus, cigarette smoking, high triglyceride levels, old age, white blood cell counting and genotype Lys/Lys were all associated with CHD. As for the interaction between gene and related exposure factors, genotype Lys/Lys strengthen the effect of hypertension and diabetes mellitus exposure (interaction index r>1.00, respectively), and weaken the effect of cigarette smoking exposure (r<1.00), both being multiplicative interaction model. However, we didnt detect the Gly241Arg polymorphism in this study. The serum sICAM-1 and hsCRP levels of the patients were significantly higher than that of the controls (P<0.05), however, among different genotypes, patients and controls respectively, there were no significant differences. Conclusions: The coding single nucleotide polymorphism at codon 469 in the exon 6 of ICAM-1 gene is positively associated with the risk of CHD and the allele Lys may be a risk factor for CHD in Chinese, and an interaction exists between this genotype and related exposure factors. There is no Gly241Arg polymorphism in Chinese.
B-31 THE SIGNIFICANCE OF HEAT SHOCK PROTEIN 70 IN PATIENTS UNDERGOING CARDIAC SURGERY H. Turner, J. McNeilly, J. Reeve, R. Peake, W. Mutch, D. Rae, P. Gibson, G. Hillis, B. Cuthbertson, B. Croal. Aberdeen Royal Infirmary, Aberdeen, United Kingdom
Introduction: Heat shock protein 70 (Hsp70) is an abundant, soluble, intracellular protein that is induced under stressful conditions such as ischaemia-reperfusion injury and may participate in myocardial protection. We assessed the significance of pre and post-operative levels of Hsp70 in predicting subsequent mortality in patients following cardiac surgery. Methods: Pre- and post-operative (6-hour) serum Hsp70 (ELISA, StressgenBioscience) were measured in 268 patients undergoing cardiac surgery. The change in Hsp70 levels following surgery was also calculated (Delta-Hsp70). Patients were followed up for mortality for 1 year. Results: Patients who died by 1-year demonstrated a higher post-operative Hsp70 (18.64 v 12.81 pg/ml;p=0.001) and higher Delta-Hsp70 (2.34 v -6.91;p=0.001), however pre-operative Hsp70 levels tended to be lower in patients who died (28.48 v 56.85 pg/ml;p=0.130). Kaplan-Meier curve survival analysis demonstrated the significance of increasing levels of Hsp70 on mortality (Figure 1). Delta-Hsp70 was predictive of mortality in a multivariate logistic regression model even when adjusted for Parsonnet and EuroSCORE (currently accepted clinical objective assessments of risk). In a similar multivariate model, patients in the lowest pre-operative Hsp70 quartile and highest post-operative Hsp70 quartile were almost four-times as likely to subsequently die (OR 3.7; p=0.002) than the other patients.
B-33 Performance characteristics of the ErennaTM cTnI assay for in vitro diagnostic use in the Singulex Clinical Laboratory. S. Karimi, P. Simpson, K. Bui, E. Quan, L. Le, S. Agee, G. Baechler. Singulex, Inc., Alameda, CA
Background: Measurement of cardiac Troponin-I (cTnI) has been designated the gold standard for diagnosis of acute myocardial infarction and as indicative of cardiac disease. Measurement of cTnI can be used for diagnosis of acute myocardial infarction and in the risk stratification of patients with non-ST segment elevation acute coronary syndromes, with respect to relative risk of mortality, myocardial infarction, or increased probability of ischemic events requiring urgent revascularization procedures. Recently a new ultra-sensitive assay for in vitro diagnostic measurement of cTnI has become available in the Singulex Clinical Laboratory (CLIA#:05D1092709, CLF#:338067). This assay utilizes the Erenna Immunoassay System, which is based on single-molecule detection. Objective: Evaluate the analytical performance of the Erenna cTnI assay for the Singulex Clinical Laboratory. Methods: Analytical accuracy and spike recovery were determined by preparing and testing spiked samples of EDTA plasma from a single donor. This plasma was spiked with cTnI standard from NIST (SRM 2921, Gaithersburg, MD) to generate a series of samples ranging from 5.2 - 52 pg/mL. Assay precision and lot-to-lot variability of assay reagents were assessed using two controls prepared by spiking two EDTA plasma samples to levels of 6 and 55 pg/mL, respectively, with a cTnI reference standard from HyTest (Turku, Finland). A reference range for the Erenna cTnI diagnostic assay was established using specimens from 153 apparently healthy blood donors (122 male, 31 female, average age 34.5 yrs, age range 19 - 63 yrs), and the 99th%, 10%CV and inter-quartile reference ranges were determined.
Conclusion: High levels of pre-operative Hsp70 may relate to a high cardioprotective state that translates to better outcome following cardiac surgery, whereas the poor prognostic finding of high post-operative Hsp70 may reflect a more significant peri-operative ischaemia-reperfusion injury. Patients with a combination of low pre- and high post-operative Hsp70 may benefit from more aggressive postoperative support and follow-up.
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Results: Analytical sensitivity of the Erenna cTnI assay was 0.2 pg/mL (0.0002 ng/L) with a LLoQ of 1.0 pg/mL with good linearity (1 - 70 pg/mL, slope = 1.01, R2 = 0.999) and spike recovery (98-103%) of the HyTest cTnI material. Analytical accuracy, determined using NIST-spiked plasma, was >85% over a range of 6 pg/mL to 50 pg/mL (y = 0.9x - 0.3261, R2 = 0.9993) with average spike recovery of 89.1% (range 85.4-91.5%). Within- and between-run precision varied between 6-8%, with acceptable lot-to-lot variability (<15%) in testing of HyTest-spiked control plasma. Using donor plasma (n=153), the distribution of cTnI concentration was Gaussian, with a mean (+/-SD) of 1.97 (+/-1.85) pg/mL, and a calculated 99th percentile value of 6.28 pg/mL (<10%CV). Conclusions: The Erenna cTnI assay shows excellent analytical performance in the Singulex Clinical Laboratory, with a 99th% cut-off value far below that of other clinically available in vitro diagnostic immunoassay systems. CLIA-regulated availability of this new, ultra-sensitive assay is an important step towards enabling early disease detection and clinical investigations into cardiac disease.

B-35 USE OF MYELOPEROXIDASE LEVELS FOR THE EARLY IDENTIFICATION OF CORONARY ARTERY DISEASE IN CHEST-PAIN PATIENTS AT THE EMERGENCY DEPARTMENT K. Makris1, C. Demponeras2, P. Petraki3, F. Zoubouloglou1, S. Potamitis1, N. Kafkas2, T. Assimakopoulou3, A. Haliassos4, I. Drakopoulos1, D. Babalis2. 1Clinical Biochemistry Department, KAT General Hospital, Athens, Greece, 2Cardiology Department, KAT General Hospital, Athens, Greece, 3Laboratory Hematology Department, KAT General Hospital, Athens, Greece, 4National External Quality Assessment Scheme in Clinical Chemistry (ESEAP), Athens, Greece
Myeloperoxidase (MPO) has been shown to play a role in the pathogenesis of coronary artery disease (CAD). It is released from activated neutrophils at sites of vascular damage and has been found increased in atherosclerotic plaques before the onset of myocardial injury. Elevations of MPO may occur independently of CRP and other markers of inflammation changes and has been suggested as a marker of risk independent of troponin elevations in acute coronary syndrome (ACS) patients. The aim of the study was to evaluate the MPO measurement for the discrimination of patients with CAD from patients admitted with acute chest pain to the emergency department (ERD) of our hospitals. Forty-four patients presented within 6 hours from the onset of chest pain included in our study. Blood samples were obtained at admission and 24 hours after the onset of chest pain. MPO, CRP, troponin-I (TnI) were measured on an immunochemistry analyzer (Abbott Architect-16200, Abbott Park, IL), interleukin-6 (IL6) with a chemiluminescent assay on Siemens-Immulite-1000 (Siemens Medical Solutions Diagnostics, Los Angeles, CA), Fibrinogen, and von-Willebrant activity (vWf) were measured on Siemens-BCS coagulation system. Using clinical evaluation (ECG) and laboratory results (Troponin-I) patients were divided into 3 groups. Group-I included patients with non-cardiac etiology chest pain (ECG and TnI-negative), group-II patients with unstable angina (ECG-positive but TnI-negative) and group-III patients with myocardial infraction (both ECG and TnI-positive). Sixteen healthy males served as controls. The following table summarizes our results: N Male/female Mean age (yrs) MPO at admission (g/L) IL-6 at admission (pg/mL) CRP at admission (mg/ dL) Fibrinogen at admission (g/L) vWf at admission (%) Group-I Group-II Group-III Controls P(anova) 10 18 16 16 7/3 12/6 11/5 16/0 54+5 56+4 55+5 51+6 358+85 1915+1084 2022+923 352+136 <0.001 3.24+0.63 8.53+6.61 10.87+5.63 2.33+0.34 <0.001 0.14+0.09 0.99+0.65 1.32+0.36 0.11+0.06 4.45+0.93 6.12+1.05 6.38+1.29 4.20+0.99 103+11 134+34 195+35 101+10 <0.001 <0.05 <0.001
B-34 NT-ProBNP, Ankle Brachial Pressure Index and Pulse Wave Velocity in the prognosis of patients presenting with Acute Coronary Syndrome J. L. Reeve1, H. Turner1, V. Mills2, J. D. McNeilly3, R. Peake1, J. Allison1, G. Hillis2, R. Soiza2, B. Croal1. 1Department of Clinical Biochemistry, Aberdeen Royal Infirmary, Aberdeen, United Kingdom, 2Department of Cardiology, Aberdeen Royal Infirmary, Aberdeen, United Kingdom, 3Department of Clinical Biochemistry, Royal Hospital for Sick Children, Glasgow, United Kingdom
Background: Risk stratification is essential to select the most suitable management strategy for patients presenting with Acute Coronary Syndromes (ACS). Pulse-Wave Velocity (PWV) and Ankle-Brachial Pressure Index (ABPI) are rapid, non-invasive techniques for assessment of arterial function. We have assessed these along with NTerminal ProBNP (NT-ProBNP) for the prediction of subsequent mortality in patients presenting with chest pain. Methods: Plasma NT-ProBNP (Siemens Immulite 2000) was measured within 48 h of admission (n=234). PWV and ABPI were successfully measured in a cohort of these patients (n=120). Patients were followed up at 6 months from admission to assess all cause mortality. Results: NT-ProBNP levels were higher in patients who died within 6 months compared to those who did not (5718 v 765pg/ml; p=0.001). Kaplan Meier survival curve analysis demonstrated a mortality of 22.4% for those patients with a NTProBNP in the upper quartile compared to a mortality of 3.4% for the rest (p<0.001). In a regression model, these patients showed a 22 times higher risk of death even when adjusted for age and sex (OR 22.7; p<0.001). PWV was not predictive of outcome (p=0.806), however those patients with an ABPI below the median had a mortality of 11.9% versus zero mortality in those with a level above the median (p=0.007). In a multivariate regression model, both NT-ProBNP and ABPI remained as independent predictors of outcome even when adjusted for each other (OR 1.147 per 1000ng/L, p=0.005; OR 0.004, p=0.014, respectively). Patients with an ABPI below the median had higher levels of NT-ProBNP (1314 v 394ng/L; p=0.007) compared to those with an ABPI above the median.
Myeloperoxidase values are statistically significantly higher in patients with CAD than in patients with chest-pain of non-cardiac etiology and controls, but no significant difference was observed in the values of unstable angina and myocardial infraction groups (t-test p=ns). We can conclude that Myeloperoxidase measurement can be used as a marker for the presence of CAD early in the diagnostic evaluation of acute chest pain patients.
B-36 A new immunoassay for determining Haptoglobin 2-2 phenotype to predict risk of mycardial infarction and cardiovascular death in individuals with Diabetes Mellitus J. Victor1, N. Levy2, R. Miller-Lotan2, S. Blum2, A. P. Levy2, W. Cheong3, J. S. Chen3. 1Synvista Therapeutics, Inc., Montvale, NJ, 2Technion University, Haifa, Israel, 3BIOCHECK, Inc., Foster City, CA
Objective: To validate the HAPTOCHEKTM immunoassay for use in predicting risk of individuals developing myocardial infarction (MI) and cardiovascular death (CV death) in patients with Diabetes Mellitus (DM). Relevance: Recently published longitudinal studies have shown that the Haptoglobin (Hp) 2-2 phenotype is predictive of cardiovascular disease in patients with diabetes. Hp is an acute serum protein which acts as an antioxidant by binding free hemoglobin, a potent oxidizing agent, and removing it from circulation. Hp exists as one of three phenotypes; Hp 1-1, Hp 2-1 or Hp 2-2. The Hp 2-2 phenotype is an inferior
Conclusions: Both NT-ProBNP concentration and ABPI are good predictors of mortality following presentation with ACS. These qualities appear to be independent from each other. Such measurements may assist in the risk stratification of these patients.
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antioxidant and the Hp 2-2-hemoglobin complex clears at a much slower rate. The Hp 2-2-hemoglobin complexes then localize with HDL which causes oxidation of HDL proteins and impaired reverse cholesterol transport. These factors are thought to lead to increased cardiovascular complications in Hp 2-2-diabetes. Methodology: We have developed an enzyme immunoassay that can determine whether a patient with DM possesses the Hp 2-2 phenotype. The assay uses a capture monoclonal antibody on microtiter strips and a horseradish peroxidase labeled monoclonal for detection. The assay requires just 10 uL of serum or plasma and can be completed in under two hours. Validation: Precision studies with the immunoassay were carried out by running 8 replicates of three Positive Controls over 10 days at two clinical sites. Intra-assay variation was < 6.7% and inter-assay variation was < 8.0% for all samples tested. Heparin, citrate and EDTA plasma and serum were collected from 26 individuals and run in the assay. The plasma results correlated well with serum values (R2 = 0.9976). When potential interferents (hemoglobin, triglycerides, bilirubin, protein, ascorbic acid, atorvastatin, niacin, pravastatin, warfarin, acetaminophen, tolbutamide, aspirin, fenofibrate, diphenhydramine, lisinopril and metformin) were tested at various doses in multiple serum samples, all but hemoglobin were within + 10% of control. As hemoglobin does elevate the readings in some samples, hemolyzed samples are not recommended. Primary Endpoint Analysis was performed on 2110 banked serum samples from the ICARE (Israel Cardiovascular Atherosclerosis Risk and vitamin E) Study whose Hp phenotype was determined by using the ICARE reference method, gel electrophoresis, and the HAPTOCHEKTM immunoassay. Agreement between both Hp phenotyping methods was > 98%. Both methods showed greater than a 2-fold increase in the Primary Composite Endpoint (MI, stroke, CV death) and with the same probability (P = 0.0002) for Hp 2-2 DM patients than for Hp 1-1 or Hp 2-1 DM patients. Both methods showed a significant risk for Hp 2-2 DM patients developing MI (P = 0.01) or CV death (P = 0.001). Log-Rank P values derived from KaplanMeier plots for both methods are virtually identical (Log Rank P = 0.0016 for gel electrophoresis and Log-Rank P = 0.0017 for the HAPTOCHEKTM immunoassay). Conclusions: The HAPTOCHEKTM immunoassay can aid in predicting the risk of developing MI and CV death in patients with DM by determining their Hp 2-2 phenotype.
Cardiac Markers
bilirubin (conjugated or free, up to 30 mg/dL), hemoglobin (up to 500 mg/dL), lipids (INTRALIPID, Fresenius Kabi AB Corporation; up to 1000 mg/dL), and rheumatoid factor (up to 1040 IU/mL). The analytical linearity range for this new method was 0.05-10 mg/L, with functional sensitivity at 0.16 mg/L. Conclusion: The data demonstrate that the CardioPhase hsCRP assay is suitable for routine laboratory use on the ADVIA Chemistry Systems from Siemens Healthcare Diagnostics.
B-38 Standardization of CardioPhase hsCRP Assay with IFCC Reference Material CRM 470 on the ADVIA Chemistry Systems from Siemens Healthcare Diagnostics M. Guo, S. Cherian, V. Streva, J. Dai. Siemens Healthcare Diagnostics Inc, Tarrytown, NY
Introduction: A new CardioPhase high-sensitivity C-reactive protein (hsCRP) method was developed by Siemens Healthcare Diagnostics on the ADVIA Chemistry Systems. This method is intended for the quantitative determination of the C-reactive protein (CRP) concentration in human serum and plasma. High-sensitivity CRP measurements may be used as an independent risk marker for the identification of individuals at risk for future cardiovascular disease and may be useful as an independent marker of prognosis for recurrent events in patients with stable coronary disease or acute coronary syndromes. Objective: This study focused on the standardization of the newly developed hsCRP assay using the IFCC reference material CRM 470. The accuracy of the standardized assay is further confirmed through the linearity study and the correlation study with the gold standard methodology, the existing nephelometric CardioPhase hsCRP method on the Siemens BN II System. Materials and Methods: This new method uses synthesized latex particles coated with polyclonal anti-human CRP antibody (rabbit). Calibrators are prepared by spiking normal human sera with recombinant human CRP. Calibrator value assignment is traceable to the IFCC reference material CRM 470. A correlation study was performed against the nephelometric CardioPhase hsCRP assay on the BN II System, and linearity was evaluated with nine equally spaced levels of samples prepared using low (0 mg/L CRP) and high (10 mg/L) linearity pools. Results: Values of the calibrators for the new CardioPhase hsCRP assay were assigned using CRM 470, and the assigned values were validated with serial dilutions of CRM 470 using two lots of reagents and two lots of calibrators. Recoveries of the undiluted and diluted CRM 470 were within 95% tolerance down to a CRP level of about 0.5 mg/L. Further confirmations of this standardization were achieved by a comparison study with the existing nephelometric CardioPhase hsCRP assay on the BN II System, and the results showed excellent correlation between the two methods using linear regression analysis (slope = 1.011, intercept = 0.013 mg/L, r = 0.9978). The linearity study showed a satisfactory linear relationship between the measured results and the expected values covering the entire analytical range (0.16 to 10 mg/L) and the extended range (0.16 to 200 mg/L). Conclusion: The newly developed CardioPhase hsCRP method on the ADVIA Chemistry Systems can measure serum or plasma CRP concentrations precisely and accurately at the low range (0.16 to 10 mg/L) and is suitable for routine clinical chemistry laboratory use.
B-37 A New CardioPhase High-Sensitivity C-Reactive Protein Assay on the ADVIA Chemistry Systems from Siemens Healthcare Diagnostics M. Guo1, C. Voyagis2, A. Bartzeliotou3, S. Cherian1, V. Streva1, I. Papassotiriou3, J. Dai1. 1Siemens Healthcare Diagnostics Inc, Tarrytown, NY, 2Siemens Healthcare Diagnostics Inc, Athens, Greece, 3Aghia Sophia Childrens Hospital, Athens, Greece
Introduction and Objectives: A prognostic value for measuring CRP has been suggested from studies with cardiac patients where elevated levels of CRP were associated with a higher risk of having a future cardiac event. Elevated levels of CRP have been associated with poor prognosis in cases of stable angina, unstable angina, and myocardial infarction. Recently, a new CardioPhase high-sensitivity C-reactive protein (hsCRP) method has been developed for the ADVIA Chemistry Systems (ADVIA 1200, 1650, 1800, and 2400) from Siemens Healthcare Diagnostics. This in vitro diagnostic method is intended to measure the concentration of CRP in serum and plasma, focusing on the low range (<10 mg/L) analyses associated with the potential prediction of cardiovascular and peripheral vascular disease. The CRP concentration measured by this new method may be used as an independent marker for the identification of individuals at risk for future cardiovascular disease. The assay is a latex-enhanced immunoturbidimetric assay; the turbidity signal is measured at 571 nm. This study evaluated the performance of this newly launched CardioPhase hsCRP assay on the ADVIA Chemistry Systems. Materials and Methods: The performance evaluation included precision, interference, linearity, and correlation. The data were collected with the ADVIA 1650, 1800, 2400, and 1200 Chemistry Systems; and method correlations were made between the existing nephelometric CardioPhase hsCRP method on the BN ProSpec system and the new method on the ADVIA Chemistry Systems. All ADVIA Chemistry Systems use the same CardioPhase hsCRP reagent packs, calibrators, and commercial controls. Results: The newly launched CardioPhase hsCRP assay on the ADVIA Chemistry Systems showed excellent low-end precision performance: at a CRP level of 0.16 mg/ L, within-run and total CVs were 5.3% and 6.8%, respectively. At the medical decision levels, ~1 mg/L and ~3 mg/L of CRP, the total CVs were 1.2% and 1.3%, respectively. The assay correlated well with the existing nephelometric CardioPhase hsCRP method on the BN ProSpec (y = 1.00x + 0.007; r = 0.998, n = 90, sample range: 0.16-8.27 mg/L). The new method also showed <10% interference with
B-39 Myeloperoxidase Improves Risk Stratification in Patients Presenting With Normal Cardiac Troponin I K. M. Schulz1, S. W. Smith1, L. A. Pearce2, R. Ler1, M. M. Murakami1, F. S. Apple1. 1Hennepin County Medical Center, Minneapolis, MN, 2Biostatistical Consulting, Minot, ND
Myeloperoxidase (MPO) is thought to play a role in the development of atherosclerosis and as a biomarker of plaque rupture. The purpose of this study was to assess the ability of MPO to identify patients presenting with ischemic symptoms suggestive of acute coronary syndrome (ACS) who would be at risk for major adverse cardiac events (MACE). EDTA plasma was collected in 400 patients presenting to the emergency department, 13% (n=50) with myocardial infarction (MI). Cardiac troponin I (cTnI) was measured by the Stratus CS System from Siemens; 99th percentile reference value of <0.1 g/L as established in the local clinical laboratory. MPO was measured by both the Dimension and Dimension Vista assays; both with a 95th percentile reference value of 633pM. Cardiac events (first of cardiac death,
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MI, need for revascularization) were assessed within 30 days and at 6 months. After adjusting for age, sex, and renal function, an increased cTnI (hazard ratio, HR = 3.9, 95% CI 2.4, 6.4) and an increased Dimension MPO (HR = 2.7, 95% CI 1.3, 5.6) drawn at admission were independently predictive of MACE within 30 days, and remained predictive at 6 months (HR = 2.4, 95% CI 1.3, 4.6). Similar results were observed for the Vista MPO assay (HR = 4.0, 95% CI 1.3, 13; and 3.4, 95% CI 1.2, 9.3, at 30 days and 6 months, respectively). Those with a normal cTnI and an increased Dimension MPO (> 633 pM) had a significantly higher cumulative MACE rate compared to those with lower MPO at 30 days (16.1% vs. 3.6%, p=0.002) and 6 months (18.1% vs. 5.0%, p = 0.002); results were similar using the Vista MPO assay (14.3% vs. 2.8%, p = 0.01; 16.6% vs. 2.8%, p = 0.007 respectively). Patients who were not diagnosed with an MI during their index hospitalization (n=350), and had an increased MPO > 633 pM (n=234), had an increased risk of MACE (HR = 3.7, p=0.03) after adjusting for age, sex and renal function. In conclusion, a combination of MPO with cTnI allowed for the identification of a greater proportion of patients at risk for MACE at both 30 days and 6 months than the use of cTnI alone. MPO, a biomarker indicative of atherosclerotic, plaque destabilization, i.e. pre-myocardial cell death, was able to identify patients presenting with ischemic symptoms suggestive of ACS and without increased cTnI, the biomarker indicative of myocardial cell death, who are at risk of a future cardiac event. 99th

the percentiles was determined along CLSI guidelines. Analytical characteristics of the assay were: LoB <0.098 pg/mL, LoD 0.091 pg/mL, and LoQ (10% CV) 0.88 pg/mL. For all subjects the 99th percentile value was 10.19 pg/mL (0.01019 g/L); mean concentration 1.45 pg/mL (95% CI 1.16 to 1.73), range 0.2 to 34.56 pg/mL. By gender, the male 99th percentile value was 16.58 pg/mL; mean concentration 1.72 pg/ mL (95% CI 1.18 to 2.27). The female 99th percentile value was 9.36 pg/mL, mean concentration 9.36 pg/mL (95% CI 0.94 to 1.55); but not statistically different than the male 99th percentile value (p=0.108). Both the male and female cTnI values were Gaussian distributions. In conclusion, cTnI measured by the high sensitive Erenna cTnI assay measures 100% of normal subjects, allowing prospective diagnostic and risk assessment studies to be performed. Such studies with high sensitive troponin-I assays are essential for early detection of cardiac disease and management of patients presenting with symptoms suggestive of acute coronary syndrome.
B-42 Assessment of Multiple Biomarkers for Diagnosis of Myocardial Infarction in Patients Presenting with Symptoms Suggestive of Acute Coronary Syndrome F. S. Apple1, R. Glaser2, W. F. Peacock3, W. L. Roberts4, A. H. Wu5. 1Hennepin County Medical Center & University of Minnesota School of Medicine, Minneapolis, MN, 2Cardiology Consultants, Villanova, PA, 3Cleveland Clinic, Cleveland, OH, 4ARUP Laboratories & University of Utah, Salt Lake City, UT, 5San Francisco General Hospital & University of California- San Francisco, San Francisco, CA
Cardiac troponin (cTn) is the preferred biomarker for detecting acute myocardial injury and infarction (MI). We studied whether multiple biomarkers of numerous pathophysiologic pathways would increase the diagnostic accuracy for detection of MI. cTnI (Abbott Architect), placental growth factor (PlGF, Abbott Architect), myeloperoxidase (MPO, Abbott Architect), BNP (Abbott Architect), plasma choline (mass spectrometry), and ischemia modified albumin (IMA, Inverness) were measured on emergency department admission (baseline, 0 h) and serially over the next 16 hours (to determine a maximum value) in 592 patients presenting with symptoms suggestive of acute coronary syndrome from four medical centers. These patients were part of the Acute Risk and Related Outcomes Assessed With Cardiac Biomarkers (ARROW) study. Thirty-five (5.9%) patients were diagnosed with MI. 86% of patients presented within 3 hours of clinical symptoms of ischemia. Acute MI sensitivities and specificities were determined based on their respective 99th or 95th percentile reference values. ROC areas under the curves were also compared. At baseline (table) sensitivity of the biomarkers ranged from 5.7% to 100%, with specificity ranging from 3.6% to 97.6%. cTnI demonstrated the highest diagnostic accuracy, and plasma choline showed the highest specificity. While the majority of biomarkers demonstrated a substantial improvement in clinical sensitivity using the maximal concentration value obtained from serial sampling, none demonstrated better diagnostic accuracy than cTnI alone (maximum area under ROC curve 0.952. In conclusion, cTnI was the single most accurate biomarker for the early diagnosis of acute MI. Additional studies to test whether a multi-biomarker approach improves clinical sensitivity or specificity are needed. ARROW Baseline Biomarker Results %Sensitivity %Specificity Biomarker Cutoff N (95%CI) (95%CI) cTnI, g/L 0.028 592 91.4 (76.9-98.2) 90.8 (88.1-93.1) PlGF, ng/L 27 549 6.2 (0.77-20.8) 91.1 (88.3-93.4) MPO, pmol/L 285 576 35.2 (19.7-53.5) 66.7 (62.6-70.7) BNP, ng/L 80 565 55.8 (37.9-72.8) 74.9 (71.0-78.5) Choline, 25 592 5.7 (0.70-19.1) 97.6 (96.0-98.7) mol/L IMA, U/mL 90 583 100 (89.7-100.0) 3.6 (2.2-5.5) Area under ROC (95%CI) 0.952 (0.905-1.000) 0.561 (0.461-0.660) 0.511 (0.403-0.619) 0.740 (0.663-0.818) 0.607 (0.494-0.720) 0.592 (0.492-0.691)
B-40 Diagnostic utility of B-Natriuretic Peptide in evaluation of diastolic dysfunction C. E. Ahnadi1, B. Essadiqi2, A. Grant1, S. Lepage1. 1Collaborative Research for Effective Diagnostics, CHUS, Sherbrooke, QC, Canada, 2Cardiology Department, CHUS, Sherbrooke, QC, Canada
Brain Natriuretic Peptide (BNP) reflects the left ventricular pressure and is known to be elevated in diastolic dysfunction. The objective of our study was to compare the BNP levels in two groups of patients with different severity of diastolic dysfunction. We measured BNP, diastolic dysfunction and clinical parameters in 121 patients (mean age: 65.1 8.4 years; 78 males, 43 females) enrolled at the cardiology laboratory and rehabilitation clinic, at the Sherbrooke University Hospital. We divided the patients in two groups according to the severity of the diastolic dysfunction: mild (group 1: E/A < 1 at rest), and moderate (group 2: E/A>1 at rest, reverse Valsalva or an abnormal tissue doppler with an E/E> 8). Patients in group 1 had a lower but not significant BNP level of 28.9 26.0 pg/mL, n = 64, in comparison to 43,9 45,4 pg/ mL, n = 57 for group 2 (p = 0.136). Patients with elevated LV end diastolic pressure, defined as E/E > 15 (n = 12) had the highest BNP levels (54.1 50.5 pg/mL), patient with E/E between 8 and 15 (n = 80) had a mean BNP level of 37.6 37.6 pg/mL and those with E/E < 8 (n = 29) had a mean BNP level of 24.0 24.6 pg/mL (p <0.05). The log-transformed BNP correlated significantly with E/Eseptal (r = 0.312, p = 0.01). Multivariate regression analysis demonstrated a significant correlation of logtransformed BNP with diastolic dysfunction (p = 0.013) regardless of age, sex and creatinine clearance. The area under the receiver-operating characteristic curve for BNP to discriminate between the two groups was 0.58 (95% CI, 0.48-0.68; p = 0.136). BNP levels show a relationship with diastolic dysfunction characteristics but does not seem to discriminate between mild and moderate diastolic dysfunction. A probable reason for this result is that BNP is not sensitive to detect diastolic dysfunction with normal filling pressure.
B-41 Serum 99th Percentile Reference Value for the High Sensitive Singulex Cardiac Troponin I Assay F. S. M. M. D. P. S. A. P. A. Simpson2, L. T. Le2. 1Hennepin County Medical Center, Minneapolis, MN, 2Singulex, Alameda, CA
New technologies are being developed that can precisely measure low concentrations of cardiac troponin in serum and plasma of normal, reference subjects. The purpose of this study was to determine the serum 99th percentile reference value for cardiac troponin I (cTnI) measured using the high sensitive Erenna cTnI assay (Singulex; Alameda, CA) which provides a linear range of 0.098 to 100 pg/mL. Serum was obtained from healthy adults (n=348); age 18 to 76 years of which 147 were male and 201 females. By health questionnaire no subject reported any known current or past history or medication for coronary artery disease or cardiac related medical condition, diabetes, hypertension, or renal disease. Non-parametric analysis for determination of
Apple1,
Murakami1,
Farris1,
Karimi2,
B-43 Performance Characteristics of Troponin I Assays Across 5 Instruments S. L. La'ulu1, W. L. Roberts2. 1ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT, 2Department of Pathology, University of Utah Health Sciences Center, Salt Lake City, UT
The aim of this study was to evaluate the performance characteristics of troponin I (TnI) assays on the following instruments: Beckman Coulter Access, Abbott
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ARCHITECT i2000SR, Abbott ARCHITECT i1000SR, Siemens ADVIA Centaur, and Ortho VITROS ECi. Testing on all methods was performed according to manufacturers instructions. Imprecision studies were performed using three concentrations of commercially available quality control materials. Two runs of duplicate testing were conducted per day, for 5 days with a minimum of 2 hours between runs. For method comparison, 110 plasma and serum samples were tested by all methods with the ARCHITECT i2000SR assay as the comparison method. Functional sensitivity was evaluated by testing 10 panels of serum pools, with values ranging between 0.010 to 0.100 ng/mL, on 2 reagent lots. Lot 1 was calibrated on the 1st day of testing. Lot 2 was calibrated on the 11th and 16th days of testing. Each panel was tested in duplicate for 10 days with each lot. To determine the 99th percentile, plasma and serum samples were drawn from 400 subjects and tested on the day of draw (fresh) for the Abbott instruments. A second frozen aliquot was tested by all methods. Total CVs ranged from 2.4 to 10.0%. Method comparison results by Deming regression had slopes of 1.03-1.51 and correlation coefficients of 0.98-1.00. A summary of functional sensitivity (10% CV) along with the calculated 99th percentile is provided (Table). Imprecision was acceptable with CVs of 10% or lower for all methods. All methods compare well to the ARCHITECT i2000SR assay. All methods showed good agreement at the 99th percentile. Overall, the ARCHITECT i2000SR is well harmonized with other methods and shows comparable results with serum and plasma. Fresh 99th percentile (confidence intervals) Frozen 99th percentile (confidence intervals) Ratio: Functional Sensitivity
Cardiac Markers
B-45 A Multimarker approach for the prediction of evolving myocardial injury in women presenting with symptoms suggestive of acute coronary syndromes M. P. Henderson1, A. R. MacRae2, A. S. Jaffe3, P. A. Kavsak1. 1McMaster University, Hamilton, ON, Canada, 2University of Manitoba, Winnipeg, MB, Canada, 3Mayo Clinic, Rochester, MN
Background: Recent data suggest that women may have fewer elevations in cTn than do men at the time of presentation. (Circulation 2004;109:580-6). This study evaluated whether a panel of cytokines, adhesion molecules, PAPP-A and a highsensitivity cTnI assay analyzed at presentation could predict those women who would have a change in cTnI concentrations. Methods: After ethics approval, a cohort of women (n=64, in 1996) who had presented to the ED 6 hours of onset of chest pain were evaluated. This cohort had EDTA and heparin plasma specimens collected and stored at-700C until 2003 when the heparin specimens were thawed for cTnI (AccuTnI) and hsCRP measurements (Beckman Coulter). In 2006, the EDTA plasma specimens were thawed and measured for IL-8, VEGF, MCP-1, EGF, IL-6, IL-10 and VCAM-1, ICAM-1, P-selectin, Eselectin, L-selectin using the cytokine and adhesion arrays, respectively (evidence investigator, Randox). In 2007, the heparin specimens were thawed for a second time for measurement with hs-cTnI and hs-PAPP-A assays (Beckman Coulter). The AccuTnI assay was used to classify a cTnI change. Briefly, an analytically significant concentration change was noted if the difference between the highest and the lowest cTn concentrations (median (IQR) = 5 (4-7) specimens/subject) observed within a subject was >3SD or >20%, consistent with the 2007 MI definition. Classification and regression tree (CART) analysis, which identifies the best analytes and cutoff values to use, was employed to identify a combination of markers from the earliest specimen to predict a cTn change using Matlab R2007b software. Results: A cTnI change was observed in 28 subjects. CART for cTnI change had a positive predictive value of 100% (95%CI:83-100) and a negative predictive value of 86% (95%CI:72-94) (see Figure).
Method
Functional Sensitivity:
TnI to Plasma Plasma Serum Plasma Serum concentration 99th (ng/mL) (ng/mL) (ng/mL) (ng/mL) at 10% CV percentile 0.018 0.013 Access 0.101 (0.015, (0.009, 5.6 0.040) 0.028) 0.014 0.017 0.020 0.016 ARCHITEC 0.076 (0.008, (0.006, (0.013, (0.010, 3.8 T i2000 0.040) 0.030) 0.040) 0.020) 0.020 0.013 0.018 0.013 ARCHITEC 0.049 (0.013, (0.011, (0.015, (0.009, 2.7 T i1000 0.042) 0.029) 0.040) 0.028) 0.018 0.013 Centaur 0.039 (0.016, (0.010, 2.2 0.040) 0.028) 0.018 0.013 ECi 0.093 (0.015, (0.009, 5.2 0.040) 0.028)
B-44 Fenofibrate treatment reduces plasma oxidized LDL and 8isoprostane Y. Dong, A. K. Tsai, M. Y. Tsai, D. K. Arnett. University of Minnesota, Minneapolis, MN
Background: Oxidative damage to lipids and proteins has a high correlation with cardiovascular events. Measurement of oxidation levels may provide an additional risk evaluation for atherosclerotic cardiovascular disease than the use of a traditional risk factors alone. We studied the effect of lipid lowering therapy with fenofibrate on two oxidative biomarkers, oxidized LDL (oxLDL) and 8-isoprostane (8-isoP). Methods: Our sample group consisted of 92 participants randomly selected from the Genetics of Lipid Lowering Drugs and Diet Network (GOLDN) study. An initial study had been done which determined that triglyceride (TG) level is not associated with changes in oxLDL. To verify our results, we selected a new sample of patients with varying baseline levels of TG. Plasma levels of oxLDL and 8-isoP were measured before and after treatment with open-label fenofibrate (160 mg/d). Plasma oxLDL was measured by ELISA (Mercodia) and plasma 8-isoP was measured by enzyme immunoassay (Cayman Chemicals) according to the manufacturers recommended protocols. Data was analyzed by paired t-tests. Results: Plasma levels of oxLDL and 8-isoP decreased significantly (p<0.001) after fenofibrate treatment in both sets of samples. While there was a significant decrease in the biomarker concentrations across all quartiles, patients with higher baseline oxLDL and 8-isoP showed a more dramatic decrease. Conclusion: Our results from the 92 GOLDN participants confirm that fenofibrate treatment significantly reduces oxLDL and 8-isoP levels, with the greatest reduction in patients with highest baseline concentrations.
Conclusion: In women, early measurements of hs-cTnI and markers of plaque instability/ inflammation may be used to predict a subsequent change in cTnI.
B-46 Preliminary Performance of a Research High Sensitive Gold Nanoparticle-Based Cardiac Troponin I Assay: Proof-of-Concept Study of the Nanosphere Verigene System M. M. Murakami1, F. S. Apple1, J. E. Hollander2, G. W. Shipp3. 1Hennepin County Medical Center, Minneapolis, MN, 2University of Pennsylvania, Philadelphia, PA, 3Nanosphere, Inc, Northbrook, IL
The goal of this study was to evaluate the performance of the Nanosphere Verigene cardiac troponin I (cTnI) high-sensitivity research assay. The LoD was 0.2 pg/mL. The linear range was 0.2 to 500 pg/mL. Imprecision (%CV) was 9.5% at 0.5 pg/mL and 9.7% at 5 pg/mL 45% of the normal subjects (181 healthy blood donors) had a measurable concentration, with a 99th percentile of 2.8 pg/mL The assay was 1 to 2 orders of magnitude lower than the predicate commercial cTnI assays When linearity data points with CVs <20% were plotted, the last cTnI concentration plotted with this precision was 1.7 pg/mL at 7%CV for the Nanosphere assay, 43.3 pg/mL at 13%CV for the Siemens Stratus CS, 28.3 pg/mL at 11%CV for the Abbott Architect, 18.5 pg/ mL at 13%CV for the Siemens Ultra, and 30.0 pg/mL at 10.0%CV for the Beckman
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Access. Correlations were excellent between assays (r=0.999 for all), but substantial differences in slopes and intercepts, respectively, were observed: Architect 2.18, 22.2; Beckman 1.34, 15.0; Siemens CS 4.03, -32.0; Siemens Ultra 5.43, -10.2. Serial serum samples from ten MI patients at 0, 90 & 180 mins after hospital presentation were measured by the Nanosphere and Siemens CS (99th percentile 70 pg/mL) cTnI assays. Two (20%) and ten (100%) of the patients were cTnI positive at presentation on the CS and Nanosphere assay, respectively. Three of the patients (30%) remained cTnI negative for >180 min on the Siemens CS. No crossreactivity was found for skeletal troponins. In conclusion, the Nanosphere Verigene System demonstrates the potential of a high sensitive cTnI assay to detect myocardial injury early after an index event through the measurement of low, abnormal cTnI concentrations at hospital presentation. Prospective clinical studies will be required to create an evidence base for the multiple clinical uses of this high sensitive cTnI assay.

B-48 Analyte stability and potential interferences in a cardiac troponin I assay S. E. Melanson, M. J. Conrad, L. Panov, P. Jarolim. Brigham and Women's Hospital, Boston, MA
Background. Our laboratory participated in the pre-market evaluation of the high sensitivity cardiac troponin I (cTnI) assay, TnI-Ultra (Siemens Healthcare Diagnostics, IL, US). We performed precision, sensitivity, accuracy and clinical utility studies. In this current study we investigated analyte stability and potential heterophilic antibody interferences. We hypothesized that the occasional reports of increasing incidence of interferences affecting various immunoassays may be associated with an increasing number of patients treated with intravenous monoclonal antibodies (MoAbs), particularly those containing imperfectly humanized mouse MoAbs. We assessed whether or not such interferences affect the TnI-Ultra assay. Methods. For the stability study, 45 positive cardiac troponin I specimens (>1.0 ng/ mL) were freshly sequestered. Five aliquots of each specimen were made immediately after initial TnI measurement. Each specimen and its corresponding five aliquots were time stamped and fifteen specimens with aliquots were stored in each of the three environmental conditions: frozen (-20C), refrigerated (+4C) and room temperature. A separate aliquot for each specimen was used to measure TnI at the following times: 3, 7, 14, 28, 56 days for frozen samples, 6, 12, 24, 48, 72 hours for refrigerated samples and 2, 4, 6, 12, 24 hours for room temperature samples. The percent recovery of TnI was collected based on the initial TnI result. We calculated the best fit curve using all timepoints for each storage temperature. We consider average decrease below 90% of the original TnI-Ultra value clinically significant. For evaluation of the potential heterophile antibody interference, we obtained from our pharmacy the list of monoclonal therapeutics administered to our patients and daily reconciled the list of these medications with the list of plasma specimens coming to our laboratory for testing. One hundred such specimens were collected, an aliquot was incubated with the Heterophile Blocking Reagent (HBR, Scantibodies Laboratory, Inc.) and both aliquots were retested in parallel. All measurements were done using the ADVIA Centaur immunoanalyzer (Siemens). Results. We observed an immediate decrease in TnI-Ultra in the first specimen of the series, most pronounced in the refrigerated specimens, followed by a slower decrease in readings. An average decrease below 90% was observed after 17 hours at room temperature and after 36 hours in refrigerator. All average readings were above 90% after 56 days of storage at -20C. In patients on previous MoAb-based therapies, there were no significant differences between TnI-Ultra results in specimens pre and post treatment with HBR. Conclusions. When using the TnI-Ultra assay, no clinically significant differences in cTnI are seen during 17 hours of room temperature storage and 36 hours of refrigerated storage. Specimens can be stored at -20C for at least 8 weeks, most likely considerably longer, without clinically significant changes in results. However, for both refrigerated and -20C storage, specimens have to be carefully thawed and/or warmed up and mixed before testing. Previous MoAb therapy did not interfere with the TnI-Ultra assay.
B-47 Myeloperoxidase levels are associated with risk of cardiovascular death and heart failure after non-ST elevation acute coronary syndrome S. E. Melanson1, D. A. Morrow1, B. M. Scirica1, M. S. Sabatine1, S. Sloan1, S. A. Murphy1, J. L. de Lemos2, P. Jarolim1. 1Brigham and Women's Hospital, Harvard Medical School, Boston, MA, 2UT Southwestern Medical Center, Dallas, TX
Background: Myeloperoxidase (MPO) is released from leukocytes and has been implicated in the pathophysiology of acute coronary syndrome (ACS). Initial studies demonstrated an association of higher MPO levels with poor cardiovascular (CV) outcomes but prospective evaluation together with contemporary biomarkers is still needed. Methods: 4516 patients randomized in MERLIN-TIMI 36 with non-ST elevation ACS had baseline samples obtained within 48 hours of last symptoms. MPO, cTnI (99th percentile = 0.07), CRP (median 5.5 mg/l, IQR 2.5, 12.8) and NTproBNP (median 213 pg/ml, IQR 58, 681) were measured using the Siemens Dimension RxL analyzer. MPO was categorized either by quartiles or by a cutpoint of 670 pM, CRP by a cutpoint of 15 mg/l, and NT-proBNP by a cutpoint of 400 pg/ml based on our prior work. Primary clinical outcomes were CV death and heart failure (HF). Results: MPO was greater than 670 pM in 1938 patients (43%). Increasing quartiles of MPO were associated with greater risk of CV death or HF (Figure). Elevated MPO (>670pM) was associated with increased risk of CV death (adjusted hazard ratio (HR) 1.8, p<0.001) and with CV death or HF after adjusting for baseline risk factors and additional biomarkers (adjusted HR 1.7, p<0.001). (Figure) This relationship was consistent in patients with normal cTnI (adjusted HR 1.9, p=0.001) or elevated cTnI (adjusted HR 1.7, p=002). Elevated MPO was associated with CV death and/or myocardial infarction at 30 days (adjusted HR 1.6, p=0.016) with an attenuated relationship by 1 year (adjusted HR 1.3, p=0.06). Conclusions: Elevated levels of MPO, a marker of leukocyte activation, are associated with a substantially higher risk of cardiac events in patients with ACS. This relationship is independent of other biomarkers and suggests that MPO offers additive information regarding cardiovascular outcomes.
B-49 Development of a novel immunoassay for characterizing the circulating form of cardiac troponin Y. Lin1, Q. Fu2, J. Zhu2, J. E. Van Eyk2. 1Axela Inc., Toronto, ON, Canada, 2Johns Hopkins University, Baltimore, MD
BACKGROUND: The detection of cardiac muscle specific troponins in blood is the current gold standard for the diagnosis of patients with acute myocardial infarction (AMI). In cardiac muscle, the troponin (cTn) complex comprises 3 tightly interacting subunits cTnI, cTnT, and cTnC. With AMI, cardiac troponin is released from the heart into the circulation where it can be detected using a variety of immunoassays that independently quantify cTnI or cTnT. cTnI is a complex analyte. Its circulating form has the potential of disease-induced post-translational modifications such as the specific and selective degradation of the N- and/or C-terminus and the possibility of presenting different ternary structures. It is accepted dogma that cTnI circulates in the blood as a cTnI-cTnC complex, yet this is based primarily on indirect evidence. There has been no clinically applicable assay that has directly characterized the circulating ternary form of these biomarkers, in part, due to a limitation in technology. OBJECTIVE: Here we describe the development of a novel immunoassay to directly characterize the primary and ternary structure of the circulating form of cTnI using diffractive optics technology (dot). Core to the dot is a diffraction grating formed from affinity reagents such as antibodies. This grating is comprised of a repetitive
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sequence of lines and generates a specific, reflected diffraction pattern when interrogated with a laser. As molecules exhibit affinity for the capture molecules that make up the grating, the diffraction efficiency is improved. METHODS: cTnI was captured using a biotinylated anti-cTnI antibody to the constant region (137-148 a.a.) that was immobilized on a pre-patterned avidin sensor and then probed i) with antibodies to either or both the N- and C-terminus to determine if cTnI was degraded, or ii) sequentially with anti-cTnT and/or anti-cTnC antibodies to determine whether cTnI existed as a monomer, dimer or trimer. The binding of the immobilized antibody, protein analyte or other antibodies was observed in real time as an increase in diffraction signal intensity. By continuously monitoring the intensity we can characterize the differences in the circulating form of cTnI. RESULTS: A novel diffraction-based immunoassays using the dotLab System was developed that allows for characterizing the primary and ternary structure of the circulating form of cardiac troponin. The new assay was able to directly probe the integrity of cTnI and determine if the protein is degraded and directly detect circulating cTnI bound to cTnC and cTnT from serum obtained from an AMI patient indicating the dimer or intact cTn (cTnI-cTnT-cTnC) is present. CONCLUSIONS: We have developed the first clinically-applicable and easy-to-use immunoassay to address the direct interactions of the subunits within the cTn complex. It directly measures protein interactions in complex samples. Real-time binding data allow a more thorough understanding of these complex interactions.
Cardiac Markers
B-51 Meeting the Challenge of the ESC/ACC Definition for Myocardial Infarction using the New Troponin I ES on the VITROS 5600 Integrated System and VITROS ECi Immunodiagnostic System S. SAW, C. Wang, S. Sethi. NATIONAL UNIVERSITY HOSPITAL, SINGAPORE, Singapore
Background: Almost 10 years after the ESC/ACC consensus for myocardial infarction many laboratories are still in transition mode for reporting troponin results that meet these criteria. An increase in cardiac troponin is a measurement >99th percentile of normal population with imprecision at 99th percentile 10%. We evaluated the performance of the new Troponin I ES reagent on both the new VITROS 5600 Integrated System and the VITROS ECi Immunodiagnostic System (Ortho Clinical Diagnostics, USA) to determine performance. Methods: Functional sensitivity was determined over ten days using ten pools of aliquoted frozen serum and run in duplicate. Routinely analysed patient requests for troponin T were used to compare the performance of both analysers with our current method. Linearity was determined using selected dilution ratios of a high and a low serum pool. A 99th percentile reference range was determined on ECi using 311 anonymised blood donor samples (age range 20-72 years); on 5600 we also used anonymised blood donor samples which were grouped into age ranges 20-29 (n=120); 30-39 (n=120); 40-49 (n=118); >50 (n=119) to determine the impact of the inclusion of significant numbers of the older age cohort in establishing the 99th percentile. The Troponin concentrations (0.039, 0.129, 0.669ug/L) were also tested in the presence of lipaemia (30mmol/L), icterus (347umol/L) and haemolysis (100-500mg/dL). Results: A 10% Functional sensitivity of 0.020ug/L on VITROS 5600 System and 0.010ug/L on ECi. The correlation for the instruments: VITROS 5600 System TnI=10.71(e411 TnT)-0.21, r=0.805; ECi TnI=6.00(e411TnT)-047, r=0.68. Linearity 5600=1.02x+0.357, r=0.999; ECi=0.975x-0.28, r=0.999. 99th percentile reference range on ECi was established as 0.039ug/L. On 5600 the reference range for each cohort were reported 0.016ug/L, 0.033ug/L, 0.026ug/L and 0.137ug/L. Taken together the 99th percentile was 0.042ug/L. No significant interference was noted for either lipaemia or icterus at the concentrations tested. However, there was a significant overrecovery (112-187%) in results at low troponin concentrations (<0.050ug/L). An excellent functional sensitivity was obtained on both instruments. On 5600 we chose to evaluate the 99th percentile reference range by age ranges to ensure that the achievement of a value less than the 10% functional sensitivity could not be compounded by inclusion of subjects of a very senior age where normal troponin values would be expected to be higher than found in the younger population. Our oldest cohort did obtain a higher 99th percentile. The results between the 2 systems are very comparable, however, age inclusion is an important factor when determining the percentile. Conclusions: Our Emergency Department (ED) is the biggest single user of troponin assay in this hospital with 34% of requests. However, like many EDs the quality of the sample received is far from optimal. Last year, about 24% of samples from ED were flagged as haemolysed. Hence, care must be used in reporting any low troponin result in the face of a haemolysed sample. In conclusion, this assay meets the laboratorys needs in terms of technical performance as well as the ESC/ACC consensus for myocardial infarction.
B-50 Analytical Evaluation of Troponin I ES Assay on the VITROS 5600 Integrated System R. Bais1, I. Gould1, D. Chesher1, J. Francis1, A. Weston2. 1Pacific Laboratory Medicine Services, Sydney, Australia, 2Ortho Clinical Diagnostics, Sydney, Australia
Objective: The VITROS 5600 Integrated System is a newly released analyser which features the integration of MicroSlide, MicroWell and MicroTip assay technologies enabling both chemistry and immunoassay to be carried out on a single system. The troponin I ES assay on the VITROS 5600 Integrated System is an enhanced chemiluminescence assay with results available within 18 minutes. We evaluated the imprecision, functional sensitivity and assay interference from haemolysis, turbidity and icterus on the VITROS 5600 Integrated System. Methods: Between run imprecision was determined by measurements of two quality control materials over a 10 day period. Functional sensitivity was determined by measuring the imprecision profile of 95 low troponin I human sample pools which were assayed 10 times in multiple runs over a 10 day period. No more than 10 samples were assayed on any one day. The coefficient of variation (CV) was then determined and plotted against the sample mean. Results were obtained for 2 different reagent lots and calibrations. Interferences were measured by adding increasing amounts of red cell haemolysate, intralipid and bilirubin to a serum pool containing low troponin I and using the MicroSensor feature on the analyser to determine the impact of the potentially interfering substance on the test result Results: Assay imprecision expressed as CV were 5.2% at 0.092 g/L and 4.6% at 0.515 g/L. The line of best fit for the functional sensitivity data was found to be an exponential curve from which the functional sensitivity determined as the 10% CV was calculated to be 0.007 g/L. When an effect greater than 10% of the initial result was considered significant, it was determined haemolysis had a positive effect at values greater than 10 g/L, lipid no effect up to 7 g/L (> 17 mmol/L) and bilirubin at values greater than 300 mol/L. Discussion: The functional sensitivity (10% CV) for all data was below the reported 99th percentile of 0.034 g/L. The level of haemolysate that results in significant interference occurs in 3.5% of samples received in our laboratory so must be considered when reporting results. This level of interference is similar to our routine troponin T assay. Interference by bilirubin is less common but should still be considered in grossly icteric specimens. Conclusion: The VITROS Troponin I ES assay is a highly sensitive and precise assay and is acceptable for clinical utilisation in acute coronary syndrome. The MicroSensor feature on the analyzer provides an excellent tool for the laboratory to estimate the effect of endogenous interferences on the troponin testing.
B-52 Integration of Cardiac Markers on a Single Platform: The VITROS 5600 Integrated System R. Khoo, C. Wang, S. Saw, S. Sethi. National University Hospital, Singapore, Singapore
Identifying Myocardial Infarction (MI) earlier is important to provide effective and timely treatment to patients presenting with symptoms suggestive of Acute Coronary Syndrome (ACS). Integration of cardiac markers in a single platform will support rapid diagnosis of those patients. VITROS 5600 Integrated System (Ortho-Clinical Diagnostics, Rochester, USA.), random access,fully automated clinical chemistry/ immunoassay analyzer serves this purpose. We evaluated technical performance of Cardiac Markers:CK,CKMB, Myoglobin (MYO),NT-ProBNP (PBNP) and TroponinI ES (TnI) assays on VITROS 5600. Performance are compared with our current Siemens Advia 2400CK, Centaur CKMB, MYO and Roche Cobase 411TNT, PBNP assays. Precision,linearity and interference studies were also done. Consolidation of assays for several cardiac markers on the VITROS 5600 optimizes
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turnaround-time and productivity allowing clinicians in rapid diagnosis and management for ACS patients especially in Emergency Department. Evaluation Data on VITROS5600 Integrated System Precision(RangeTeste Test Correlation r n Linearity d/Total%CV) 54-1436U/L(1.45- V5600=0.9924Adv- 0.99 12 y=1.0022x+ CK(U/L) 3.86%) 6.5817 07 0 8.9801,r= 0.9994 CKMB(ng 0.74-239ng/mL(3.08V5600= 0.98 12 y=1.0045x-1.96, /mL) 14.63%) 0.8827e411+3.4409 56 0 r=0.9997 MYO(ng/ 18.9-117ng/mL(2.06- V5600=0.8878Cent 0.98 y=0.9981x42 mL) 3.56%) aur+3.7953 85 37.475,r=9986 NPBNP(p 93-29060pg/mL(2.27- V5600=0.9508e411+ 0.99 12 y=0.9889xg/mL) 3.68%) 190.48 49 0 552.82,r=0.9981 V5600TNI TNI(ng/ 0.021-66.7ng/ 0.80 12 y=1.015x+0.357, =10.706e411TNTmL) mL(3.99-10.00%) 54 0 r=0.9992 0.2108 Levals of Interferents(in our study) with <10% interference Anal Concentr Triglycerides Bilirubin( Haemoglobi Claim Claim( Claim ytes ation (mmol/L) umol/L) n(mg/dL) (TG) TBIL) (Hb) CK 71,973U/L 7.9,9.2 612,630 0,100 9 684 150 CKM 5.71,50.4n 24.7,24.7 388,398 500,500 9 684 150 B g/mL 59,879.9n MYO 36.2,30.8 372,368 500,500 33.9 342 500 g/mL NPB 909,10500 31.8,32.3 395,397 500,500 33.9 342 500 NP pg/mL

B-54 EVOLUTION OF COEFFICIENTS CKMM3/MM1, CKMB2/MB1 AND LD1/LD2 IN PATIENTS WITH ACUTE MYOCARDIAL INFARCTION R. Sanchez Navarro, S. Blanco Martn, T. De Haro, M. Zafra, J. Mora. Hospital Universitario San Cecilio, Granada, Spain
OBJECTIVE: Measuring the activity of plasma isozymes of lactic dehydrogenase (LDH), MM1, MM2 and MM3 of creatin kinase isoforms (CKMM) and MB2 and MB1 of CKMB, in a group of patients with acute myocardial infarction (AMI). To study the evolution of the ratios CKMM3/MM1, CKMB2/MB1 and LD1/LD2 during the 24-48 hours post-infarction and their relationship with other cardiac markers to check its effectiveness as biochemical markers of coronary revascularization. MATERIALS AND METHODS: Were studied 20 patients treated at our center, with symptoms and signs of AMI. Samples were collected from venous blood at regular intervals from the onset of symptoms (0-4, 4-8, 8-12, 12-18, 18-24 and 24-48 hours); samples for isoforms CKMM and CKMB protected with EDTA, to a final concentration of 5 mmol/L in order to avoid the transformation in vitro of a isoform to another. CK were measured, LD, AST and CKMB, myoglobin and troponin; LD isoenzyme and CK isoforms CKMM and CKMB, separated by electrophoresis on agarose gel quantified and expressed as a percentage of the total activity. MM3/MM1 ratios were calculated, MB2/MB1 and LD1/LD2. Was estimated coefficient of correlation between variable. RESULTS: The ratios MM3/MM1 and MB2/MB1 increased significantly in the first 4 hours after AMI (p<0.001) and normalization past 24 hours coinciding with favourable results. Correlation coefficient between the two ratios r = 0.96 (p <0.05), with the myoglobin r = 0.98 and r = 0.97 (p<0.05) respectively. The isoenzyme LD1 began to rise in the first few hours after AMI, but the ratio LD1/ LD2 remained within the normal range until 8-12 hours, reaching its maximum value at 24 hours (p<0.001) from which began his normalization. CONCLUSIONS: We propose identifying LD isoenzyme and the isoforms CKMM and CKMB as markers of AMI valuing especially the interest of serial measurements of these parameters to assess the kinetics of standardization in the blood, therefore, the relationship appears to exist between serum values and the extent of the injury might be given an important value these markers for the diagnosis of myocardial reperfusion after fibrinolytic therapy.
B-53 Clinical performance of a new high-sensitivity cardiac troponin I assay M. Plebani1, M. Mion1, E. Novello1, M. Zaninotto1, L. Babuin2, S. Iliceto2. 1Department of Laboratory Medicine, University-Hospital, Padova, Italy, 2Department of Cardiac Thoracic and Vascular Sciences, UniversityHospital, Padova, Italy
The ESC/ACCF/AHA/WHF guidelines for the universal definition of myocardial infarction (MI) recommend cardiac troponins as the biochemical gold standard for the detection/exclusion of MI. The 99th percentile of the biomarker values in a reference population and 10% coefficient of variation (CV) are indicated respectively as the decision limit for myocardial necrosis and its corresponding maximal allowable total imprecision. The objective of this study was the performance evaluation of a recently developed cardiac troponin I (cTnI) assay applied to the new analyzer Dimension VISTA (Siemens Healthcare Diagnostics, USA) (cTnI-V). In particular, the analytical sensitivity, the imprecision characteristics and the clinical usefulness were studied in comparison to those observed for the routinely used cTnI assay applied to the Dimension RxL analyzer (Siemens Healthcare Diagnostics, USA) (cTnI-R). The cTnI-V method is an homogeneous sandwich chemiluminescent immunoassay based on LOCI (Luminescent Oxygen Channelling Immunoassay) technology. cTnI values are expressed in g/L. Data from manufacturer: analytical sensitivity, 0.015; analytical measurement range, 0.015-40.000; range ecompassing 99th percentile (n=199), 0.000-0.045; 10% CV, <0.040. During the study all cTnI concentrations were determined in lithium-heparin plasma samples. The analytical sensitivity, calculated as mean+2SD from n=20 replicates of the zero calibrator was 0.0054. The imprecision profile was calculated testing cTnI concentrations in 13 pools following NCCLS (CLSI) EP5-A protocol. cTnI concentration corresponding to 10% CV, obtained from the imprecision profile, was 0.048. In the method comparison study cTnI was measured in 116 samples from laboratory routine analysis. Concentration range: cTnI-R = <0.04-31.15, cTnI-V = <0.015-31.800. Linear regression analysis: cTnI-V=1.065 x cTnI-R - 0.018, r=0.99. Altman Bland test: bias, 95%CI= 0.131, 0.023-0.239. For the clinical study cTnI concentration was measured in the admission sample of a selected patients population: 41 non consecutive patients (31 males, 10 females; age: median, range=65, 33-86 years) presenting to the emergency department (ED) with chest discomfort (time from chest pain to ED presentation: median, range=120, 30-240 min), admitted to hospital and discharged with MI diagnosis (ST segment elevation MI=STEMI vs non-STEMI: 19 vs 22). cTnI decision limit adopted for MI diagnosis (10% CV): 0.15 (cTnI-R), 0.048 (cTnI-V). cTnI-R was higher than cut-off in 2 out of 41 patients (4.9%) and cTnI-V in 24 out of 41 (58.5%). The analytical performances studied were satisfactory. The higher analytical and clinical sensitivity of the evaluated cTnI assay suggests its clinical usefulness not only as specific but also as sensitive marker for an accurate, timely and rapid (time to first result 11 vs 20 min) management of critically illness ED patient suspected for having an acute MI.
B-55 Cross-reactivity with endogenous proBNP in three commercial BNP assays in heart failure patients B. CAULIEZ. Hospital, ROUEN, France
INTRODUCTION: B-type natriuretic peptide (BNP) and NT-proBNP, two sensitive markers of cardiac dysfunction, are derived from a common precursor, proBNP1-108. Non cleaved proBNP is secreted into the blood at high level; this is particularly marked in heart failure patients. Several studies have demonstrated that (i) this precursor is the major immuno-reactive form in in house BNP assays and (ii) commercial BNP assays perform differently for measurement of exogenous proBNP and BNP. However, it remains unclear whether these assays demonstrate crossreactivity with endogenous proBNP in heart failure patients. METHODS: We used EDTA plasma samples from four heart failure patients, separated by gel filtration-fast protein liquid chromatography. BNP (Triage BNP assay on Access; AxSYM BNP assay; SHIONORIA BNP assay) and proBNP (BioPlex 2200 proBNP assay in development; Bio-Rad) were measured in eluted fractions. RESULTS: ProBNP was the major immuno-reactive form in the commercial assays tested, whereas the low molecular weight immuno-reactive form (BNP) represented less than 40 % of the immuno-reactivity. This percentage was dependant not only on the patient tested but also on the assay used. As shown on the representative chromatogram, the AxSYM BNP assay detected twice the amount of BNP in the minor peak than the Triage BNP assay. The proBNP assay demonstrated a high specificity for proBNP as no low molecular weight reactive form was detected.
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Inflammatory Markers Remodelling after Cardiac Resynchronization Therapy A. E. STANCIU1, M. DOROBANTU2, R. VATASESCU2, C. IORGULESCU2, M. STANCIU3. 1"Prof.Dr.Al.Trestioreanu" Institute of Oncology, BUCHAREST, Romania, 2Clinic Emergency Hospital Bucharest, Bucharest, Romania, 3University "Politehnica" of Bucharest, Bucharest, Romania
Background: In chronic heart failure (CHF) patients, cardiac resynchronization therapy (CRT) leads to reverse left ventricular (LV) remodelling. Our objective was to evaluate whether changes in levels of some inflammatory markers (interleukins IL1, IL-6, IL-8, tumor necrosis factor TNF-, matrix metalloproteinases MMP-2, MMP-9) correlate with CHF severity and response to CRT, using N-terminal probrain natriuretic peptide (NT-proBNP) as a reference standard. Methods: 21 patients (13 males, 8 females) with CHF (III-IV NYHA functional class) were investigated for immune activation before (T0) and 1 hour (T0p), 1 week (T1), 3 (T2), 6 (T3), 12 (T4) months after CRT treatment. The serum levels of MMP2, MMP-9, interleukins IL-1, IL-6, IL-8, TNF- (R&D Systems) and NT-ProBNP (Biomedica) were measured at the same time by an ELISA method. Results: In all patients, a significant decrease in circulating levels of NT-proBNP, IL6, IL-8 and MMP-2 was observed at follow-up. Figure 1 presents the averages of decrease rates of NT-proBNP (from 100% before CRT to 87.60% 17.75%, p<0.05 at 1 hour after CRT - T0p and 19.69% 34.98%, p<0.05 at 12 months after CRT - T4), IL-6 (from 100% to 72.51% 41.28%, p<0.05 at T0p and 7.55% 18.77%, p<0.05 at T4), IL-8 (from 100% to 83.44% 43.78%, p<0.05 at T0p and 6.28% 15.7%, p<0.05 at T4) and MMP-2 (from 100% to 92.68% 15.89%, p<0.05 at T0p and 34.10% 28.32%, p<0.05 at T4). MMP-9, IL-1 and TNF- levels were unchanged. A significant correlation was observed between Nt-proBNP and IL-6, IL-8, MMP-2 (r>0.95, p<0.05).
CONCLUSION: we confirmed that BNP is detected as a minor immuno-reactive form in different commercial assays measuring BNP and demonstrated that detection of this minor immuno-reactive form in heart failure patients depends greatly on the assay used. This could be explained by different assays configurations. Consequently, specific measurement of proBNP may be useful as an alternative or in addition to the BNP measurement.
B-56 Influence of non-cardiac surgery on highly sensitive troponin T (hsTnT) T. C. Aw, Z. Pei, Q. Liu, S. Zhou, H. Y. Goh, S. P. Tan. Changi General Hospital, Singapore, Singapore
Objective: We evaluated whether troponin T (TnT) is increased during non-cardiac surgery using a new high-sensitive (hsTnT) assay (Roche). Methods: Eighty consecutive inpatients in whom sera was available before and up to 96 hours post-surgery (orthopaedic - 40, abdominal 27, urologic - 6, others - 7) were studied. Both TnT (assay sensitivity 0.01 ng/mL) and hsTnT (assay sensitivity 3 pg/ mL) were measured using electro-chemiluminescence (Cobas 6000, Roche); suggested cut-points for myocardial injury (interassay cv < 10%) is 0.3 ng/mL for TnT and 15 pg/mL for hsTnT. Eight patients whose pre-operative TnT was >0.01 ng/ mL were excluded. A difference of < 5 pg/mL between post- and pre-op hsTnT values was considered unchanged. Results: A total of 70 subjects (32 men, 38 women), aged 23-97 years, were studied and divided according to their pre-op hsTnT: Group A (n=14, 20.0%) - undetectable (<3 pg/mL), Group B (n=45, 64.3%) - 3.1-14.9 pg/mL, and Group C (n=11, 15.7%) - myocardial injury (> 15 pg/mL). Post-op hsTnT values were: Group A - undetectable in 10 and unchanged in the other 4 subjects. Group B - < 14.9 pg/mL in 38 subjects (B1) and > 15 pg/mL in the other 7 (B2). The pre- and post-op hsTnT in B1 were unchanged although minor increases of between 1.3-5 pg/mL were noted in 21 subjects. In B2 the pre- and post-op hsTnT were unchanged in 2 and elevated in 5 subjects (between 5-8 pg/mL in 3 people and more than 3-fold difference in the other 2: 9.6-33.1 and 16.8-32.4 pg/mL). Group C - decreased by 20 pg/mL in 1 person, unchanged in 8 (with minor increases of between 3.4-4.4 pg/mL in 3), and increased in the remaining 2 patients (2-fold: 1632 and 3-fold: 21-61 pg/mL). Summary: In pre-operative subjects hsTnT was undetected in 14 (10 remained undetectable after surgery) and myocardial injury was detected in 11. While hsTnT is unchanged in 52 cases after non-cardiac surgery minor increases of 1.3-4.9 pg/mL were noted in 26. Post-surgical elevations in hsTnT > 5 pg/ml were seen in 7 subjects. Conclusion: Troponin remains undetectable in some inpatients even with the new hsTnT assays. The significance of minor changes in hsTnT (< 4.9 pg/mL) remain unclear. Thus more studies and new guidelines are required on what constitutes cutpoints for myocardial injury when hsTnT assays are used. It may be prudent to screen hsTnT pre-operatively in at-risk subjects especially the elderly to provide clearer guidance in the evaluation of myocardial injury in the postoperative period.
Conclusions. At 12 months follow-up, CRT was associated with reverse LV remodelling and a significant decrease in IL-6, IL-8, MMP-2 and NT-proBNP levels. This suggests an important role of immune activation in the process of reverse LV remodelling in patients responding to CRT.
B-58 Performance of STAT Troponin-I Immunoassay on the ARCHITECT i1000SR and i2000SR Instrument Platforms J. Waterston1, J. Hoffman2, C. Kramer2, R. Taylor2, H. Pietschmann2, K. Ford2, A. Newhart3. 1Thermo Fisher Scientific, Middletown, VA, 2Abbott Laboratories, Abbott Park, IL, 3Thermo Fisher Scientific, Middletowwn, VA
Background: Cardiac Troponin is the preferred biomarker for the diagnosis of myocardial infarction (MI) and risk stratification of patients with acute coronary syndromes with respect to relative risk of increased probability of ischemic events, myocardial infarction, or mortality. Objective: The goal of this study was to demonstrate equivalent Troponin-I assay performance across the ARCHITECT i1000SR and on the i2000SR instrument systems. Methods: Total Precision was performed on sample concentrations of 0.013, 0.500, and 15.000 ng/mL for 20 days
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following the CLSI procedure EP5-A2. The Total Precision estimate reflects the summation of within-run, between-run, and between-day variance components for one reagent lot on two i1000SR instruments and two reagent lots on two i2000SR instruments. Functional Sensitivity was evaluated across multiple systems per platform with guidance from the IFCC. (M. Panteghini et al Chin Chem 2004:50(2):327-332). The 99thpercentile determination for the Reference Range was determined by testing four hundred thirty (430) specimens with guidance from CLSI document C28-A2. Method Comparison followed guidance from CLSI document EP9-A2. The correlation study used two i1000SR instruments and one i2000SR instrument each with one hundred forty four (144) specimens ranging from 0.017 to 33.285 ng/mL on the i2000SR. Results: Total Precision: The Total Precision range observed was from 4.3 to 5.5% for the i1000SR and from 2.7 to 4.6% for the i2000SR. Functional Sensitivity: Variation can exist between systems and instruments; though, the median 10%CV for the i1000SR and i2000SR instruments in this study were 0.036 and 0.043 ng/mL, respectively. Reference Range: For the population studied, both instrument systems had an upper reference limit of 0.02 ng/mL for the 99th percentile. Method Comparison The regression line was i1000SR = 1.07*(i2000SR) -0.32 where the 95% CI for the intercept is from -0.65 to 0.00 and the 95% CI for the slope is from 1.05 to 1.10. Using Passing-Bablok methodology the regression line was i1000SR = 1.08*(i2000SR) -0.19 where the 95% CI for the intercept is from -0.28 to -0.10 and the 95% CI for the slope is from 1.06 to 1.09. The correlation coefficient was 0.989 with the lower one-sided 95% CI at 0.985. Conclusion: The Abbott ARCHITECT STAT Troponin-I assay delivers precise and equivalent results across the ARCHITECT ifamily (i1000SR and i2000SR), thus supporting flexible and reliable cardiac solutions within single system laboratories as well as in larger networks that can utilize both instruments together with confidence and high standards of quality.

B-60 Performance evaluation of an automated immunoassay for the determination of cardiac Troponin I on the Olympus AU3000i Immunoassay System D. Fitzgerald, C. Kiely, H. Kelly, N. Shine, M. McNulty, S. Gaston. Olympus Life Science Research Europa (GmbH), Co.Clare, Ireland
Since its introduction cardiac troponin I (cTnI) has proven to be one of the most specific and sensitive markers of acute myocardial infarction (AMI), perioperative myocardial infarction, cardiac contusion and other kinds of myocardial tissue damage. The Olympus Troponin I and Troponin I short assays are paramagnetic particle, chemiluminescent immunoassays for the quantitative determination of cTnI levels in human serum and plasma using the Olympus AU3000i Immunoassay System. Reported here are results from the development and evaluation of these automated assays for cTnI on the Olympus AU3000i Immunoassay System. Both assays are referenced to the NIST SRM2921 (Standard Reference Material). Limit of detection and functional sensitivity (at a total CV of 10%) was determined to be 0.01ng/mL and 0.02ng/mL respectively for both assays. The 99th percentile was determined to be 0.03ng/mL for both assays. Assay imprecision was characterized over 20 days (80 reps/instrument) according to CLSI protocol EP5-A2. Total coefficients of variation (CV) for low (0.03ng/mL-1ng/mL), medium (2ng/mL-50ng/mL), and high (50ng/mL100ng/mL) human serum pools were less than 6% for both assays. A method comparison performed against the Abbott ARCHITECT using 161 serum samples yielded a correlation coefficient of 0.95, with a slope of 0.4 and intercept of 0.004ng/mL. Methods comparison between the long and short versions of the cTnI assays on the Olympus AU3000i system demonstrated an acceptable slope (0.98), intercept (0ng/mL) and correlation coefficient (0.99). Linearity was demonstrated over the concentration range 0.04ng/mL to 86.5ng/mL. No significant interferences were detected from bilirubin, hemoglobin triglycerides and Intralipid. Reagents were optimized to negate RF and HAMA interference. No significant cross-reactivity was observed with skeletal TnI, cardiac TnC and cardiac TnT. Based on our evaluation, we conclude that the Olympus Troponin I and Troponin I short assays are exceptionally sensitive, precise, and specific methods that meet the current NACB guidelines for measuring cTnI in human serum and plasma.
B-59 Assessment of the 99th Percentile and Functional Sensitivity of the ARCHITECT STAT Troponin-I Immunoassay with Serum, Lithium Heparin and EDTA Plasma Specimens J. Waterston1, J. Hoffman2, C. Kramer2, R. Taylor2, K. Ford2, A. Newhart1. 1Thermo Fisher Scientific, Middletown, VA, 2Abbott Laboratories, Abbott Park, IL
Background: The ESC/ACC and NACB recommend that troponin levels elevated above the 99th percentile (%ile) of normal can provide prognostic information to identify patients at increased risk of future cardiac events or mortality. Heparinized plasma and serum specimens may be used with the ARCHITECT STAT Troponin-I assay, although heparinized plasma is recommended to allow faster turnaround times and robust specimen integrity. Since EDTA plasma is the preferred tube type for other cardiac markers (i.e., BNP), the ability to utilize this specimen type for troponin testing would provide increased laboratory flexibility. Ideally, troponin cutoffs should be equivalent among specimen types, though often this parameter is not studied and may vary by test method. Objective: The goals of this study were to determine the 99th %ile of normal using matched serum, lithium heparin and EDTA plasma specimens and to determine the functional sensitivity (10% CV level) using troponin panels prepared in serum, lithium heparin, and EDTA plasma for the ARCHITECT STAT Troponin-I assay. Methods: A total of 449 normal subjects (224 males; 225 females) were tested and the 99th%ile was calculated with guidance from CLSI document C28-A2. Functional sensitivity was evaluated across multiple systems per platform with IFCC guidance (M. Panteghini et al Chin Chem 2004:50(2):327-332) by use of a 10member panel for each specimen type. These panels were prepared by supplementing a negative matrix pool with endogenous troponin-I to target concentrations. Panels were tested in replicates of 2, spanning 10 days utilizing 2 reagent lots and 3 calibrations on ARCHITECT i1000SR and i2000 SR instruments. Total %CV as a function of mean concentration was fitted to a reciprocal curve to estimate the concentration corresponding to the 10%CV. Results: The 99th%ile and functional sensitivity (10% CV level) are comparable between serum, and lithium heparin, and EDTA plasmas. For the population studied, the upper reference limit (99th%ile) is 0.023 ng/mL for serum, 0.025 ng/mL, EDTA plasma, and 0.028 ng/mL for lithium heparin plasma. The functional sensitivity (10% CV level) result for serum, and EDTA and lithium heparin plasmas was 0.027, 0.023, and 0.027 ng/ml, respectively for the three specimen types. Conclusion: This study demonstrates the 99th %ile and functional sensitivity for the Abbott ARCHITECT STAT Troponin-I assay are comparable between serum, lithium heparin and EDTA plasma specimens. This is especially important for laboratories that utilize more than one specimen type so clinicians can interpret troponin values without differentiation by specimen type.
B-61 Performance evaluation of an automated immunoassay for the determination of BNP (B-type Natriuretic peptide) on the Olympus AU3000i Immunoassay System D. Fitzgerald, C. O'Donnell, S. Gaston. Olympus Life Science Research Europa (GmbH), Co.Clare, Ireland
B-type natriuretic peptide (BNP) is a cardiac hormone that regulates haemodynamic equilibrium and alleviates ventricular stress. In patients with chronic heart failure, BNP levels increase in proportion to the severity of clinical symptoms and degree of decreased left ventricular ejection fraction. BNP has clinical utility in the evaluation, management, and prognosis of patients with heart failure. The Olympus BNP assay is a paramagnetic particle, chemiluminescent immunoassay for the quantitative determination of BNP levels in human plasma using the Olympus AU3000i Immunoassay System. Reported here are results from the development and evaluation of the automated assay for BNP on the Olympus AU3000i Immunoassay System. Lowest detectable level was determined to be 0.96pg/mL. Assay imprecision was characterized over 20 days (80 reps/instrument) according to CLSI protocol EP5-A2. Total coefficients of variation (CV) for low (<50pg/mL), medium (50pg/mL - 500pg/mL), and high (>500pg/mL), samples were 3% at all concentration ranges tested. A method comparison performed against the Siemens ADVIA Centaur using 137 plasma samples yielded an acceptable correlation coefficient of 0.97, a slope of 1.0 and an intercept of 0pg/mL. Reagent onboard and calibration stabilities were determined to be at least 28 days. Reagent shelf life was determined using an accelerated stress protocol, assessing recovery bias after recalibration. Changes in recoveries of control materials using stressed reagent was typically <10%. Linearity was demonstrated using 3 patient samples at concentrations between 127pg/mL and 3788pg/mL, and yielded linearities with average biases <5%. No significant interference was detected from bilirubin, hemoglobin, triglycerides and Intralipid. Reagents were optimized to negate RF and HAMA interference. No significant cross-reactivity was observed with the other natriuretic peptides, ANP, CNP, VNP, and DNP. Based on our evaluation, we conclude that the Olympus BNP assay is a sensitive, precise, and specific method for determining BNP in human plasma.
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B-62 Multicentre Evaluation of a High Sensitive Elecsys Troponin T Assay R. Beyrau1, S. Braun2, A. Dolci3, H. Freidank1, E. Giannitsis4, B. Handy5, A. Jaffe6, P. Jarolim7, H. Katus4, S. Melanson7, C. Mller1, M. Panteghini3, P. Venge8, A. Saenger6, J. Jarausch9, et al. 1Universittsspital, Basel, Switzerland, 2Deutsches Herzzentrum, Mnchen, Germany, 3Azienda Ospedaliera Luigi Sacco, Milano, Italy, 4University Hospital, Heidelberg, Germany, 5MD Anderson Cancer Center, Houston, TX, 6Mayo Clinic, Rochester, MN, 7Brigham and Womens Hospital, Boston, MA, 8University Hospital, Uppsala, Sweden, 9Roche Diagnostics GmbH, Penzberg, Germany
Background The universal definition of acute myocardial infarction (AMI) requires the use of the 99th percentile reference limit as cutoff for troponin positivity. Imprecision at that concentration should not exceed 10% CV. This performance is not achieved with most of the current troponin assays. Therefore, there is the need for increased assay sensitivity. Methods Measurements were performed with a new generation of the Elecsys TnT assay with increased analytical sensitivity. The new formulation of the assay uses the capture antibody from the 4th generation TnT assay. The detection antibody from the 4th generation assay was genetically re-engineered by replacing the constant C1 region in the mouse monoclonal antibody by a C1 region from human IgG. This modification was done to further limit the interference by human anti-mouse antibodies (HAMAs) in patient samples in addition to other means limiting interference by HAMA already used in the 4th generation assay. The imprecision was evaluated in a study according to the CLSI EP5-A2 guideline including measurements from a total of 72 individual serum/ plasma pools determined on 21 days (63 or 84 replicates). Comparison to the 4th generation Elecsys TnT assay was performed in 1500 samples from the hospitals routine. Reference values were determined in a prospective study with 546 apparently healthy volunteers of both genders aged between 20 - 71 years. The sensitivity in the diagnosis of AMI at admission to the hospital was determined in 199 patients presenting with chest pain, including 56 patients diagnosed as AMI according to the universal definition. Statistical calculations included regression analysis according to Passing Bablok and the Wilcoxon test. Results The between-day imprecision was < 10% CV for TnT concentrations < 12 ng/ L. Method comparison to the 4th generation TnT assay showed slopes between 0.95 and 1.05, intercepts between 12 and 26 ng/L and correlation coefficients between 0.991 and 0.997. Due to the different assay detection limits, a limited comparability was observed at low TnT concentrations (<100 ng/L). The upper reference limit (99th percentile) was determined at 14 ng/L. The highest test result in the reference population was 24.9 ng/L. The distribution of reference values depended on age and gender, with statistical significant differences (p < 0.05) between the same decades of age in males and females and between the lowest and highest decade in both genders. The sensitivity of the high sensitive TnT assay (cutoff: 14 ng/L) in samples taken within 30 min from admission to the hospital was 93% (95%CI: 83 - 98%). In the same population, the sensitivity of the commercially available 4th generation TnT assay (cutoff: 30 ng/L) was 71% (95%CI: 58 - 83%), which was significantly lower (p <0.001). Conclusions The new high sensitive TnT assay provides excellent analytical performance at concentrations present in apparently healthy people. More importantly, the assay seems to display a higher sensitivity for the detection of myocardial necrosis on admission to the hospital.
Cardiac Markers
hs-CRP, biochemical analytes (Total Cholesterol (TC), LDL-C, HDL-C, Triglycerides (TG), Blood Glucose, Creatinine) and CBC were measured in samples collected at admission. TnI was measured at admission (cTnI0) and every four hours until a peak value was reached (cTnI peak). LVEF was determined by Doppler echocardiography, during the hospitalization. During 6 months follow-up the rate of mortality (), new myocardial infarct (re-MI), and chronic hearth failure (CHF) were recorded. For the evaluation of the significance of predictors of end points (; re-MI; CHF) at 6 months, a T-test and a logistic regression analysis were performed with the following variables: age; gender; dyslipidemia (TC, LDL-C, HDL-C, TG); diabetes; smoking; comorbid conditions; LVEF; BNP; cTnI0 and cTnI peak, hs-CRP, Blood Glucose, Creatinine, CBC (WBC and Plts). Continuous variables were categorized according to clinical established cut-offs or, where lacking or not fully recognized, to cut-offs from ROC analysis (age 74 years; BNP 224 ng/mL; LVEF 40%; WBC 10.5/L). All variables were analyzed for prognostic power by calculating hazard ratio (HR) and Fisher test: those with a p<0.20 were entered in a logistic regression model. Results. In univariate analysis (Fisher test) the events (n. 112; CHF 44%, 35%, reMi 21% ) were associated in NSTEMI population with age (p<0.0001), gender (p 0.06), dyslipidemia (p<0.0001), diabetes (p<0.0001), comorbid conditions (p<0.0001), BNP (p: <0.0001), TnI peak (p< 0.001), hs-CRP (p<0.0001), Blood Glucose (p<0.0001), Creatinine (p<0.0000), WBC (p<0.0001); in STEMI population with age (p<0.0008), gender (p 0.0024), dyslipidemia (p0.0322), comorbid conditions (p<0.0001), LVEF (p<0.0001), BNP (p<0.0001), hs-CRP (p 0.0002), Blood Glucose (p0.0427), Creatinine (p<.0095). In multivariate analysis (logistic regression) many factors resulted not independently associated with end-points events. The independent predictors were: in NSTEMI, age (HR 1.86; 95%CI 1.12-3.05), comorbid conditions (2.11; 1.28-349), BNP (6.44;3.88-10.69), Blood Glucose (1.83;1.08-3.11), Creatinine (2.08;1.20-3.63); in STEMI population, gender (HR 2.11; 95%CI 1.10-4.03), comorbid conditions (2.98; 1.28-349), LVEF (4.13; 1.48-11.51), BNP (4.30; 2.148.64). Conclusions. In our NSTEMI and STEMI populations, the predictive power of several commonly used variables is not the same. In multivariate analysis only BNP and comorbid condition were confirmed as strong independent predictors of total adverse events (death, re-MI, and CHF) at long term (6 months) in both ACS populations. The non -significance of cTnI values (at admission and at peak) could depend on the number of patients with a surgical rivascolarization.
B-64 Performance of Siemens Advia High Sensitivity and Wide Range CReactive Protein Methods A. Tucker-Epps, L. M. Bachmann, W. G. Miller. Virginia Commonwealth University, Richmond, VA
C-Reactive Protein (CRP) is measured at concentrations below 10 mg/L as a marker for risk of cardiovascular disease. Concentrations of 1 and 3 mg/L represent decision points in clinical practice guidelines. The objective of this study was to evaluate the performance of the new Siemens Advia CardioPhase high sensitivity (hsCRP) assay compared to the former, wide-range (wrCRP) measurement procedure. Both are turbidimetric immunoassays based on aggregation of polystyrene latex particles coated with anti-CRP antibody. Both assays were performed using a Siemens Advia 1800 analyzer according to manufacturers instructions. The assays were calibrated weekly using manufacturer provided calibrators. Imprecision was estimated according to CLSI EP5 protocol using three pooled human serum samples. Within run CVs at 1.24, 3.28 and 8.36 mg/L were 0.82%, 1.20% and 0.64% for the hsCRP assay, and 1.44%, 1.45% and 0.54% for the wrCRP assay. Total CVs at the same concentrations were 1.35%, 1.50% and 0.84% for the hsCRP assay, and 6.41%, 4.05% and 0.64% for the wrCRP assay. The analytical measurement range was evaluated by making 9 admixtures from 2 sera with hsCRP concentrations of 0.28 and 9.45 mg/L. Using CLSI EP6 statistical procedures, the hsCRP assay exhibited deviations from linearity from -8.2% to 5.1%, and the wrCRP assay deviated from linearity from -18.9% to 8.0%. Fifty individual serum samples with hsCRP concentrations from 0.14-9.00 mg/L were measured using the hsCRP and wrCRP assays. A non-linear relationship was observed at concentrations below 0.5 mg/dL. The Deming regression equation for values above 0.5 mg/L (N = 43) was: hsCRP = 0.83*wrCRP +0.52 mg/L, Sy-x = 0.07 mg/dL. The same 50 sera were measured using a Roche Immuno-chemiluminometric high sensitivity CRP assay with a Roche Integra analyzer and Roche calibrators. Deming regression gave hsCRP = 1.07*Roche -0.05 mg/dL, Sy-x = 0.17 mg/dL and a linear response over the complete concentration range. When wrCRP was compared to the Roche assay, a non-linear relationship was observed below 0.5 mg/dL. Deming regression for values above 0.5 mg/L (N = 43) gave: wrCRP = 1.29*Roche -0.68 mg/ L, Sy-x = 0.20 mg/dL.
B-63 RISK STRATIFICATION IN ACS: same predictors in STEMI and NSTEMI populations? P. Cappelletti1, D. Rubin1, P. Bulian2, M. Cassin3, G. Nicolosi3. 1Clinical Pathology AOSMA, Pordenone, Italy, 2CRO, Aviano, Italy, 3Cardiology AOSMA, Pordenone, Italy
Objective. We evaluated the performance of cardiac markers, other biochemical measurements, and clinical risk indicators to predict the risk of adverse events at long term (6 months) in both populations of NSTEMI and STEMI patients. Relevance. In ACS (acute coronary syndromes), identification of high-risk patients might be helpful for selection of more aggressive pharmacological or interventional treatment. It is debatable if the variables maintain the same prognostic value in different population of ACS, namely ST or non-St elevation ACS. Materials and Methods. In 900 patients (67% males; age range 26-100, median 68 years), consecutively admitted in CCU - 601 NSTEMI and 299 STEMI - cTnI, BNP,
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Cardiac Markers
The influence of potentially interfering substances was examined by adding known concentrations to each of two serum pools with 3.24 and 6.43 mg/dL hsCRP. Intralipid at triglyceride-equivalent concentrations of 600 and 1000 mg/dL caused biases of 2.8% to -6.5% for hsCRP and -3.4% to -8.6% for wrCRP. Ditauro-bilirubin at concentrations of 20 and 50 mg/dL resulted in biases of 2.4% to 6.6% for hsCRP, and 1.1% to 9.0% for wrCRP. Hemolysate from red blood cells with hemoglobin concentrations of 250 and 500 mg/dL resulted in biases of -0.8% to -3.8% for hsCRP, and -1.2% to -5.9% for wrCRP. In conclusion, the hsCRP assay had acceptable performance for measurement of low CRP concentrations required for cardiac risk assessment. The wrCRP assay exhibited poor performance at lower concentrations.

B-66 Peripheral blood cells transcriptome to study new biomarkers for myocardial infarction follow up V. N. Silbiger1, A. D. Luchessi1, R. D. Hirata1, A. Carracedo2, M. Brin3, L. G. Lima-Neto1, C. P. Pastorelli1, J. Dopazo4, D. Montaner4, F. Garcia4, M. P. Sampaio5, M. P. Pereira5, E. S. Santos5, D. Armaganijan5, M. H. Hirata1. 1Faculdade de Cincias Farmacutica - Universidade de So Paulo, So Paulo, Brazil, 2Universidad de Santiago de Compostela, Santiago de Compostela, Spain, 3Complexo Hospitalario Universitario de Santiago de Compostela, Santiago de Compostela, Spain, 4Bioinformatics Department- Centro de Investigacin Prncipe Felipe, Valencia, Spain, 5Instituto Dante Pazzanese do Estado de So Paulo, So Paulo, Brazil
Introduction: The main clinical manifestation of atherosclerosis is the acute myocardial infarction (MI), which is a medical emergency that requires a prompt diagnosis and efficient therapy. New markers to predict MI have been scarcely described in the last decade. Objectives: A transcriptomic experiment was carried out to study the global gene expression in peripheral blood cells (PBC) to study surrogate biomarkers for the early diagnosis of myocardial infarction. Methods: 5 MI patients and 6 healthy individuals were selected in a coronary emergency facility. MI patients were followed up 8h after MI (T1), after angioplasty (T2) and each 12 h during 48 h (T3 to T6). The control group (GC) was compose by healthy individuals and this 5 MI after one year past of the event. RNA was extracted by PAXgene system (Qiagen). PBC mRNA expression profiles were assessed by Affymetrix GeneChip Human Exon 1.0 ST array. Signals were quantile-normalized using iterPLIER algorithm, filtering (IQR, DABG and CROSS HYBR) and bayesian adjusted t-statistics from the linear models for Microarray data (limma) package interfaced on oneChannelGUI was applied to evaluate the differential gene expression. The comparisons of interest where each time of MI versus GC, and the evolution of the genes selected in the first moment during the time investigated. The functional interpretation in terms of biological process, molecular interaction and disease processes were performed using Ingenuity Systems program. Results: After normalization and filtering were available 16869 genes. 25 genes were differently expressed between MI T1 and GC with Benjamini & Hochberg adjusted pvalue < 0.05 and fold change 1.5, follow by 596, 138, 24 and 6 in T2, T3, T5 and T6, respectively. No differences were found after 24 hours (T4). Only 13 genes were common of the first comparison when T2 (after angioplasty) was compared with CG and 3 genes after 12 h of MI (T3), which were involved in cellular growth/proliferation and lipid metabolism. Nine genes involved with cell-mediated immune response were selected only in T1. Conclusion: The PBC transcriptome of MI follow up displayed significant variations on expression of genes involved in immune-mediated response, growth/proliferation and lipid metabolism. Immune response-related genes selected in this study may predict the initial response to the MI lesion.
B-65 New antibodies for dPAPP-A immunoassays T. I. Solovyeva1, A. B. Postnikov1, D. V. Serebryanaya2, N. N. Tamm1, A. V. Kharitonov2, A. G. Katrukha1. 1HyTest Ltd., Turku, Finland, 2Shool of Biology, Moscow State University, Moscow, Russian Federation
Pregnancy associated plasma protein (PAPP-A) is a high-molecular weight glycoprotein initially identified in blood of pregnant women as heterotetrameric (2:2) complex (htPAPP-A) with proform of eosinophil major basic protein (proMBP). Recently it was revealed that PAPP-A is also expressed in unstable atherosclerotic plaques and can be used as a blood marker of adverse outcome in acute coronary syndrome (ACS) patients. It was shown that atherosclerotic form of PAPP-A was free from proMBP and had lower molecular weight than htPAPP-A. Thus it was suggested that PAPP-A from plaques as well as PAPP-A from blood of ACS patients is a homodimeric form of PAPP-A (dPAPP-A). Immunoassays that do not discriminate htPAPP-A and dPAPP-A are used now for dPAPP-A measurements in blood of ASC patients. However, low but appreciable levels of htPAPP-A in normal and ACS patients blood could significantly influence dPAPP-A measurements by these nonspecific assays. Thus, design of novel dPAPP-A-specific immunoassay seems to be very promising for accurate marker quantification. The aim of our study was to generate monoclonal antibodies (MAb) specific to dPAPP-A and to design the prototype immunoassays suitable for dPAPP-A measurements in ACS patients blood. After mice immunization with dPAPP-A purified from human atherosclerotic coronary vessels we have obtained nine hybridoma cell lines producing monoclonal antibodies, specific to endogenous dPAPP-A and recombinant dPAPP-A (produced in mammalian cell line) and having no cross-reaction with htPAPP-A. All new antibodies were tested in pairs with panel of PAPP-A antibodies able to recognize all three forms of PAPP-A - htPAPP-A, endogenous dPAPP-A and recombinant dPAPPA. Out of 300 tested two-site combinations two (pairs PAPP8-PAPP24 and PAPP52PAPP30) were selected for the development of dPAPP-A specific sandwich immunoassays. In these assays one antibody (MAb PAPP8 or MAb PAPP30) was specific to dPAPP-A (without cross-reaction with htPAPP-A), whereas another one (MAb PAPP24 or MAb PAPP52) was able to recognize all three forms of PAPP-A. Detection MAbs PAPP24 and PAPP30 were labeled with stable Eu(3+) chelate. Both assays recognized endogenous and recombinant PAPP-A and showed very low cross-reactivity (< 1%) with htPAPP-A. The analytical detection limit of assays was less than 2 mIU/L, when purified recombinant dPAPP-A (HyTest, Finland) was used as a calibrator. dPAPP-A level in plasma samples of 43 ACS patients (acute myocardial infarction, unstable angina, 3-20 hours after chest pain onset) and 34 nonACS patients control group was measured. dPAPP-A level in plasma from ACS patients being measured by PAPP8-PAPP24 and PAPP52-PAPP30 assays was 2.60 and 2.54-fold, respectively (p<0.01), higher than in plasma samples of control group. Conclusions: here for the first time we are describing new generation of dPAPP-A specific MAbs and new type of immunoassay suitable for direct measurement of dPAPP-A in ACS patients blood.
B-67 New insights into human proBNP processing: the evidence for furinmediated proBNP processing in vitro. A. G. Semenov1, A. B. Postnikov1, N. S. Karpova2, K. R. Seferian1, N. N. Tamm1, D. V. Serebryanaya2, A. G. Katrukha1. 1HyTest Ltd., Turku, Finland, 2Department of Biochemistry, Moscow State University, Moscow, Russian Federation
Brain natriuretic peptide (BNP) is a peptide hormone that acts to decrease systemic vascular resistance and central venous pressure and to increase natriuresis. BNP is secreted into the circulation by cardiomyocytes. Active BNP-32 hormone (32 amino acid residues, a.a.r.) is formed from the precursor molecule, proBNP (108 a.a.r.), along with N-terminal fragment (NT-proBNP; 76 a.a.r.), by specific enzyme cleavage. The cleavage site of the proBNP molecule is located between amino acid residues Arg76 and Ser77. At present 2 proprotein convertases, furin and corin, are discussed as possible candidates responsible for proBNP processing, but still it is not clear which of these two enzymes is responsible for proBNP cleavage. The data obtained by our group enable us to suggest that proBNP processing is mediated by furin. Recently we have demonstrated that processing of human proBNP is suppressed by O-glycosylation (Semenov et al. 2009). Of several mutant proBNP forms expressed in HEK 293 cells, only the proBNP variant with substitution Thr71Ala71 (T71A
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variant) was effectively processed in cells due to the lack of O-glycosylation at Thr71, located close to the cleavage-site region, as substitution of alanine for serine prevents O-glycosylation. The fact that proBNP processing is inhibited by O-glycosylation enables us to use T71A variant of proBNP in further experiments on proBNP processing. To elucidate the role of furin in proBNP processing, we expressed proBNP in furindeficient human cell line LoVo and estimated the level of its processing by measurements of NT-proBNP/(proBNP+NT-proBNP) ratio in conditioned media of transfected cells by in-house immunoassays. It was shown that processing of recombinant proBNP (T71A variant) expressed in LoVo cells was inefficient. However, when T71A variant of proBNP was coexpressed in LoVo cells with furin, it was effectively cleaved to form NT-proBNP and BNP-32. To show that reduction of furin level in HEK 293 cells (normally expressing furin) or inhibitor-dependent reduction of furin activity in the cells leads to the reduction in proBNP processing level, the cells expressing recombinant proBNP were transfected with furin-specific siRNA or treated with furin inhibitor and the level of proBNP processing was estimated. We showed that siRNA-mediated furin suppression as well as inhibition of furin activity led to significantly impaired proBNP processing in HEK 293 cells. To demonstrate that proBNP cleavage by furin is site-specific and results in formation of BNP-32 molecule, we analyzed the products of in vitro proBNP processing by mass-spectrometry. MALDI-MS analysis revealed that furin-mediated cleavage of recombinant proBNP expressed in E.coli as well as of endogenous proBNP (affinitypurified from plasma of heart failure patients) led to the specific formation of BNP32. So we conclude here that the data obtained in the present study confirm the hypothesis of furin-mediated proBNP processing.
Cardiac Markers
B-69 Analytical Performance of an Optimized ELISA for Galectin-3 A. Smith1, A. H. Wu1, G. Abel2, R. H. Christenson3, S. Wang4. 1University of California, San Francisco, San Francisco, CA, 2Lahey Clinic, Burlington, MA, 3University of Maryland, Baltimore, MD, 4BG Medicine, Waltham, MA
Objectives: We evaluated the analytical performance of an optimized galectin-3 ELISA (Galectin-3 Assay, BG Medicine, Waltham, MA). The galectin-3 (gal-3) isotype is a 26 kDa protein that binds -galactosidase. Relevance: Gal-3 plays an important role in post-inflammation fibrosis and adverse cardiac remodeling leading to post-MI development of heart failure and heart-failure progression. Recent clinical studies have shown that gal-3 blood levels may be useful in the management of heart failure (HF) patients. Methodology: Analytical sensitivity was determined according to CLSI EP17-A. Dynamic range, defined as the range within which gal-3 can be reliably measured, was determined by recovery of spiked samples, and imprecision over the dynamic range (20-day CLSI EP5-A2 protocol). Serum and EDTA-plasma were compared and cross-reactivity towards other galectins, -galactoside binding lectins, collagens, HF drugs, and common interferents was determined according to CLSI EP 7-A2. EDTAplasma samples from healthy subjects (n=1,092) and patients with HF (n=1,160) were also tested. Results: The analytical measurement range (AMR) was up to 100 ng/mL. Both limit of detection (LoD) and limit of quantitation (LoQ) were 1.77 ng/mL. Imprecision and spike recovery testing results are shown in Table 1. There was no interference of gal-3 in the presence of bilirubin (recovery 108%), lipids (100%), albumin (109%), cholesterol (103%), and creatinine (98%), and there was <0.5% cross-reactivity towards other isoforms (galectin 1, 2, 4, 7, 8, 9, and 12) and collagen (I and III). Sample hemolysis was shown to significantly affect measured gal-3. No interference from 34 common drugs was observed (recovery was 91.7% - 108.1%). Summary imprecision and spike recovery of the galectin-3 ELISA Imprecision: Low Medium High CV (%) (5.95 ng/ml) (20.13 ng/mL) (68.01 ng/mL) Within-run 5.03 2.86 9.27 Run-to-run 5.25 4.12 12.75 Total 7.65 5.84 15.77 Spike recovery results Samples tested Range, % recovery Mean, % recovery EDTA plasma 5 88.3-108.1 98.9 Serum 5 96.4-107.7 102.1 Conclusions: The analytical performance of this galectin-3 assay is acceptable for routine laboratory testing. Automated and point-of care assays are needed to provide broad access to gal-3 testing for clinical use.
B-68 Stability of Cardiac Troponin I in serum S. Duh, R. H. Christenson. University of Maryland School of Medicine, Baltimore, MD
Objective: We investigated the stability of cardiac troponin I (cTnI) in serum upon storage under different conditions as well as after several freeze-thaw cycles. Relevance: Cardiac troponin is the preferred biomarker for myocardial infarction diagnosis and risk stratification. Despite the importance of this analyte, cardiac troponin methods demonstrate heterogeneous characteristics and are neither standardized nor harmonized. Further, due to commutability issues with some assays and the troponin reference material, use of human serum matrix is necessary for moving toward standardization. cTnI stability data in serum stored at 4 C and at -70 C with multiple freeze and thaw cycles prior to pooling is not available. Methodology: Three serum pools with targeted cTnI concentrations at around 1 ng/ mL, 5 ng/mL, and 10 ng/mL were prepared using left-over specimens no more than 3 days old. The cTnI value of each pool was measured on day 0 on the Stratus CS (Siemens, Gasgow, DE) and then immediately subjected to refrigerated conditions of 4 C. One aliquot from each pool was removed for cTnI measurement on the 7th and 11th days. All analyses were performed in duplicate and the mean used for data analysis. After 11 days of refrigerated storage, aliquots were frozen and maintained at -70 C for 4 months. Aliquots were then removed and thawed for 5 minutes in a 30 C water bath and centrifuged before analysis. Aliquots were subjected for up to 4 freezethaw cycles, each after at least two more days of storage and cTnI was measured in duplicate after each thaw. Results: At day 0 the serum pools had measured cTnI concentrations of 1.19 ng/mL, 6.13 ng/mL, and 9.63 ng/mL for the low, medium and high pools, respectively. After 7-day storage at 4 C, the respective serum pools retained 96.2%, 99.3%, and 97.0% of their initial cTnI value. After 11 days, recovery of the 3 pools was 91.1%, 95.7%, and 92.5%, respectively. After 4 months of storage at -70 C, the three serum pools recovered 97.3%, 96.6%, and 97.6% of their initial cTnI. After 2 more freeze-thaw cycles the low, medium, and high serum pools retained 97.0%, 97.0%, and 96.3% of their cTnI value, respectively. After 4 cycles, recover of cTnI was 93.6%, 94.5%, and 93.0%. Conclusion: cTnI values in serum remain stable at 4 C for 7 days with an average recovery of 97.5%, and for 11 days refrigerated with an average recovery of 93.1%. cTnI in serum stored at -70 C is stable for 4 months with an average recovery of 97.2%. When stored at -70 C, cTnI measurement showed an average recovery of 93.7% after 4 freeze-thaw cycles. cTnI in serum is a robust analyte that yields high recovery after refrigerated or cold storage conditions, and after several freeze-thaw cycles.
B-70 Performance of the StatusFirst CHF NT-proBNP point-of-care device for patients suspected of heart failure using plasma and whole blood E. N. Shea, A. H. Wu. UCSF, San Francisco, CA
Objective: NT-proBNP is a biomarker useful in the diagnosis of congestive heart failure. As this test becomes more popular, turnaround times for this assay have become shorter. We measured NT-proBNP levels in fresh human EDTA whole blood and plasma samples using StatusFirst CHF NT-proBNP (Nanogen, Inc.) in conjunction with the DXpress Reader in a laboratory setting to determine the degree of correlation between these measurements. Methods: Data was collected from 339 unique individuals enrolled at UCSF (n=319) and the San Diego VA (n=20). Patients were classified as having CHF (n=126) or CHF was ruled out (n=213). EDTA specimens were collected from all subjects, and matching lithium heparin samples were collected from 99 subjects. Method comparisons were performed using Passing-Bablok regression analyses to examine the correlation between whole blood vs. plasma, and lithium heparin vs. EDTA. Nonlab health care operators (simulating a POCT environment) obtained measurements over 20 days to estimate the total precision. Also compared were whole blood vs. plasma results from trained technologists vs. non-lab personnel. The in vitro stability of NT-proBNP was assessed on whole blood and plasma samples after 24 and 48 hours of ambient and refrigerated temperature storage compared to results tested immediately after collection. Results: The table shows the method comparison summaries for different specimen types and between operators. The total precision was 14.9-21.7%CV for NT-proBNP
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of 47.8-1489 pg/mL. No significant difference was found in normalized NT-proBNP concentrations in EDTA whole blood and plasma when stored at ambient and refrigerated temperatures (p>0.10). Lithium heparin was stable in all cases except for refrigerated whole blood. Table1-Results for method comparison and nave operator Comparator Slope Intercept experience operator method comparision EDTA plasma vs. EDTA whole blood 1.017 2.8pg/mL Lithium Heparin whole blood vs. EDTA whole blood 0.966 -1.0pg/mL Lithium Heparin plasma vs. EDTA plasma 0.966 6.6pg/mL nave vs. experienced operator EDTA whole blood vs. EDTA whole blood 1.001 2.6pg/mL Lithium Heparin whole blood vs.Lithuim Heparin whole 0.985 -2..6pg/mL blood EDTA whole blood vs. EDTA plasma 0.976 -15.7pg/mL Conclusions: NT-proBNP measurements obtained from EDTA, lithium heparin, and whole blood specimens are interchangeable using the StatusFirst CHF NT-proBNP point-of-care device. Minimally trained operators can obtain similar results to those obtained from qualified technicians.

immunosorbent assay (ELISA) (Galectin-3 Assay; BG Medicine, Inc., Waltham, MA). RESULTS: The figure demonstrates that Galectin-3 values in this reference population were normally distributed; mean value=12.4 ng/mL, SD=4.4 ng/mL; median = 12.0 ng/mL; central 95% reference interval, 3.8 - 21.0 ng/mL. All subjects had detectable galectin-3 levels (lower limit of detection for the Galectin-3 Assay is <0.12 ng/mL). Correlation coefficients were calculated between galectin-3 and a variety of variables. Minor but significant correlation (<.05) was observed between galectin-3 and age, kidney function and BMI, and by gender and race strata; no significant correlation was observed between galectin-3 and ALT, AST, GGT, LDH, glucose and electrolytes.
B-71 The Use of a Bulk Protein Depletion Strategy to Improve Performance of Troponin I ELISA H. Li, T. Sanderson, L. Bradbury. Pall Life Sciences, Woburn, MA
Objectives: Demonstrate the potential value of a simple bulk protein depletion strategy for improved performance of antibody based assays. Background: it is well known that the performance of antibody based assays like ELISAs is improved when the sample is less complex. For example, a target protein spiked into PBS vs. serum will result in two very different standard curves. A simple depletion strategy utilizing chromatography media is evaluated using human cardiac Troponin I (cTn I) complex as a model since it is a commonly used biomarker for cardiac care. Materials and Methods: Undiluted pooled serum was subjected to protein depletion (250uL serum + 150uL packed resin) using traditional chromatography (ion exchange) and several affinity ligands (Protein A, HSA-specific, IgG-specific, Blue Trisacryl). Whole and depleted serum samples were then spiked with multiple concentrations of cTn I complex and cTn I concentrations were measured by ELISA (CalBiotech). Total protein was determined using a BCA assay (BIORAD). Samples were analyzed on 4 12% SDS-PAGE (BIORAD) stained with GelCode Blue (Pierce). Results: Reduction in total protein after exposure to the various chromatography media ranged from ~2085%, depending on the media chosen. This was reflected in the SDS-PAGE results. As a result of this sample complexity reduction, cTn I detection by ELISA improved significantly, ranging from ~20% increase to ~150% increase relative to signal without depletion. Conclusions: Data from this cardiac Troponin I spike & recovery model system demonstrates that a simple chromatography based method to remove a significant amount of protein from serum results in a dramatic improvement in detection sensitivity of a commercial ELISA kit. This type of strategy could be easily incorporated into spin, vacuum or flow through devices. Although other proteins might require different chromatography media, screening is relatively simple. CONCLUSIONS: The central 95% normal reference interval of galectin-3 measured by the Galectin-3 Assay is 3.8 - 21.0 ng/mL. Galectin-3 was detectable in the plasma of all healthy individuals by this optimized ELISA and exhibited a normal distribution. Common demographic and biochemical parameters had no or minimal effects on Galectin-3 results and will not impact clinical utility.
B-73 Troponin T fails to detect low levels of myocardial necrosis as detected by Troponin I E. Fernandez, S. Garcia, D. Gago, C. Barros, B. Laborda. Hospital Cabuenes, Gijon, Spain
Background: The clinical interpretation of minor elevations of troponin poses a common clinical problem in patients presenting to an emergency room (ER) to rule out Acute Coronary Syndrom (ACS). Despite this, few studies have been carried out to elucidate the different sensitivity between troponin I and troponin T when interpreting minor increases in troponin of questionable significance. Aim: To compare the sensitivity of troponins T and I in detecting ACS with less than 2 hours of evolution since the onset of symptoms. Materials and methods: Forty consecutive AMI patients with minor increases in troponin I at (ER) presentation and less than 2 hours of evolution of symptoms were recruited. Diagnosis of AMI was established according to the European Society of Cardiology (ESC) and the American College of Cardiology (ACC) guidelines for the redefinition of myocardial infarction. All the patients showed a clear and sharp rise and fall of cardiac markers following the initial troponin determination. Troponin T was measured in samples collected in both heparin lithiums and EDTA-K3 tubes in order to remove the possible effect of anticoagulant. Troponin I and troponin T were measured with Dimension Dade Behring and third-generation Roche reagents, respectively. Stratus (Dade Behring) was used as confirmatory test for troponin I (values 0.09 ng/mL were considered as positive). Seventeen patients showed ST segment elevation. Results: The range of troponin values was the following: Troponin I: 0.10 (p99th)-0.26 (10%CV); troponin T: <0.010-0.274 ng/mL; Troponin I (Srratus): 0.09-0.22. Only in 12 out of 40 (30%) AMI patients troponin T yielded positive results (>0.010 ng/mL), thus showing a sensitivity of 30% for detecting early AMI positive-troponin I presenters (within 2 hours of onset of symptoms) at ER. No differences were found in troponin T values according to the type of anticoagulant. Conclusions: Our results clearly show that third-generation troponin T fails to detect minor increases associated to early AMI presenters, that can otherwise be detected even by first generation troponin I assays.
B-72 Reference interval for plasma galectin-3 in healthy subjects age 55 years and older P. Muntendam1, A. Adourian1, R. Christenson2. 1BG Medicine, Inc., Waltham, MA, 2University of Maryland Medical Center, Baltimore, MD
OBJECTIVE: To establish a reference interval for plasma galectin-3, a carbohydrate-binding protein with a role in the path physiology of cardiac remodeling and heart failure progression. To determine the association between plasma galectin-3 and demographic and biochemical variables. RELEVANCE: The reference interval for an optimized galectin-3 assay and the relationship of values with demographic and biochemical parameters are unknown. Knowledge of the reference interval for galectin-3 is necessary for proper interpretation. METHODOLOGY: Blood specimens were collected from participants in the BioImage cardiovascular risk study. This cohort consisted of 1,092 healthy volunteers; males (n=520), age 55-80; females (n=572), age 60-80; overall mean age=68.1 years, SD=6.3. Common laboratory tests were performed and demographics including race and body mass index (BMI) were collected. EDTA plasma samples were analyzed using an optimized enzyme-linked
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from Siemens did reagent comparison studies. Only linear regression was done initially. Subsequently (responding to JCAHO recommendations), the initial validation data was reassessed. Comparison [n=61] of PT data [ISI = 1.96 (X)] and [ISI = 1.0 (Y)] with Table Curve 2D software (Systat; San Jose, CA) suggested a nonlinear relationship with a best fit equation given by: Y = .8 + .1 X 1.8 (r-sq = .994) [1] A linear regression model was the 27th best fit with r-sq = .968. When the data was converted to INRs the relationship became: Y = .08 + .9 X .91 (r-sq = .993). [2] The exponent in [2] is near 1.0 and this equation can be well approximated by a linear regression with Y = .29 + .713 X (r-sq = .992). [3] When POC INR (Y) was compared with instrument results (ISI = 1.0) the best fit equation was given by: Y = -6.14 + 6.48 exp (.12X) [n = 254; r-sq = .868] [4]. For 32 values of the POC INR =/> 3.5, 28 (.875) were greater than instrument values. When POC INR values (Y) were compared to instrument results [ISI = 1.96] (X) the best fit regression equation was given by: Y = -.16 + 3.37(LN X) [n = 50; r-sq = .57] [5] and for 49 values of POC INR =/> 3.5 only 14 (.286) were greater than the instrument value [z score = 10.5, p<.001]. The increased scatter and lower value of rsq seen in [5] as compared to [4] is related to the limited range of the data as only higher values of the INR were verified at the CL. Although equations [4] and [5] used two different instruments these came from the same manufacturer and used an optical end-point. Therefore reagent difference was likely a more important source of result variability than machine difference. The validation data suggests that INR varies with the ISI of the TR being used. However the relationship between INR values obtained with different ISI reagents is approximately linear in contrast to the underlying PT data. Also the relationship of POC INRs to CL INRs is non-linear and will vary with the ISI of the reagent being used in the CL.
Tuesday PM, July 21

Poster Session: 2:00 pm - 4:30 pm Hematology/Coagulation
B-74 Reference values for 67 hematocytometry parameters as determined by the Sysmex XE-5000 analyser. J. M. Pekelharing1, O. Hauss2, R. de Jonge3, J. Lokhoff1, J. Sodikromo1, M. Spaans1, R. Brouwer3, R. Hinzmann2, S. de Lathouder3. 1Reinier de Graaf Ziekenhuis, Delft, Netherlands, 2Sysmex-Europe, Norderstedt, Germany, 3Erasmus Medisch Centrum, Rotterdam, Netherlands
Introduction: Compared with other hematology analysers and with earlier versions of this instrument, the new Sysmex XE-5000 analyser determines 67 parameters in blood. (It also measures 9 parameters in other body fluids than blood, but that was not part of this study). It combines a number of electronical and optical measurements in a diluted blood suspension using a flow cytometry aperture technique. In order to establish the value of all new parameters for use in the diagnosis and treatment of patients a clinical setting, it is necessary to know what results can be expected when analysing samples from healthy persons. To answer this question, blood samples from 309 apparently healthy hospital employees were analysed using the Sysmex XE-5000. Methods: After a publicity campaign in our clinic, we obtained voluntary blood samples from 133 male and 176 female hospital employees. The samples were analysed in full mode (including differentials and reticulocyte measurements). ESR was measured in the same tube. In serum taken at the same time some selected chemistry tests were done: LD, Bilirubin and Ferritin. A few questions (age, sex, use of therapeutic drugs) were asked using a questionaire. Results: The 21,000 results obtained were analysed using standard statistical methods. We describe for the first time the reference values for a number of new parameters. A summary of some of the new parameters for males (m) and females (f) follows here: - IRF (immature reticulocyte fraction) m/f: 1.6-10.5 %. - Ret-He (Hb filling of reticulocytes) m: 2025-2406 amol, f: 1953-2409 amol. - D-He (differential Hb filling of erythrocytes and reticulocytes): m: 271-453 amol, f : 239-451 amol. - IPF (immature platelets) m/f: 0.8-6.3 % or 2.3-12.7 x 10E9/l. - Neut-X (degranulation) m/f: 128-138 ch. - IG ("bands") m/f: <0.06 % or <0.06 x 10E9/l. For many parameters we found that different reference ranges should be used for males and females. In our study we did not take a sample from the general population, but from healthy and working hospital employees. This no doubt leads to more ideal and narrower reference ranges for some parameters, than when blood samples from the general population were used. Conclusion: Reference ranges for 67 hematology parameters were established. Since half of them are new and promising, the establishment of their exact reference values will help in the process of acquiring experience with them in the clinical setting. It is to be expected that our extensive study of the hematological situation of healthy persons using the new Sysmex XE-5000 analyser, will lead to a faster and better diagnosis and monitoring of patients.
B-76 Evaluation of Sysmex UF-1000i Automated Urine Sediment Analyzer J. Ben-Ezra, K. Rainbow, R. McPherson. Virginia Commonwealth University, Richmond, VA
Background: Microscopic identification and quantification of cellular elements is frequently performed to aid in the diagnosis and monitoring of urinary tract disorders. The Sysmex UF1000i is a second generation automated urine sediment analyzer with enhanced capabilities for discriminating cellular elements from debris. Objective: To evaluate the Sysmex UF-1000i for automated measurement of elements in urinary sediment in healthy and sick individuals. Materials and Methods: Urine specimens were selected from those submitted to our hospital central laboratory for routine analysis. Normal specimens (N=58) were defined as being totally negative by dipstick analysis. Abnormal specimens (N=137) had at least one abnormal dipstick marker. All specimens were analyzed within four hours of collection on both the UF-1000i and the UF-100, which was in routine use. Results: The 58 normal urine specimens showed the following ranges of cellular elements (number/L): RBCs 0- 27.6, WBCs 0.2- 24.2, Epithelial Cells (ECs) 0-36.9, Casts 0- 3.19, Bacteria 0- 1313.4. Recoveries of cellular elements after dilutions of 1:2 and 1:4 ranged from 93- 103% for RBCs and WBCs, 78-108% for ECs, 19-51% for Casts, and 132-154% for bacteria. Element RBCs WBCs ECs Within Run Imprecision Level (/uL) 1.07 199 0.62 189 1.81 12.7 2.8 39.3 11.6 189 CV 0.47 0.02 0.44 0.03 0.23 0.10 0.19 0.12 0.44 0.08
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What is the True INR?

Bacteria
D. Lantz1, E. S. Pearlman2. 1East Boston Neighborhood Helath Center, East Boston, MA, 2Boston Medical Center, Boston, MA
When EBNHC introduced a POC assay for INR (Coagacheck; Roche Diagnostics: Indianapolis, IN) in its anti-coagulation clinic, a validation study compared 254 INR results assayed on the POC system and at an external laboratory [Quest Diagnostics; Cambridge, MA] using a CA-7000 coagulation analyzer (Siemens; Deerfield IL) and a thromboplastin reagent [TR] with ISI near 1.0. The POCT was calibrated at the factory using a TR with ISI of approximately 2.0. Central laboratory (CL) verification using TR [ISI = 1.96] was required when POC INR value =/> 5.0 and sporadic for lower values. In May 2008, the (ISI =1.96) TR was replaced with an (ISI = 1.0) TR on our CA-530 coagulation analyzer (Siemens; Deerfield, IL) A technician
Deming regression analysis of cell counts between the two instruments showed slopes close to 1.0 for RBCs, WBCs, and ECs; for Casts the slope was > 2.0, possibly due to interference from mucus, while for Bacteria the slope was 0.10, perhaps due to a new detection algorithm on the UF-1000i that better discriminates bacteria from debris. Conclusion: The UF-1000i provides accurate counts of RBCs, WBCs, ECs, and Casts as judged by direct comparison with the UF-100. Improved cast counts may be achieved by dilution of interfering mucus. Precision of counting sedimentary elements is fully acceptable for clinical use. In addition, the UF-1000i produces lower bacteria counts than the UF-100, which may be related to improved discrimination of bacteria from debris.
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B-77 SOLUBLE ENDOGLIN AND ITS INTERACTION WITH ADHESION MOLECULES IN PATIENTS WITH THALASSEMIA INTERMEDIA I. Papassotiriou1, C. Lazaropoulou1, M. Siopi1, A. Kattamis2. 1Department of Clinical Biochemistry, Aghia Sophia Childrens Hospital, Athens, Athens, Greece, 2First Department of Pediatrics, Athens University Medical School, Athens, Athens, Greece
Endoglin (CD105) is an accessory protein of the transforming growth factor- receptor system expressed on vascular endothelial cells. Mutations on the endoglin gene are associated with hereditary hemorrhagic telangiectasias (Osler-Weber-Rendu syndrome) and, thus, endoglin has been extensively studied in the context of this disease. Endoglin is highly expressed on endothelial cells in healing wounds, developing embryos, inflammatory tissues and solid tumors. It is a marker of activated endothelium, while its vascular expression is limited to proliferating cells. Previous studies have shown that the endothelial function is impaired in patients with Thalassemia Intermedia (TI). Oxidative damage resulting from hemolysis and iron load, leads to increased expression of the intercellular and vascular adhesion molecules-1 (ICAM-1 and VCAM-1) and impaired nitric oxide (NO) bioavailability. As endoglin plays a critical role in angiogenesis and dysregulation of its expression and/or activity has been implicated in multiple vascular diseases, we aimed to investigate the expression of endoglin and its correlation with factors of endothelial dysfunction in patients with TI. Thirty adult patients with TI were included in the study, while 20 healthy individuals served as controls. Soluble endoglin, soluble forms of adhesion molecules ICAM-1, VCAM-1, E and P-Selectins, thrombomodulin, von Willebrand factor, as well as NO and vascular endothelial growth factor (VEGF), were measured in patients and controls, by immunoenzymatic methods. The main results of the study are: a) levels of endoglin, E-selectin, thrombomodulin, VCAM-1, ICAM-1 and VEGF in patients with TI (5.50.4 ng/ml, 91.420.0 ng/ml, 48.816.5 ng/ml, 1413.7176.1 ng/ml, 658.834.6 ng/ml and 619.1227.8 pg/ml, respectively) were higher compared to controls (4.90.3 ng/ml, 10.32.2 ng/ml, 4.00.6 ng/ml, 328.343.8 ng/ml, 107.626.5 ng/ml, 81.358.1 pg/ ml), (p<0.01), b) endoglin levels in patients with TI correlated positively with concentrations of ICAM-1 (r=0.760, p<0.003), VCAM-1 (r=0.520, p<0.05), ESelectin (r=0.790, p<0.0020, P-Selectin (r=0.530, p<0.04), while these correlations were absent in normal individuals. Angiogenesis is a highly coordinated process in which VEGF, endoglin and hypoxia inducible factor 1 (HIF-1) play a pivotal role by coordinating interaction between endothelial cells, extracellular matrix and the surrounding cells. Taking into assumption that endoglin is a protective-repair tissue protein, our findings support the hypothesis that patients with TI exhibit increased degree of angiogenesis and endothelial regeneration, which are probably compensatory mechanisms in response to tissue hypoxia and damage.

between regions, but CAR and Benin are predominant and 20% of the black population is heterozygous for alpha-thalassemia. Thus, the association between sickle cell disease and alpha-thalassemia is frequent. This association has as its consequence, a lesser degree of hemolysis, higher hemoglobin and lower rates of leg ulceration, cerebral vascular accident and retinal abnormalities. There are no reports of the incidence of mutations in the factor II and V genes in the Brazilian population with sickle cell disease. Considering the high incidence of sickle cell disease in Bahia (1 case for every 750 born alive per year), and the importance of characterizing these patients phenotypically and molecularly, this study has as its objective, research into alpha-thalassemia, factor V Leiden, prothrombin gene mutation and beta-globin haplotypes in 236 children, between the ages of 2-6 years, diagnosed with sickle cell disease during neonatal screening, APAE, Bahia, Brazil, between January 2002 and June 2005. METHODS: Sickle cell disease phenotype was determined by the study of hemoglobin through HPLC, apparatus: Variant I (Bio Rad) and kits: Sickle cell e beta-thalassemia. The haplotypes were characterised using PCR and restriction site analysis utilizing endonucleases (RFLP technique). Alpha-2-thalassemia was determined by detection of the 3.7 kb deletion, by allele-specific PCR technique and of mutations in factors II (G20210A) and V (R506Q) using PCR followed by analysis of fragments by RFLP. RESULTS AND CONCLUSION: 145 cases of SC and 91 cases of SS were diagnosed. 23.4% of SS patients and 29.5% of SC patients had alpha-thalassemia, mostly in the heterozygous state. The incidence of the G20210A mutation and factor V Leiden, both in heterozygosity, was 1.3% and 1.9% respectively. The most frequent haplotypes were CAR/I and Benin/I for the SC and CAR/Benin for the SS. The Senegal haplotype was identified only in SC patients, in heterozygous form with haplotypes I and II. Three chromosomes with haplotype III were found. This is a novel study of great importance to the clinical follow-up of patients with sickle cell disease with early diagnosis.
B-79 INCIDENCE OF DEFICIENCY OF G6PD IN SCREENING IN BRASILIA, CAPITAL OF BRAZIL NEWBORN
S. F. FONSECA1, L. F. ABDALLA2, S. S. COSTA2, A. S. CABRAL2. 1UnB, BRASILIA, Brazil, 2LABORATORIO SABIN, BRASILIA, Brazil
Glucose-6-phosphate dehydrogenase (G6PD) deficiency affects approximately 400 million people around the world. The most affected populations are African Americans (12-15%), the people of the Mediterranean (2.5-25%), Asians and Middle Easterners. People with this deficiency may be asymptomatic or may present with acute or chronic haemolytic anaemia. Data regarding the incidence of G6PD deficiency in Brazil are scarce and variable, varying between 1% and 10% according to the ethnic origin of the population studied. Neonatal testing for this enzymopathy has not been obligatory in this country. The Federal District (FD) has an unusual population, consisting of individuals from the various regions of Brazil who arrived in this State in waves of varying intensity, together with those from different areas who migrate steadily to this State. The objective of this study was to verify the incidence of deficiency of G6PD among babies born in FD, who were tested neonatally in the Sabin Laboratory, Brasilia, Federal Capital of Brazil, in the period from January 2007 to January 2008. METHODS: A total of 1933 blood samples collected on filter paper were analysed. Quantification of G6PD activity was performed by an enzymatic colorimetric method normalised to haemoglobin. Newborns were considered normal if they presented with a result in excess of 2.1 g/g Hb. For children considered abnormal, a new blood sample was collected, in a heparin tube, for confirmation of deficiency through quantification of the enzyme by the enzymatic method (RV: 4.613.5 U/g Hb). RESULTS AND CONCLUSION: 18 newborns were indicated as deficient in G6PD in initial testing, of which 5 were confirmed by enzymatic quantification. The index of positivity in the sample was 0.3% (5/1933), and the percentage of false cases after retesting was 72%. The results of this study point to an incidence of G6PD deficiency, in the population studied, lower than the figure previously published in the Brazilian population, which may be explained by factors such as reticulocytosis, present in many newborns with neonatal jaundice, or perhaps it may be the true incidence of this deficiency in District Federal.
B-78 INCIDENCE OF ALPHA-THALASSEMIA, FACTOR V LEIDEN, PROTHROMBIN GENE MUTATION AND DETERMINATION OF HAPLOTYPES OF BETA-GLOBIN GENE IN CHILDREN WITH SICKLE CELL DISEASE DIAGNOSED BY NEONATAL SCREENING, BAHIA, BRAZIL. S. F. FONSECA1, T. AMORIM2, R. RIBEIRO3, N. BOA SORTE3, A. PURIFICAO3, W. V. SANTOS4, C. C. SOUZA4, M. S. GONCALVES4. 1UnB, BRASLIA, Brazil, 2APAE, SALVADOR, Brazil, 3APAE, BAHIA, Brazil, 4FIOCRUZ, BAHIA, Brazil
Since 2001 in Brazil, newborn screening for sickle cell disease has been mandatory, and the state of Bahia, northeast Brazil, has the highest incidence of this hemoglobinopathy in the country. The illness has variable levels of severity and a high mortality rate. Genetic, clinical and laboratory factors may account for this clinical heterogeneity. Among the genetic factors are the phenotype of the hemoglobinopathy, haplotypes of the beta-globin gene, presence of alpha-thalassemia, and of mutations in the prothrombin and factor V genes. Regarding the phenotype, individuals with sickle cell anemia (SS) and those with S thalassemia are more severe, while the ones with SC hemoglobinopathy (SC) and with S+ thalassemia have a better outcome. Haplotypes are important prognostic factors. The Benin, Senegal, Central African Republic (CAR), Cameroon and Indian-Arabic haplotypes are most widely found, with Senegal being associated with a more benign illness and CAR with the greatest severity and precocious death. In Brazil, the frequency of haplotypes is heterogeneous
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B-80 Clinical performance of an automated coagulation analyzer, Coapresta 2000, for analyzing blood samples with a prolonged activated partial thromboplastin time A. Ohsaka1, K. Ishii2, T. Yamamoto2, Y. Kuno2, T. Horii2, Y. Tabe1. 1Juntendo University School of Medicine, Tokyo, Japan, 2Juntendo University Hospital, Tokyo, Japan
Background: Assessment of blood samples with a prolonged activated partial thromboplastin time (APTT) requires mixing studies with normal plasma, and an additional sample may need to be collected from the patient for further analysis. Aims: The aim of this study was to evaluate the performance of an automated coagulation analyzer, the Coapresta 2000 (CP2000) from Sekisui Medical (Tokyo, Japan), which is a fully automated multiparameter coagulation analyzer equipped with a photo-optical clot detection unit. The CP2000 is designed to perform coagulation, chromogenic, and immunologic assays with continuous loading of samples, reagents, and disposables. Its chief advantage is automatic dilution for mixing studies using normal plasma, with a plot of the results being generated. Methods: The CP2000 analyzer was evaluated with respect to its technical characteristics during the determination of routine coagulation parameters and was compared with a mechanical clot detection-based analyzer (STA-R). To evaluate the clinical performance of the CP2000 for analyzing sample with prolonged APTT, 10 laboratory staff members performed mixing studies by three methods: (1) manual dilution and analysis, (2) manual dilution and automatic analysis with the STA-R, and (3) automatic dilution and analysis with the CP2000. The time from starting analysis to generating the plot of the results and the sample volumes needed for mixing studies with each method were determined. Mixing studies were also performed with the CP2000 analyzer using factor-deficient plasma samples or patient plasma samples, and the plots of the results were categorized into three curve patterns, which were the deficiency pattern (concave), the inhibitor pattern (convex), and the suspected inhibitor pattern (straight). Results: The intra-assay and inter-assay coefficients of variation for the CP2000 analyzer were <5% for all of the parameters tested in both the normal and pathological ranges. No significant interference was demonstrated when analyzing hemolytic, icteric, and lipemic samples. The results of the coagulation assays obtained using the CP2000 analyzer were well correlated with those obtained using the STA-R analyzer, with the correlation coefficients ranging from 0.871 to 0.993. The time from the start of analysis to reporting the results and the sample volumes required for mixing studies (seven-point dilution) were 28.2 +/- 2.4 minutes and 1.05 mL with manual analysis, 23.2 +/- 2.1 minutes and 1.40 mL with the STA-R analyzer, and 8.5 +/- 0.1 minutes and 0.175 mL with the CP2000 analyzer, respectively. Thirty-two patient samples with a prolonged APTT (>50 seconds) were categorized into three types as follows: deficiency pattern in 15, inhibitor pattern in 6, and suspected inhibitor pattern in 11. All 6 inhibitor samples and 9 out of 11 suspected inhibitor samples were positive for lupus anticoagulant. Conclusion: The CP2000 analyzer achieved a shorter time from analysis to reporting, a smaller blood volume for mixing studies, and less operator-dependent variation compared with manual analysis. The CP2000 analyzer may be useful for assessment of blood samples with a prolonged APTT and can rapidly provide data on the probable cause, even at a patient's first visit.
One of the reasons to order hemoglobin identification by HPLC is that a hemoglobin variant is noted on hemoglobin A1c chromatograms. Only a few hemoglobin variants have been identified by the Bio-Rad Variant Turbo A1c HPLC methodology by their specific elution times, and almost all abnormal hemoglobins automatically are labeled with the generic comment variant. The A1c measurement has an assay run time of only 1.400 minutes and has not been optimized by the identification of hemoglobin variants. Recently we have identified a hemoglobin variant distinct from hemoglobin A eluting at 0.872 minutes and measuring 21.1% of the total hemoglobin present on our A1c methodology. However, when we used the Bio-Rad Variant II to identify the hemoglobin, no abnormal hemoglobin was obvious, and the chromatogram was interpreted as consistent with a normal study. Upon subjecting the specimen to electrophoresis (Helena Laboratories, Beaumont TX), a hemoglobin variant migrating as hemoglobin S was present on alkaline gel, and a split hemoglobin A peak was present on acid gel. An abnormal alpha or beta chain abnormality was not identified when the specimen was subjected to capillary electrophoresis (CAPILLARYS 2, Sebia, Norcross, GA). Subsequent studies identified the hemoglobin as Hemoglobin G-Georgia (95(G2) ProLeu). This specimen was from a 63 year old female of middle-east extraction who had a complete blood count (CBC) that demonstrated anemia of chronic disease. In our review of the literature, we did not find a hemoglobin variant that eluted as hemoglobin A when the Bio-Rad Variant II HPLC instrument used for hemoglobin identification, but was present on the Turbo A1c chromatogram. Bio-Rad technical support personnel world-wide reported to us they were unaware of a hemoglobin variant that eluted as hemoglobin A on the hemoglobin chromatogram, but was obvious on the A1c chromatogram. We believe this is the first case of such a paradoxical finding. We recommend that unusual variants noted on A1c chromatograms be identified in order that A1c levels are interpreted correctly and patients are provided with proper genetic counseling for their abnormal hemoglobin variant.
B-82 Instability study of abnormal hematologic sample with Bayer ADVIA 120 P. Wu, Y. Li, F. Li, J. Chen. Department of Clinical Laboratory, Renmin Hospital of Wuhan University, Wuhan ,Hubei, China
Objective Abnormal hematologic sample is unstable and easy to change over time, the analysis of which is much accounted of in clinical diagnosis. As the instability may differ from normal sample, the aim of this study was to investigate the instability of abnormal hematologic sample and normal hematologic sample by comparison. Methods We investigated the effect of storage time on abnormal sample stability as well as normal sample stability. All the values were generated by ADVIA 120, an automated hematology analytical system. Abnormal hematologic samples that obtained from 90 patients were grouped into three types (abnormal red blood cell, abnormal white blood cell and abnormal platelet). Normal hematologic samples were obtained from 30 healthy volunteers. All the samples were stored at 4 C in order to conduct analysis at different time points (0 h, 4 h, 8 h, 12 h, 24 h) up to 24 h after blood was drawn. Sample instability was assessed by the graphical truncated normal sequential-test. A parameter was considered unstable when the change of the mean value was more than one coefficient of variation CV (%) of the analytical method used, with 5% risk of error for the decision. Results White blood cell (WBC) counts, red blood cell (RBC) counts and platelet (PLT) counts at 4C were most stable for at least 24 h in abnormal group as well as normal group. Neutrophil (NEU) counts, hemoglobin (HGB) and mean corpuscular volume (MCV) were less affected by storage time in both two groups. Mean platelet volume (MPV) changed over time, we can see a significant increase after 4h in abnormal group while it changed after 12 h in normal group. The instability of Large unstained cell (LUC) and Lymphocyte (LYM) counts in abnormal group was larger than that in normal group, LUC changed after 12 h with an increase, LYM showed an increase at first and decreased after 8 h. Conclusions The instability of abnormal hematologic sample is demonstrated difference from normal hematologic sample. Some parameters, which are key factors directing clinical diagnosis, exhibit considerable change in short time. Hence, to shorten the hours from sampling to detection can be much accounted of in clinical laboratory medicine.
B-81 HPLC Results For Hemoglobin G-Georgia: The First Hemoglobin Paradoxically Observed on A1C But Not On Hemoglobin Identification Chromatograms. P. J. Howanitz1, J. H. Howanitz1, Y. P. Chauhan2. 1SUNY Downstate and Kings County Hospital Center, Brooklyn,, NY, 2Kings County Hospital Center, Brooklyn,, NY
High Performance Liquid Chromatography (HPLC) has become the most common methodology for hemoglobin identification in the U.S., in part, because separation of hemoglobin variants is superior to previously-used hemoglobin electrophoresis methods. Of the HPLC instruments that are used, the Bio-Rad Variant instruments (Bio-Rad, Hercules CA) are the most widely used. Hemoglobin variants are identified by the appearance of abnormal peaks and the percentages and elution times of the abnormal hemoglobins over the 5.30 minute assay run time. The Bio-Rad On-Line Library for hemoglobin variants contains over 700 chromatograms of over 110 unique hemoglobins that have been reported by customers. Hemoglobin electrophoresis in most large laboratories now is relegated to confirming or assisting in the identification of abnormal hemoglobins.
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B-83 Transfusion-Induced D-Los Angeles Hemoglobinopathy. J. H. Howanitz1, A. A. Igbokwe1, P. J. Howanitz1, Y. P. Chauhan2. 1SUNY Downstate and Kings County Hospital Center, Brooklyn, NY, 2Kings County Hospital Center, Brooklyn, NY
Hemoglobin D-Los Angeles, sometimes known as D-Punjab, (22 121 glu gln) is one of the five most common hemoglobin A -chain variants, and when combined with hemoglobin S, causes patients to have sickle cell disease. However, because patients who are heterozygous for hemoglobin D-Los Angeles usually are asymptomatic, this hemoglobinopathy routinely is not be detected in volunteer blood donors. Following blood transfusions, patients have been transfused with red blood cells from heterozygotic donors who had hemoglobin S, C, or O-Arab, thereby potentially causing misdiagnoses in the recipient. To prevent the recipient from being identified incorrectly as having a hemoglobinopathy, it is important to determine if transfusions were the cause of the hemoglobin abnormality detected. Here we report the first case of transfusion-induced D-Los Angeles hemoglobinopathy. We identified a specimen that contained an abnormal hemoglobin eluting at 4.05 minutes accounting for 4.8% of the total hemoglobin using the Bio-Rad Variant II and corresponding Beta Thal column (Bio-Rad, Hercules CA). Comparison with chromatograms in the Bio-Rad Online Library revealed that the elution time of 5 hemoglobin D-Los Angeles variants ranged between 4.09-4.19 (mean 4.15) minutes, and these hemoglobin D-Los Angeles variants accounted for 25.4-38.7 (mean 35.0) % of the total hemoglobin. Chromatography of blood from four donor segments of blood infused into our patient revealed three to have been hemoglobin AA and one hemoglobin AD-Los Angeles. The occurrence of hemoglobin AD-Los Angeles from the recipient was confirmed by alkaline and acid gel electrophoresis (Helena Laboratories, Beaumont TX). On the HPLC chromatogram, the D-Los Angeles variant eluted at 4.10 minutes and accounted for 37.1% of the hemoglobin in the donor unit. Dilutions of the donor segment of the AD-Los Angeles unit and two other AD-Los Angeles specimens with AA blood of similar hemoglobin concentrations and subsequent chromatography revealed that as D-Los Angeles percentages decreased from a mean of 35.6% to 0.5%, the corresponding baseline mean elution time of 4.10 minutes decreased to 3.97 minutes. Similar decreases in the percentage of hemoglobin variants and the associated elution times of other heterozygous variants such as hemoglobin C were also demonstrated (N= 30, Baseline mean 35.5%, 5.16 minutes: Upon dilution, mean 1.0%, 5.04 minutes). We suggest that because the D-Los Angeles donor was asymptomatic and the predonation hematocrit was within the reference range during predonation screening, the donors blood was collected and made available for transfusion. We informed our blood bank and our blood supplier of the unit of blood containing D-Los Angeles to assure that the donor was notified. We report the first case of transfusion-induced hemoglobinopathy associated with hemoglobin D-Los Angeles. Because transfusion-induced hemoglobinopathies occur with hemoglobin variants at lower percentages than is seen in heterozygotes, corresponding elution times of these variants in donor specimens occur earlier and interpretations of the abnormal hemoglobins must take the concentration-related elution time into consideration. We recommend that donors whose units of blood are identified as the source of transfusion-induced hemoglobinopathies be notified of the variant so that appropriate counseling can occur.

NGSP values were analyzed. Both the inter-day and the intra-day precision were evaluated. HbF and CHb interference on HbA1c was also assessed. Results: Over a cartridge lifetime of 2500 injections, 10 minutes were spent in replacing the TURBO-2.0 prefilter compared to 1.5 hours in replacing the guard cartridge in the previous method. The reduction in time is due to the TURBO 2.0 only requiring priming and calibration with the analytical cartridge installation rather than at every guard cartridge change. Regression analysis of 40 samples with NGSP values gave a slope of 1.044, intercept of -0.334 and a correlation coefficient of 0.987. The Bland-Altman assessment of agreement between the TURBO 2.0 and the NGSP values met the 0.75% acceptance criteria for level 1 laboratory certification with low and high 95% confidence limits of -0.45 and 0.50 respectively. Intra-day precision showed a CV of 0.0% and 0.3% for low and high controls respectively. Inter-day precision showed a CV of 1.3% and 1.2%. HbF levels of up to 29.8 % showed 0.3% difference from an unspiked control. No clinically significant interference was seen from CHb. The bias attributable to AE, AD, AS and AC on HbA1c results was less than the clinically significant criteria of 10% generally applied in recent publications. Conclusion: The enhanced TURBO 2.0 has several workflow advantages and allows for more accurate quantitation of HbA1c in the presence of abnormal hemoglobin variants and interfering adducts.
B-85 Increased Levels of Anaphylatoxin (C5a) and Bradykinin in End-Stage Renal Disease Patients on Maintenance Hemodialysis. Potential Relevance to Heparin Mediated Hemodynamic Responses D. A. Hoppensteadt, C. Adiguzel, J. Cunanan, E. Litinas, J. Fareed. Loyola University Medical Center, Maywood, IL
Introduction: In addition to upregulation of inflammatory mediators, patients maintained on hemodialysis because of end stage renal disease (ESRD) are subjected to periodic exposure to heparin and contact activation due to procedural settings. Recently the presence of a heparin contaminant, oversulfated chondroitin sulfate was linked with the adverse reactions and deaths in these patients. Materials & Methods: We measured C5a anaphylatoxin and bradykinin levels in ESRD patients prior to and after maintenance hemodialysis using sandwich ELISA methods. A control group comprised of 40 normal healthy individuals was included to establish the normal level of these mediators. Results: Both C5a and bradykinin levels were elevated in pre-dialysis samples from ESRD patients (C5a: 14.2 4.6 vs. 3.20.6 ng/ml, bradykinin: 9.32.4 vs. 41.8 ng/ ml). Dialysis itself produced an increase in both the C5a and bradykinin levels. The postdialysis samples were further increased, suggesting that dialysis and heparinization itself result in the up-regulation of these mediators. Supplementation of heparin, contaminated heparin or isolated contaminant to the plasma also resulted in the generation of C5a and bradykinin. The plasma samples included in these studies were obtained from patients who were not treated with contaminated heparin. Conclusions: These results suggest that both C5a and bradykinin are up-regulated in ESRD patients and this level can be further augmented by dialysis and heparinization. Therefore, additional factors may have contributed to the complex adverse reaction profiles and deaths in patients administrated with contaminated heparin.
B-84 Evaluation of the VARIANT II TURBO HbA1c - 2.0 Kit D. Bartling. Bio-Rad Laboratories, Hercules, CA
Background: Bio-Rad is introducing the new VARIANT II TURBO HbA1c -2.0 kit (TURBO 2.0). This new kit has several workflow advantages including minimizing priming and calibration frequency and eliminating temperature optimization. The cartridge lifetime has been extended to 2500 injections. The guard cartridges are replaced with newly designed prefilters that have an extended lifetime of 500 injections. The combination of new buffer formulation, new prefilters and new cartridge improves resolution and minimizes interference from hemoglobin variants: such as HbD, HbE, HbS and HbC, and adducts such as Labile A1c (LA1c) and Carbamylated Hb (CHb). The interference threshold for HbF has also been increased dramatically. Objectives: The objective of the study was to evaluate the enhanced TURBO-2.0 kit. Methods: A set of 2500 samples were run on a single analytical cartridge for a period of 10 days. The time savings potential compared to the previous kit was determined. A correlation of 40 samples with values determined by a National Glycohemoglobin Standardization Program (NGSP) Secondary Reference Laboratory (SRL) was perfomed. Also, panels of 35 HbAE, 15 HbAD, 20 HbAS and 20 HbAC samples with
B-86 Upregulation of Procalcitonin in Acute Coronary Syndrome, End Stage Renal Disease and Heparin Induced Thrombocytopenia as an Indicator of Inflammation D. A. Hoppensteadt, V. Bansal, C. Adiguzel, J. Cunanan, J. Fareed. Loyola University Medical Center, Maywood, IL
Introduction: Increased levels of procalcitonin (PCT) and other calcitonin precursors are associated with systemic inflammatory responses in patients with burns, trauma or sepsis.Since acute coronary syndrome (ACS), atrial fibrillation (AF), end stage renal disease (ESRD) and heparin-induced thrombocytopenia (HIT) represent syndromes involving inflammatory responses, we hypothesized that PCT may also be elevated inpatients with these pathologic conditions. Materials & Methods: Apparently sepsis-free blood plasma samples collected from normal donors (n=50) and patients with ACS (n=30), AF (n=40), ESRD(n=67) and HIT (n=40) were retrospectively analyzed for circulating PCT levels using an immunocolormetric assay (ILMA PCT,BRAHMS, Berlin Germany). This test utilizes two monoclonal antibodies that are directed to the calcitonin and katacalcin portions of PCT. The capturing antibody is immobilized on the surface of the coated tube and a labeled anti-calcitonin
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is used as a tracer. Diluted plasma samples incubated and both antibodies react with the PCT to form a sandwich complex. The excess tracer antibody is discarded after incubation. The amount of residual tracer is measured in a luminometer. The intensity of luminescence is proportional to the amount of PCT in the sample. Results: Normal plasma samples contained PCT levels of 0.240.11 ng/ml, whereas higher levels were measured in ACS (0.670.23 ng/ml), AF(0.710.35 ng/ml), ESRD (0.930.36 ng/ml) and HIT (0.650.41ng/ml). Although wide ranges in the PCT values were observed, the levels were much higher than normal. Conclusions: These studies suggest that patients with ACS, AF, ESRD or HIT may exhibit elevated levels of PCT which may not be due to sepsis. The pathophysiologic role of procalcitonin in these syndromes requires further investigation.
B-89 Optimal Dosage for Hemodilution In Vitro Fibrinogen Substitution After Severe
R. J. Molinaro, D. Bolliger, F. Szlam, J. Levy, K. Tanaka. Emory University, Atlanta, GA

OBJECTIVE: Replacement of fibrinogen is presumably the key step in the management of dilutional coagulopathy. We performed an in vitro thromboelastometric study to determine the optimal fibrinogen concentration in diluted blood to achieve clot formation normalization. RELEVANCE: Massive hemorrhage in surgical and trauma patients is primarily treated by administration of crystalloid and colloid fluids to maintain normovolemia. However, the administration of crystalloid or colloid fluids and red blood cell (RBC) concentrates for managing massive hemorrhage causes clotting factors including fibrinogen to decrease. Critically decreased fibrinogen concentrations (<100 mg/dL) tend to occur earliest, whereas critical concentrations of the other coagulation factors as well as critically low platelet counts are reached later, during the course of massive blood loss and fluids substitution. Various experimental and clinical studies suggested that replacing fibrinogen is presumably the key step in managing dilutional coagulopathy. We have determined the optimal fibrinogen concentration in diluted blood at which the rate of clot formation becomes normalized. METHODOLOGY: Blood samples from six healthy volunteers were diluted with saline to 80% keeping hematocrit at 24% using red cell concentrate. We measured coagulation factors and thrombin generation in plasma at baseline and after dilution. Thromboelastometry was used to evaluate 1) speed and quality of clot formation in diluted blood samples supplemented with fibrinogen 50 to 300 mg/dL, and 2) clot resistance to fibrinolysis. Diluted and undiluted samples with no added fibrinogen served as controls. VALIDATION: Coagulation parameters (PT, fibrinogen, ATIII, Factors II, VII, IX, and X) and platelets were reduced by 74 to 85% after dilution. Peak thrombin generation was reduced by 56%. Adding fibrinogen led to a dose-dependent improvement of all thromboelastometric parameters. EC50 for fibrinogen replacement in haemodiluted blood was calculated to be 125 mg/dL. Adding tissue plasminogen activator, 0.15 g/ mL, led to a decrease of clot firmness and lysis time. CONCLUSIONS: Normalizing fibrinogen levels improved dilutional coagulopathy as demonstrated by the improvement of thromboelastometric parameters. Target plasma concentration for fibrinogen replacement might be greater than 200 mg/dL as only these concentrations optimized the rate of clot formation. This concentration of fibrinogen is twice the level suggested by current transfusion guidelines. Although improved, clots were still prone to fibrinolysis indicating that the efficacy of fibrinogen therapy may be influenced by co-existing fibrinolytic tendency occurring during dilutional coagulopathy.
B-87 Activation of Prekallikrein by Contaminated Heparins, Isolated Contaminant and Related Hypersulfated Chondroitin Sulfate Preparations: Pharmacologic Implications D. A. Hoppensteadt, L. Meyers, M. Clark, C. Adiguzel. Loyola University Medical Center, Maywood, IL
Introduction: Use of heparins contaminated (CH) with oversulfated chondroitin sulfate (OSCS) has been associated with severe adverse events. Studies were designed to determine the effect of OSCS on the generation of kallikrein (K) in plasma and whole blood systems. Materials & Methods: CH, a contaminant free heparin (CFH), three OSCSs isolated from different heparins, two hemisynthetic chondroitin sulfate preparations (HCS), chondroitin sulfate (CS), dermatan sulfate (DS), and mucopolysaccharide polysulfate (MPS) were compared at concentrations of 0-1000 g/ ml following supplementation to human plasma or whole blood (citrated or hirudinized ). K activity was measured using a specific synthetic chromogenic substrate (chromozyme PK) following incubations up to 30 minutes. Results: At a concentration of 100 g/ml, in citrated plasma OSCS preparations produced the strongest activation of prekallikrein (>0.75/15 mins). CH produced a weaker activity (0.32/15 mins) that was comparable to that produced by CFH (0.24/15 mins). MPS also produced strong activation of prekallikrien into K. In citrated whole blood HCS-1, HCS-2, the three HCs and MPS produced a strong generation of K (>1.5/5 mins). CH produced a weaker effect (1.3/5 mins) whereas CFH was much less (0.25/ 5 mins). DS also produced a measurable generation of K (1.2/ 5 mins). CS produced weaker effects (0.6/ 5 mins). In hirudinized whole blood, all agents produced sizable generation of K (>1.5/5 mins). The contaminants and OSCS preparations were relatively stronger whereas CS was much weaker (0.2/ 5 mins). Conclusions: These studies demonstrate that the generation of K is a nonspecific effect of sulfated glycosaminoglycans.
B-88 Fibrinokinetic Deficit in Chronic Kidney Disease and End Stage Renal Disease Patients Contributes to the Hemostatic Abnormalities C. Adiguzel, J. Cunanan, D. Hoppensteadt, V. Bansal, J. Fareed. Loyola University Medical Center, Maywood, IL
Introduction: Increased bleeding is observed in patients with chronic kidney disease (CKD) or end stage renal disease (ESRD) despite a normal coagulation profile and fibrinogen level. The hemostatic deficit in these patients may due to defects in fibrin formation. Materials & Methods: The fibrinokinetic profile of CKD (n=50) and ESRD patients on hemodialysis was measured. Citrated plasma was supplemented with thrombin and CaCl2. The rate of fibrin formation was measured by monitoring the optical density (OD) at 405 nM. After reaching steady state, urokinase was added to measure the fibrinolytic profile. Forty normal male and female individuals were also analyzed. Results: Fibrinokinetic profiles for normals showed strong clot formation (average OD = 1.20.3;range 0.7-1.4). In the CKD patients a much weaker clot was formed (average OD of 0.210.13;range 0.05-0.41). In the ESRD patients on maintenance hemodialysis, the pre-dialysis sample showed a weaker fibrinokinetic profile reaching near normal levels with a clot density of 1.30.4 (range 0.8-1.6). In the urokinase induced fibrinolysis assay, CKD patients plasma exhibited a much stronger fibrinolytic index compared to the normal population (80% vs. 20% lysis). In ESRD patients, clot lysis was weaker compared to the CKD patients. Conclusions: These results are contrary to previous reported observations that dense clot resistant to fibrinolysis is formed in CKD and ESRD patients. Furthermore, the clots observed in these patients were highly susceptible to lysis. Maintenance hemodialysis results in improving the fibrinokinetic and fibrinolytic profile in the ESRD patients and may contributes to improved hemostasis in ESRD patients.
B-90 Evaluation of a New High Throughput Hemostasis Analyzer M. McInerney, N. Kelly, E. Kearney, J. Landowska, J. Grace. Trinity Biotech, Bray, Ireland
Introduction: The performance of a new high throughput analyzer (DestinyMax) for the measurement of Hemostasis tests was evaluated at Trinity Biotech. The analyzer performs testing in Clotting, Chromogenic and Immunoturbidometric modes, has cap piercing capability and performs approximately 350 Prothrombin Time tests per minute. Materials and Methods: Method Comparison: Normal and patient plasma samples were run on the new analyzer and on a second analyzer (DestinyPlus). Results for a Clotting test, Prothrombin Time - PT (TriniCLOT PT Excel S), a Chromogenic test, Antithrombin - AT (TriniCHROM Antithrombin IIa) and an Immunoturbidimetric test, D-dimer - (TriniLIA D-dimer), were compared on both analyzers. Precision: Within-run and Total precision were determined for each test on the new analyzer using control material in Optical and Mechanical modes. Four vials of each level of control were run in triplicate on five days (n=60). Results: Method Comparison: Results from both analyzers were compared for each test using the slope of the linear regression (m) and the correlation coefficient (r): PT (sec) Optical/Mechanical: m=1.01/1.13, r=1.00/1.00, n=75/78. AT (%): m=0.95, r=0.99, n=80. D-dimer (ng/mL): m=1.12, r=0.98, n=79. Precision: See Table 1
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Table 1: Within-run and Total Precison (%CV) Test Wthin-run %CV Total %CV PT Level 1 1.9 Opt, 1.7 Mech 2.2 Opt, 2.0 Mech PT Level 2 1.7 Opt, 2.1 Mech 2.7 Opt, 3.0 Mech PT Level 3 1.1 Opt, 2.4 Mech 2.5 Opt, 4.1 Mech AT Level 1 2.7 6.4 AT Level 2 4.7 5.3 D-dimer Level 1 9.9 13.5 D-dimer Level 2 3.1 5.0 Conclusion: The performance of the new analyzer (DestinyMax) is determined to be equivalent to that of the second analyzer (DestinyPlus). All three test types had slopes and correlation coefficients close to 1.0. Within-run and Total %CVs demonstrate good precision performance for the new instrument with each test type.

B-92 Comparative determination of selected hemostaseological parameters in a case of severe hypofibrinogenemia caused by snakebite of Deinagkistrodon Acutus using the electromechanical clot detection and optical end point analysis G. Kramp, M. Schifflers, R. Driesch, I. Peredniene, A. M. Gressner, O. A. Gressner. RWTH-University Hospital, Aachen, Germany
Introduction: Deinagkistrodon is a venomous pitviper species, which includes Deinagkistrodon acutus (D. a.), also called 100-paces-snake, predominantly found in Southeast Asia. The venome of this snake displays a complex mixture of several highly potent hemotoxic ingredients, such as metalloproteinases, alpha fibrinase, serine proteases and nucleotidases, which are responsible for its powerful inhibitory effect on coagulation. Aim: We report of case of a patient bitten by D. a. who was admitted to our hospital. During hospitalization, we performed comparative measurement of fibrinogen (Clauss method and immunological method), Thrombin clotting time (TCT) (Prothrombin time (PT), and activated partial thromboplastin time (aPTT) with the Sysmex CA7000TM optical end point analyser (Siemens Medical Solutions, Nuremberg, Germany) and the STA-R EvolutionTM electromechanical clot detection analyser (STAGO/ Roche, Paris, France) in order to evaluate their analytical reliability in such severe hemostaseological disorders. Case: Within four hours after snake bite of D. a. in the right hand, a severely prolonged PT/INR and aPTT together with a dramatic decrease in fibrinogen concentration (Claus method) was observed. On the next day, after receiving the polyvalent Calloselasma antivenom as salvage therapy, an immediate partial recovery from the hemostatic disturbances lasting for 36 h was observed. In addition a slightly normalization in fibrinogen concentration occurred. Three days later the patient received the monovalent antivenin against D. a.. Within 72 h after application, a normalization of all mentioned hemostaseological parameters including fibrinogen concentration (Clauss method) was observed. Results: A plasma mixing study for the assessment of the fibrinogenolytic activity of the snake venom illustrates a progressive fibrinolysis following incubation of standard human plasma with patient plasma. Good correlations between the CA7000 and STAR analyzers were also observed for the Prothrombin time (PT) (r=0.9822) and the activated partial thromboplastin time (aPTT) (r= 0.8315).Thrombin clotting time (TCT) (r=0.8414) and fibrinogen (r=0.9827) as determined using the Clauss method showed good correlations between the CA7000 and STA-R analyzers. Also, on both analyzers, fibrinogen (Clauss) correlated very well with the immunologically determined fibrinogen on the Siemens Medical Solutions BN2 nephelometer (correlations= STA-R: r=0.922; CA7000: r=0.9122). However, the CA7000 frequently determined fibrinogen (Clauss) concentrations below the analytical cut-off while the concentrations determined by the STA-R were still in the analytical range. Also, the STA-R displayed a better correlation between fibrinogen (Clauss) and TCT (STA-R: r=0.6249; CA7000: r=0.5110). Conclusion: Even though most parameters tested display a good correlation between both analyzing systems (CA7000 and STAR), electromechanical clot detection analysis seems to be superior to optical end point analysis in situations of extreme hypofibrinogenemia, as it displays less tendency towards extreme values of TCT and fibrinogen (Clauss) and better correlations between TCT and fibrinogen (Clauss) or fibrinogen (immunological). Furthermore, electromechanical clot detection seems to be more precise for the determination of (low) fibrinogen concentrations.
B-91 Automatization of glucose 6 phosphate dehydrogenase and 6phosphogluconate dehydrogenase determination. G. L. Salvagno, G. Lippi, M. Gelati, F. Bellorio, V. Vandelli, M. Montagnana, O. Ruzzenente, C. Brentegani, G. Guidi. Laboratorio di Biochimica Clinica, University of Verona, Italy
Background. Glucose-6-phosphate dehydrogenase (G6PD) is a cytosolic enzyme in the pentose phosphate pathway, a metabolic pathway that supplies reducing energy to cells by maintaining the level of the co-enzyme nicotinamide adenine dinucleotide phosphate (NADPH). Since G6PD deficiency, an X-linked recessive hereditary disease characterized by abnormally low levels of the enzyme, is very common worldwide and might cause acute hemolytic anemia in association with some foods and medications, the automatization of its determination is advisable to cope with the growing request of tests, especially in developing countries. Materials and Methods. The conventional assay for manual determination of G6PD and 6-phosphogluconate dehydrogenase (6PGD) (a two-phase enzymatic reaction, the former transforming glucose-6-phosphate with NADP+and G6PD into 6-phosphogluconate and NADPH and H+ and the latter transforming 6-phosphogluconate and NADP+ and 6phosphogluconate dehydrogenase into Ribulose-5-P and nADPH+H+ and CO2) has been adapted to use on a Roche Cobas Mira. After sample extraction, all assay steps have been automated, including reagent addition, incubation and data collection. The increase in absorbance is finally read at 340 nm. Results. The intra-assay imprecision (n=10) of the automated assay for both G6PD and 6PGD was remarkably better than that of the manual assay (G6PD: 0.8% vs 5.1%; 6PGD: 1.2% vs 7.3%, respectively). The correlation with the manual assay was also excellent, being: y = 0.862x + 1.81 (r=0.927; p<0.001) for G6PD, and y=0.730x + 4.02 (r=0.839; p<0.001) for 6PGD, respectively. The overall bias between assay as estimated by Bland-Altman plots analysis was satisfactory (Figure 1). Sensitivity and specificity of both assays, as compared with the reference manual assays, were 100%. Conclusion. The automatization of the manual assays for G6PD and 6PGD not only provides improved analytical performances over the traditional (and reference) manual assay, but it is also allows faster processing of the samples and a much higher throughput.
B-93 Evaluation of the VARIANT II TURBO HbA1c - 2.0 Kit* T. Sanborn1, J. Palm1, S. Tanaka2, K. Matsumoto2. 1Memorial Medical Hospital, Modesto, CA, 2Bio-Rad Laboratories, Inc., Hercules, CA
Background: Bio-Rad is introducing the new VARIANT II TURBO HbA1c -2.0 kit (TURBO 2.0). This new kit has several workflow advantages including minimizing priming and calibration frequency and eliminating temperature optimization. The cartridge lifetime has been extended to 2500 injections. The guard cartridges are replaced with newly designed prefilters that have an extended lifetime of 500 injections. The combination of new buffer formulation, new prefilters and new cartridge improves resolution and minimizes interference from hemoglobin variants: such as HbD, HbE, HbS and HbC, and adducts such as Labile A1c (LA1c) and Carbamylated Hb (CHb). The interference threshold for HbF has also been increased dramatically. Objectives: The objective of the study was to evaluate the enhanced TURBO 2.0 kit.
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Methods: A set of 2500 samples were run on a single analytical cartridge for a period of 10 days. The time savings potential compared to the previous kit was determined. A correlation of 40 samples with values determined by a National Glycohemoglobin Standardization Program (NGSP) Secondary Reference Laboratory (SRL) was perfomed. Also, panels of 35 HbAE, 15 HbAD, 20 HbAS and 20 HbAC samples with NGSP values were analyzed. Both the inter-day and the intra-day precision were evaluated. HbF and CHb interference on HbA1c was also assessed. Results: Over a cartridge lifetime of 2500 injections, 10 minutes were spent in replacing the TURBO 2.0 prefilter compared to 1.5 hours in replacing the guard cartridge in the previous method. The reduction in time is due to the TURBO 2.0 only requiring priming and calibration with the analytical cartridge installation rather than at every guard cartridge change. Regression analysis of 40 samples with NGSP values gave a slope of 1.044, intercept of -0.334 and a correlation coefficient of 0.987. The Bland-Altman assessment of agreement between the TURBO 2.0 and the NGSP values met the 0.75% acceptance criteria for level 1 laboratory certification with low and high 95% confidence limits of -0.45 and 0.50 respectively. Intra-day precision showed a CV of 0.0% and 0.3% for low and high controls respectively. Inter-day precision showed a CV of 1.3% and 1.2%. HbF levels of up to 29.8 % showed 0.3% difference from an unspiked control. No clinically significant interference was seen from CHb. The bias attributable to AE, AD, AS and AC on HbA1c results was less than the clinically significant criteria of 10% generally applied in recent publications. Conclusion: The enhanced TURBO 2.0 has several workflow advantages and allows for more accurate quantitation of HbA1c in the presence of the above described abnormal hemoglobin variants and interfering adducts. * Pending 510(k) Clearance
contribute significantly to the macromolecular substrate recognition of Pro by prothrombinase, and 2) fVa controls the rate of Pro activation by fXa within prothrombinase.
B-95 Teststrip-based genotyping to assist in the prediction of anticoagulant dose requirement H. Puehringer1, G. Klose2, B. Schreyer2, W. Krugluger3, R. M. Loreth2, C. Oberkanins1. 1ViennaLab Diagnostics GmbH, Vienna, Austria, 2Clinical Haemostaseology, Westpfalz-Klinikum GmbH, Kaiserslautern, Germany, 3Institute of Laboratory Medicine, Donauspital, Vienna, Austria
Coumarin derivatives (warfarin, phenprocoumon) are the most widespread oral anticoagulant drugs for the prevention and treatment of thromboembolic disorders. However, these vitamin K antagonists have a narrow therapeutic range and a wide interindividual variability in dose requirement. Despite adjustment for clinical variables, adverse events are frequently encountered during the initial phase of therapy. Genetic polymorphisms in the drug-targeted vitamin K epoxide reductase complex 1 (VKORC1) and in the drug metabolizing enzyme CYP2C9 have been reported to account for the majority of variations in the therapeutic response to warfarin. Aims and Methods: A genetic test (StripAssay) for the simultaneous detection of two VKORC1 polymorphisms (-1639G>A, 3730G>A) and the functionally defective CYP2C9 variants *2 (430C>T) and *3 (1075A>C) was developed. The protocol is based on multiplex PCR and reverse-hybridization of biotinylated amplification products to allele-specific probes on membrane teststrips. The new StripAssay is currently being used in an ongoing clinical study to classify patients into high, intermediate and low dose responders to coumarin anticoagulants. Results: Preliminary data based on more than 170 patients treated with phenprocoumon (Marcumar) indicated a considerably lower stable dosage required for therapeutic anticoagulation in carriers of a combined VKORC1 -1639A and CYP2C9 *2 or *3 genotype compared to carriers of a single variation or wildtype alleles. The VKORC1 3730G>A polymorphism seemed to have no additional predictive power for phenprocoumon dose variability. Summary and Conclusions: The new diagnostic assay and the results obtained during our study will assist clinicians to achieve a safer and more individualized anticoagulant therapy. (oberkanins@viennalab.co.at)
B-94 The Function of Acidic Amino Acid Region 659-663 of Factor Va on Prothrombinase during Prothrombin Activation J. Hirbawi, M. Kalafatis. Cleveland State University, Cleveland, OH
There are an estimated eighty million Americans who suffer from various forms of cardiovascular disease. A deep understanding of the molecular mechanisms involved in the coagulation cascade is essential in the development of efficient drugs that can help patients treat their disease without suffering the usual side effects from various drugs. Following vascular injury, the process of hemostasis facilitates the generation of thrombin, which in turn allows the formation of a fibrin clot and the arrest of hemorrhage. The proteolytic conversion of prothrombin (Pro) to thrombin is catalyzed by the prothrombinase complex composed of the enzyme, factor Xa (fXa), the cofactor, factor Va (fVa), assembled on a membrane surface in the presence of divalent metal ions. Activation of human Pro occurs following two sequential cleavages by membrane-bound fXa (Arg271 followed by Arg320, with prethrombin 2 as intermediate). The incorporation of fVa into the prothrombinase complex results in a 300,000-fold increase in the catalytic efficiency of fXa for thrombin generation. This complex catalyzes the activation of Pro following the opposite pathway (cleavage at Arg320 followed by Arg271, with meizothrombin as intermediate). Thus, the activity of factor Xa is controlled by fVa. It has been demonstrated that the COOH-terminal region of fVa contains cluster of acidic amino acids that are crucial for its cofactor activity. More specifically, we have shown that amino acid region 695-698 from fVa heavy chain regulates the rate of cleavage of prothrombin at Arg271 by prothrombinase. The COOH-terminal portion of the heavy chain also contains another cluster of acidic amino acids (encompassing residues 659-663). In the present study we have investigated the role of amino acid region 659-663 from the COOH-terminus of fVa heavy chain. Site-directed mutagenesis was performed to generate, factor V (fV) molecules containing point mutations where the five amino acids were all mutated to either lysine (V5K), or alanine (fV5A). We have also constructed a mutant molecule with region Asp659-Asp663 deleted (fV659-663) and a molecule with both regions, Lys680-Arg709 and Asp659-Asp663 deleted (fV). These recombinant molecules along with wild type fV (fVWT) were transiently expressed in COS7L cells, purified to homogeneity and assessed for cofactor activity. Two-stage clotting assays revealed that all recombinant mutant molecules had reduced clotting activities compared to fVaWT. Kinetic analyses studying prothrombinase assembled with the mutant molecules demonstrated diminished kcat values, while the affinity of all the mutant molecules for fXa was similar to fVaWT. Gel electrophoresis analyses demonstrated delayed cleavage of prothrombin at both Arg320 and Arg271 by prothrombinase assembled with the mutant molecules however, cleavage at Arg271 was severely impaired resulting in lingering of meizothrombin throughout the activation process. Overall the data demonstrate that acidic amino acids from the COOH-terminus of the factor Va heavy chain play a preeminent role in proper prothrombinase complex assembly and function, and are consistent with the interpretation that 1) acidic regions from the COOH-terminus of fVa heavy chain
B-96 Biomarker Profiling of Plasma Samples from Suspected HeparinInduced Thrombocytopenia Patients using a Protein Chip Array Method J. Fareed, H. Zhu, J. M. Walenga, J. Cunanan, D. Hoppensteadt. Loyola University Medical Center, Maywood, IL
Introduction: Heparin-induced thrombocytopenia (HIT) represents a catastrophic adverse event associated with the use of this anticoagulant. While the pathophysiology of this syndrome is complex, inflammatory processes are contributory to the overall pathogenesis of this syndrome. Various markers of inflammation such as CRP, MCP-1, and interleukins are up regulated. The up regulation of the inflammatory process is reported to be associated with the generation of unique biomarkers in other disorders such as the acute coronary syndrome and end stage renal disease. This study was designed to determine the biomarker profile of samples obtained from patients suspected of HIT. Methods: 138 blood samples from patients suspected of HIT syndrome were analyzed using a gold chip on Ciphergens system employing surface-enhanced laser desorption ionization (SELDI) technology. The biomarker profile was determined in the range of 0 - 150 kDa. Parallel controls consisted of blood samples form 50 healthy normal male and female individuals. All of the samples were also analyzed for the presence of HIT antibodies using a commercially available ELISA method form GTI Diagnostics. 14C-serotoin release was measured in all samples. Results: Of the 138 samples analyzed, 102 samples exhibited a unique biomarker peak at 11.9 kDa with varying intensities, whereas the peak was absent in samples from normal individuals. Of the 138 samples, 116 tested positive for HIT antibodies in the ELISA. Only 70 samples were positive in the 14Cserotoin release assay. Conclusion: The generation of HIT antibodies is consistent with the generation of a unique biomarker at 11.9 kDa in patients suspected of HIT syndrome.
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B-97 Molecular and Functional Heterogeneity in Contaminants Isolated from Recalled Heparin. Impact on Anticoagulation and Potential Adverse Reactions J. Fareed, W. Jeske, C. Adiguzel, D. A. Hoppensteadt, J. Walenga. Loyola University Medical Center, Maywood, IL
Introduction: The primary contaminant in recalled batches of unfractionated heparin (UFH) is reported to be oversulfated chondroitin sulfate (OSCS). It has been assumed that different heparin batches contained similar forms of OSCS. Materials & Methods: Non-heparin contaminants were isolated from four batches of contaminated UFH and two batches of LMWH by digestion of heparin followed by alcohol precipitation and ion-exchange chromatography. Anticoagulant activities were measured using whole blood and plasmatic assays. Thrombin generation inhibition and protamine/PF4 neutralization studies were carried out in human plasma. Each contaminants interaction with AT and HCII was characterized. Results: The contaminated UFHs did not exhibit major differences in molecular weight profile (14.8-15.6 kDa), USP potency or anticoagulant actions. There were differences in their anti-Xa:anti-IIa ratios (0.93-1.24) and in the amount of heparinase resistant material (14-30%). Two heparins also contained significant amounts of dermatan sulfate. Each isolated contaminant exhibited distinct neutralization profiles with PF4 and protamine. The LMWHs were comparable in molecular weight and biologic actions, but differed in heparinase-1 digestion profile. The molecular weight of the contaminant isolated from LMWH was lower (12.8 vs. 14.1-16.8 kDa). The contaminants also exhibited differences in thrombin generation inhibition. The contaminants isolated from heparin and LMWH had potencies of 28-46 USP U/mg and 38-46 USP U/mg, respectively. Conclusions: Contaminants isolated from recalled batches of heparin are heterogenous. Moreover, the contaminants obtained form LMWHs may exhibit additional structural and biologic differences. The variations observed in the adverse reactions with recalled heparins may be due to compositional variations in the contaminants.

B-99 Prospective Validation of Siemens INNOVANCE D-Dimer Test Negative Predictive Value in Outpatients Suspected of Venous Thromboembolic Events L. Lewis1, G. Banet1, C. Aguilar2, A. Duncan3, E. Lindhoff-Last4, J. R. Wu5, R. Porche-Sorbet6, C. S. Eby6. 1Department of Emergency Medicine, Washington University School of Medicine, St. Louis, MO, 2Hospital General Santa Barbara, Soria, Spain, 3Clinisys Associates Ltd, Atlanta, GA, 4Departrment of Internal Medicine, Division of Vascular Medicine and Haemostaseology, University Hospital, Frankfurt, Germany, 5Duke University Medical Center, Durham, NC, 6Department of Pathology & Immunology, Washington University School of Medicine, St. Louis, MO
Relevance to Clinical Laboratory Medicine: The accuracy of diagnosing pulmonary emboli (PE) and deep vein thromboses (DVT) based on symptoms and signs is poor, but can be improved by using clinical scoring systems to assign pretest likelihoods of venous thromboembolic events (VTE). D-dimers are circulating fragments of fibrin generated during thromboses by plasmin degredation of crosslinked fibrin polymers. Quantitiative D-dimer immunoassays have the potential to aid in the exclusion of VTEs in patients with low and intermediate VTE likelihood scores, but due to differences among commercial assays, each one must be evaluated to determine whether a cut-off value with sufficient sensitivity and specificity can be used prospectively to rule out VTEs in selective patients suspected of VTE. Objective: To establish an aids in diagnosis claim and validate a previous cut-off value for a new formulation of INNOVANCE D-dimer assay. Methodology: We conducted an observational study involving 902 patients. Eligible patients suspected of VTE provided a sample of citrated whole blood and were assigned a pretest VTE probability score followed by a protocol directed laboratory and imaging diagnostic evaluation. Patients diagnosed with VTEs were anticoagulated and patients without a VTE were contacted 3 months later to identify anyone with delayed VTE diagnoses. Platelet poor plasma was prepared and frozen for later thawing and batch D-Dimer testing using Siemens INNOVANCE D-dimer reagents and Siemens BCS automated coagulation analyzers. We used a D-dimer cutoff of > 0.5 mg/L FEU for positive results. Results: D-Dimer sensitivity 97% specificity 42% NPV 98% PPV 32% VTE prevalence 21.8% INNOVANCE D-dimer + D-dimer Total VTE + 191 6 197 VTE 412 293 705 Total 603 299 902
B-98 Biomarker Profiling of Plasma Samples From End Stage Renal Disease Undergoing Maintenance Hemodialysis Using Anionic (SAX2) and Gold Chips J. Fareed, H. Zhu, D. A. Hoppensteadt, C. Adiguzel, V. Bansal. Loyola University Medical Center, Maywood, IL
Introduction: Dysregulated protease activation in end stage renal disease (ESRD) results in the activation of coagulation, fibrinolysis, complement and the kallikreinkinin pathways. This process results in the generation of several cleavage products, which are characteristic of this syndrome. To profile the protease cleavage products, surface enhanced laser/desorption ionization (SELDI) mass spectrometry was used. We hypothesized that because of the dysregulation of proteases, plasma of patients with ESRD may exhibit unique biomarker patterns that are characteristic of the disease. Materials & Methods: Plasma samples (pre and post) from 22 patients undergoing maintenance hemodialysis and control individuals (40) were collected and analyzed in parallel to characterize the biomarker profile of the ESRD patients pre and post hemodialysis. The Gold and SAX2ProteinChip Array methods were utilized according to the manufacturers specifications. Results: Both the ESRD patients and control plasma samples provided similar biomarker profiles in the 25 - 125 kDa range. The SAX2 and Goldchips provided comparable results. However, there were differences in the biomarker profile below 25 kDa. The control plasma exhibited two distinct peaks at 17.8 and 14.3 kDa. The ESRD patient plasma exhibited both of these peaks and in addition showed two unique peaks at 15.4 and 16.3 kDa. In addition the ESRD patient samples demonstrated minor components at 10.1, 10.9 and 11.9 kDa. Post hemodialysis samples did not show any major differences in the biomarker profile with reference to the peaks at 14.3, 15.4, 16.3 and17.8 kDa; however, the peaks at 10.1, 10.9 and 11.9 kDa were diminished. Conclusions: This data suggests that protease activation in the ESRD results in the generation of specific biomarker peaks, which may be used to risk stratify these patients. Characterization of these unique peaks may provide newer information on the pathogenesis of ESRD with particular reference to protease activation.
Conclusion: An INNOVANCE D-dimer cut-off of 0.5mg/L when performed on a BCS coagulation analyzer is a sensitive biomarker for acute VTE. When the prevalence of VTE is low, as seen in this population, the negative predictive value of a INNOVANCE D-dimer < 0.5 mg/L approaches 100%.
B-100 Comparison of STA Satellite and Compact Automated Coagulation Analyzers R. Porche-Sorbet1, J. Hughes2, P. Barnes2, G. Cutsforth3, K. Whelchel3, C. S. Eby1. 1Washington University School of Medicine, St. Louis, MO, 2Barnes-Jewish Hosptial, St. Louis, MO, 3Diagnostica Stago Inc, Parsippany, NJ
Relevance to clinical laboratory medicine: High quality patient care requires rapid turn around times for an increasing number of coagulation tests even when test volume does not justify a fully automated, expanded menu analyzer. This niche is filled by the STA Satellite coagulation analyzer introduced in the USA in December, 2008. Objective: Determine the Satellites precision and accuracy compared to the STA Compact analyzer which has an expanded test menu and faster through-put. The Satellite analyzer is a fully automated analyzer (H 27.4, W 21.1, D 25.5 in) with 20 samples, random access capacities and performs clot-based and photometric assays. Test menu includes: PT, aPTT, fibrinogen, quantitative D-dimer, unfractionated (UFH) and low molecular weight heparin (LMWH), and antithrombin(AT). Sample Collection: Residual plasma samples were collected from: out-patients with normal PT and aPTTs or attending anticoagulation clinic, in-patients, intensive care patients, and patients treated with therapeutic doses of UFH or LMWH. The
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Washington University Human Research Protection Office approved the protocol. Methodology: STAGO normal and abnormal control reagents used for intra and inter assay precision determinations. PT, aPTT, fibrinogen, D-dimer performed on fresh plasmas; UFH, LMWH, and AT performed on plasmas frozen at -200 C, thawed in 370 C water bath, gently mixed. Testing performed in singlet. Results: Intra, inter assay coefficients of variation within acceptable ranges. Correlations and mean percent differences excellent: Test PT aPTT fibrinogen D-dimer UFH activity LMWH activity antithrombin activity Compact v. Satellite (R2 ) percent mean difference 0.996 -3.7 0.991 3.5 0.984 8.8 0.999 4.3 0.984 4.0 0.988 1.9 0.920 2.6
B-102 Eight novel VKORC1 mutations cause hereditary warfarin resistance C. Geisen1, M. Watzka2, K. Sittinger1, G. Spohn1, E. Seifried1, J. Oldenburg2. 1Institute of Transfusion Medicine and Immunohematology, Goethe-University, Frankfurt, Germany, 2Institute of Exp. Hematology and Transfusion Medicine, University, Bonn, Germany
Background: VKORC1 is the key enzyme of the vitamin K cycle and the molecular target of coumarins, which represent the most commonly prescribed drugs for therapy and prevention of thromboembolic conditions. The vitamin K epoxide reductase enzyme complex recycles vitamin KO to vitamin KH2, an essential cofactor for the post-translational gamma-carboxylation of several blood coagulation factors. Six heterozygous missense mutations in VKORC1 leading to variable degrees of coumarin resistance (Val29Leu, Asp36Tyr, Val45Ala, Arg58Gly, Val66Met, Leu128Arg) have been reported earlier. In this study we present 15 additional unrelated patients with mutations in the VKORC1 gene causing hereditary warfarin resistance. Methods: After exclusion of acquired reasons for an increased coumarin requirement 15 candidates for hereditary coumarin resistance were sequenced for all 3 exons and flanking intronic regions of the VKORC1 gene. Results: In 15 unrelated patients with variable degrees of coumarin resistance ranging from partial to complete resistance genetic testing revealed different causative missense mutations in the VKORC1 gene (His28Gln, Val29Leu, Asp36Gly, Asp36Tyr, Ser52Trp, Ser56Phe, Trp59Cys, Val66Gly, Val66Met, Ile123Asn, Tyr139His). The missense mutations are located either in the first cytoplasmatic loop or in the close vicinity of the coumarin binding motif of VKORC1 thus indicating that not only one specific epitope seems to be critical for coumarin action. Conclusions: Various missense mutations in VKORC1 cause partial and complete coumarin resistance. Here we present the data in 15 unrelated patients revealing 11 different missense mutations, eight of which have not been reported previously. Our data may contribute in identifying further functional epitopes of VKORC1 and may provide a basis for a rational design of novel anticoagulants targeting VKOR in the future.
Conclusion: The STA Satellite coagulation analyzer accurately performs all coagulation tests needed for immediate patient management while economizing on size and minimizing complexity of operation. It is an ideal analyzer for a small acute care hospital or ancillary laboratory in a large medical center.
B-101 Adaptation of a D-Dimer Assay on an Automated High-Throughput Chemistry Analyzer to Reduce Turn-Around Times A. C. Chin, C. R. Fantz, J. C. Ritchie. Emory University, Atlanta, GA
OBJECTIVE: To compare method performance of an automated chemistry to an automated coagulation D-dimer assay. RELEVANCE: Pulmonary embolism is characterized by dyspnea and thoracic pain which are common clinical symptoms observed in patients admitted to emergency care centers and requires prompt diagnosis by the clinical team. Quantification of fibrin degradation products which include the small D-dimer fragment is performed to exclude pulmonary embolism. However, current methods to quantify D-dimer concentration are not sufficient in delivering more rapid turn-around times. Furthermore, cardiac troponin I assays are commonly ordered to differentiate from acute coronary syndrome. Currently in our laboratory, assays for D-dimer concentration is quantified from plasma in sodium-citrate blue-top tubes on the DadeBehring BCS coagulation analyzer while cardiac troponin I is assessed from plasma in lithium-heparin green-top tubes on the Beckman LX-20 chemistry analyzer. Development of a high-throughput automated method that measures both D-dimer and cardiac troponin I concentrations would prove to be advantageous in improving turn-around times and patient management. METHODS: The D-dimer assays from Roche Diagnostics Inc. and from Diagnostica Stago Inc. are developed for use on the Dade-Behring BCS and the Beckman LX-20, respectively. The Roche/LX-20 method interassay and intraassay imprecision was judged according to an allowable error of 20% (American Association of Bioanalysts Table of Grading Limits). Method comparisons were performed on 50 citrated-plasma (blue-top) samples. Next, comparisons between 50 patient-matched citrated and heparinized (green-top) plasma samples were performed on the Roche/LX-20 assay. RESULTS: Inter-assay precision for the low (429 D-Dimer Units or DDU ng/ml) and high (1815 DDU ng/ml) controls were 5.32% and 1.15%, respectively. Intra-assay precision for the low (429 D-Dimer Units or DDU ng/ml) and high (1815 DDU ng/ ml) controls were 2.12% and 2.02%, respectively. Linearity was assessed by dilution of the manufacturers calibrator (range 0-4290 DDU ng/mL). Initial evaluations between the LX-20/Roche method (y) and the BCS/Stago (x) on sodium-citrated (blue-top) plasma samples yielded a linear correlation of y=0.7191x+75.584, r=0.9032, n=42. CONCLUSION: The assay on the chemistry analyzer is fully automated and exhibits good performance characteristics regarding inter- and intra-assay precision, and acceptable correlation with the coagulation analyzer. Adaptation of a D-dimer assay on a chemistry analyzer allows simultaneous testing with cardiac markers which should lead to better turn-around times and ultimately, accurate and quicker patient diagnosis.
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Lipids/Lipoproteins

Materials and Methods: 136 CRF patients and 80 healthy control (group 1) were inclided in this study. Out of 136 patients,44 were on regular hemodylysis (group II) and 92 were being managed conservatively (group III). Blood sample (5.0ml) were collected after a 12 hour fasting and serum were seperated and stored at -200C until analyzed. Lipid profile (TC,TG, HDL-C, LDL-C) were estimated by sphectrophotometric enzymatic method using commercial kit manufactured by Bio Lab. Result:-TC was highier (4.370.24) in CT than HD (3.920.75). HDL-C was significantly lower (1.0 0.35) in CRF than controls (1.20 0.29), p-value-0.033. Among control, 55% have TG<1.32 mmol/ltr and 45% have >1.32 mmol/ltr which is among CRF, only 47.1% of have <1.32 mmol/ltr TG and 52.1% have >1.32 TG. TG level was significantly higher in patients on CT than in HD, p-value-0.012. Patients with CRF, DM, and HTN have high total cholesteerol (4.80.91) and LDL-C (3.110.30) as compared to CRF only (TC=3.90.75, LDL-C=2.330.14), pvalue<0.05. Similarly, CRF and DM have high toatal cholesterol (4.6 1.71) as compared to CRF only (3.90.75), p-value<0.05. Conclusion:- Lipid prifile abnormality is present in patients with chronic renal failure mainly in the pateints on conservative treatment than hemodialysis. Diabetes and hypertension may be the risk factor in increasing lipid profile in patients with chronic renal failure.
Tuesday PM, July 21

Poster Session: 2:00 pm - 4:30 pm Lipids/Lipoproteins
B-103 Evaluation of New Homogeneous Assay Kit to Determine HDL-C with a High Reactivity with Cholesterol in Various Types of HDL Y. Katayama, H. Soya, M. Fujinaka, H. Mori, A. Tomita, N. Kayahara. Kyowa Medex, Shizuoka, Japan
Object: In development of homogeneous assay to determine cholesterol in highdensity lipoprotein (HDL-C), high specificity to HDL-C is essential. Out of changes of HDL metabolism, there are various types of HDL such as non-esterified cholesterol-rich HDL and ApoE-rich HDL, which often increase quantitatively. Since these various types of HDL are classified as HDL, precise determination of the cholesterol as HDL-C is also required. In particular, because inhibitors against cholesteryl ester transfer protein (CETP), recently expected to be drugs for treatment of atherosclerosis, make ApoE-rich HDL increase significantly and an amount of the HDL is linked to the therapeutic effect of inhibitors, precise determination of cholesterol in the HDL is required even for evaluation of inhibitors. So, we have studied a new assay to determine HDL-C with a high specificity to HDL-C and a good reactivity with cholesterol in various types of HDL such as non-esterified cholesterolrich HDL or ApoE-rich HDL, and have developed the new assay kit to determine HDL-CMetabolead HDL-C. The assay kit, used for the enzymatic determination of HDL-C in samples, is comprised of the two liquid reagents, and the specificity to HDL-C is based on an electrostatic interaction of a polyanion and a cationic substance (without a divalent metal ion) with lipoproteins. In this study, we evaluated the new assay using the kit. Method: The determination of HDL-C in a sample was carried out using the Hitachi 7170S auto analyzer with usual measurement parameters. As a sample, we used human serum samples obtained from healthy individuals, TG-rich 46 serum samples, 24 serum samples containing non-esterified cholesterol-rich HDL, and 28 serum samples containing ApoE-rich HDL. Results: i) Lower limit of detection and linearity: The lower limit of detection was 1 mg/mL, and linearity was obtained up to 180 mg/dL. ii) Imprecision: Within-run imprecision (CV) at 70 mg/dL and run-to-run imprecision (CV) at 50 mg/dL were 0.68% (n=20) and 0.70% (n=7), respectively. iii) Accuracy: The performance of the new assay was compared with that of the Designated Comparison Method (DCM) as a standard method in CRMLN (Cholesterol Reference Method Laboratory Network), using 97 serum samples from healthy individuals. A correlation between these two methods (Y=0.9855X+0.68, r=0.9983) was obtained. Furthermore, the performance of the new assay was compared with that of the ultracentrifugation method, using 46 TG-rich serum samples, and a correlation (Y=1.0502X-1.44, r=0.9892) was obtained. iv) Reactivity with cholesterol in HDL: The performance of the new assay was compared with that of DCM, using 24 serum samples with enriched non-esterified cholesterol in HDL, and a correlation (Y=0.9505X+3.20, r=0.9982) was obtained. Furthermore, the performance of the new assay was compared with that of the 13% polyethylene glycol precipitation method, using 28 serum samples with ApoE-rich HDL, and a correlation (Y=0.9454X+1.83, r=0.9950) was obtained. Conclusion: We evaluated the new assay kit to determine HDL-C. It proved that the new assay has a high specificity to HDL-C and a high reactivity with cholesterol in various types of HDL, such as non-esterified cholesterol-rich HDL and ApoE-rich HDL.
B-105 Correspondence of Paraoxonase 1 gene Q192R polymorphisms and lipid profile in ethnic Tatars settled in Bashkortostan, RUSSIA L. Zueva1, I. Tuktarova2, V. Pauk2, A. Gilmanov1, O. Mustafina2. 1Bashkir State Medical University, Ufa, Russian Federation, 2Institute of Biochemistry and Genetics, Regional Research Center of Russian Academy of Sciences, Ufa, Russian Federation
Paraoxonase 1 (PON1) is a plasma enzyme closely linked with high density lipoproteins (HDL). It also plays an important role in preventing LDL particles from oxidation and thus holds a protective role against atherosclerosis. Several studies showed genetic variants of PON1 associated with the risk of coronary heart disease (CHD). It is established that PON1 gene Q192R polymorphism is associated with diminished PON1 concentration, and increased CHD risk in RR genotype subjects. The aim of the present study was to evaluate an association of PON1 gene Q192R polymorphism and serum lipid levels in ethnic Tatars settled in the Republic of Bashkortostan, Russia. We investigated 980 blood samples from unrelated ethnic Tatars aged 12 to 109 yrs. Blood cell DNA was isolated from 8 ml of venous blood by phenol-chloroform extraction, the PON1 alleles were determined by Restriction Fragment Length Polymorphism Polymerase Chain Reaction (RFLP-PCR). The levels of plasma triglycerides, total cholesterol and HDL cholesterol (HDL-C) were determined by enzymatic method using Vector-Best test kits (Russia), the LDL cholesterol (LDL-C) concentration was calculated by Friedewald equation. Statistical analysis was performed with Statistica 6.0 for Windows software, statistical significance was assumed at P < 0.05. Our results show that QQ, QR, and RR genotype frequencies in ethnic Tatars were 46.23%, 45.23% and 8.54%, and Q and R allele frequencies were 68.84% and 31.16%, respectively. Statistically significant differences in lipid levels between RR genotype carriers and QQ and QR genotype carriers were observed. No significant changes of triglyceride levels were found in carriers of different genotypes. RR genotype carriers were of lower HDL-C and higher LDL-C levels compared to noncarriers of respective genotype, and the CHD risk in this group was generally increased.
B-106 Association Between Apolipoprotein(a) Phenotypes, Pentanucleotide Repeat Polymorphism And Lipoprotein(a) levels in Young CAD Patients And Their Children J. K. GAMBHIR1, H. Kaur1, U. Pati2, D. S. Gambhir3. 1University College of Medical Sciences, Delhi, India, 2Department of Biotechnology, Jawaharlal Nehru University, Delhi, India, 3Department of Cardiology, Kailash Heart Institute, Noida, Delhi, India
Background: High plasma lipoprotein(a) [Lp(a)] levels are a genetic risk factor for premature coronary artery disease (CAD). This work was planned to study the influence of apo(a) isoform size (K4 repeats) and pentanucleotide repeat polymorphism (PNRP) on plasma Lp(a) levels in CAD patients and their children.
B-104 Lipid profile in patients with chronic renal failure undergoing conservative treatment and hemodialyisi. B. K. Yadav, S. Ghimare, B. JHA. IOM,TUTH, Kathmandu, Nepal
Introduction: Chronic renal failure (CRF) is a gradual and progressive loss of the ability of the kidneys to excrete wastes, concentrate urine, and conserve electrolytes. Cardiovascular disease is the principle cause of morbidity and mortality in patients with chronic renal failure. Diabetic nephropathy, hypertension and gromerulonephritis are the most common cause of renal failure. Dyslipidemia in CRF leads to progression of the renal injury.
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Methods: The study was carried out in angiographically assessed CAD patients (age < 40 yrs; n=44) their spouses (n=44) and children (n=100). Plasma Lp(a) levels were determined by ELISA, apo(a) isoform size by Western blotting and PNRP by the amplification of 5 control region followed by PAGE and silver staining. Results: Mean plasma Lp(a) levels were significantly higher in patients (father) as compared to their healthy spouses and children (p<0.01). Combined influence of both polymorphisms was studied by dividing the subjects according to low molecular weight and high molecular weight apo(a) isoforms (LMW < 22; HMW > 22 K4 repeats, respectively) which were further stratified according to low PNR and high PNR ( < 8 and > 8 PNR). The patients having a combination of < 22 K4 repeats and < 8 PNR had the highest plasma Lp(a) concentrations. Sons inheriting the same phenotype also had significantly higher Lp(a) levels as compared to daughters (p <0.05). Conclusions: These results demonstrate a strong inverse effect of low PNR along with LMW apo(a) isoforms leading to high Lp(a) levels in patients and their children, particularly sons. Further it is suggested that sons in particular, might inherit atherogenic genes with respect to plasma Lp(a) levels from their affected father. Therefore, studies on Lp(a) levels and apo(a) polymorphism in offsprings may help to identify children having high risk of premature CAD.
Lipids/Lipoproteins
B-108 Quantitative determination of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in serum and plasma by gas chromatography- mass spectrometry. M. R. Al-Turkmani, A. Shkreta, L. Fischer, T. Law, M. D. Kellogg. Children's Hospital Boston and Harvard Medical School, Boston, MA
OBJECTIVE: To evaluate performance characteristics of a gas chromatography-mass spectrometry (GC-MS) method for quantitative determination of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in human serum and plasma. INTRODUCTION: EPA and DHA are anti-inflammatory n-3 fatty acids present at relatively high concentrations in fish oil. Consumption of EPA and DHA has been associated with improved outcomes in several diseases including cardiovascular disease, diabetes, inflammatory diseases, mental disorders, and some cancers. Studies have suggested that plasma EPA and DHA concentrations could serve as a risk marker for coronary heart disease or sudden cardiac death. METHODS: Lipids were extracted from 100 L serum or plasma, and fatty acids were methylated using boron trifluoride and methanolic base. The resulting fatty acid methyl esters were extracted with hexane and analyzed by GC-MS. Heptadecanoic acid (17:0) was used as an internal standard, and one point calibration was employed for EPA and DHA quantification. Within-run (n=10) and between-day (n=22) precision studies were performed on three serum pools with different EPA and DHA levels. Linearity was determined using samples with concentrations ranging from 0 to 4000 M. EPA and DHA recoveries were determined at 50, 200, 400, and 800 M. Functional sensitivity was the lowest concentration at which the coefficient of variation (CV) was less than 15% (n=10). Serum EPA and DHA levels were compared to those obtained from EDTA-anticoagulated and heparin-anticoagulated plasma samples that were drawn simultaneously (n=5). Method comparison was conducted using 30 serum samples against a different GC-MS method. RESULTS: Within-run CV values were 7.0, 6.3, 8.8 % at 57, 95, 178 M EPA, and 6.6, 7.8, 8.3 % at 120, 160, 262 M DHA, respectively. Between-day CV values were 13.7, 9.3, 11.7 % for EPA, and 8.8, 9.5, 9.4 for DHA, respectively. When EPA and DHA concentrations were expressed as mole % of total C12-C22 fatty acids, withinrun CV values were less than 5% and between-day CV values were less than 6.7%. The assay was linear throughout the entire tested range for both EPA and DHA (0 4000 M). The average recovery was 101.3 % for EPA and 99.7 % for DHA, and the functional sensitivity was 10 M for EPA and 20 M for DHA. No significant differences in EPA and DHA concentrations were found among different sample types (serum, heparin plasma, and EDTA plasma). Split sample comparison with another GC-MS method indicated good correlation (EPA: slope = 0.73, intercept = -1.21, r2 = 0.99; DHA: slope = 1.03, intercept = -15.25, r2 = 0.96). CONCLUSION: We have developed a reliable GC-MS method for quantitative determination of EPA and DHA in serum and plasma.
B-107 Is One Friedewald Type Equation (FTE) Enough? L. Casey1, E. S. Pearlman2. 1Dorchester Huse Multiservice Center, Dorchester,, MA, 2Boston Medical Center, Boston, MA
It is standard in many clinical laboratories to estimate LDL-cholesterol (eLDL-C) as a function of total cholesterol (TC), HDL-C and triglyceride (TG) concentrations in a FTE of the form:eLDL-C = TC - HDL-C - kTG [1] where k is a simple fraction that is usually taken to be (1/5) or sometimes (1/6) and the eLDL-C is considered valid for TG<400 mg/dL. The lab at DHMSC (a community health center) was measuring LDL-C (mLDL-C) on all lipid profile specimens irrespective of TG concentration. For financial and TAT reasons use of an eLDL-C was felt desirable. We were however, concerned to find the best FTE and also wished to ascertain whether more than one FTE was desirable as a function of TG concentration. All chemical analyses were done on the Xpand Plus analyzer (Siemens; Deerfield IL) and statistical analysis used multiple linear regression (MLR) with the Linest function on Excel software (Microsoft; Redmond WA) and the two-tailed nonparametric sign test. We initially assessed the results on 628 consecutive specimens with TG<250 mg/dL. Using the first half of this data and MLR with mLDL-C as the independent variable an equation was derived: mLDL-C = .975TC - 1.115HDL-C .092TG [2] Ratios of TC:HDL-C and TC:TG were calculated for each of the 314 specimens and using the median values for these ratios, equation [2] took the form of a FTE: mLDLC = TC - HDL-C - .18TG [3] Equation [3] suggested that the value of k in equation [1] was between (1/6) and (1/5) for TG<250 mg/dL. Using the second group of 314 consecutive samples we found that the median absolute difference (MAD) between eLDL-C and mLDL-C was 5.95 mg/dL for k=(1/6) and 6.55 mg/dL for k=(1/5) [p=NS]. But the MAD for k=(1/8) and k=(1/4) [ 6.75 and 9.5 mg/dL respectively] was significantly greater than for k=(1/6) [p<.01]. We now repeated this procedure for 275 consecutive specimens with TG between 250 and 399 mg/dL. Using the first 138 specimens the analogous equation to [2] was: mLDL-C = .784TC - .448HDL - .063TG [2a] and the analogous equation to [3] was: mLDL-C = TC - HDL - .138TG [3a] Equation 3a suggested a value for k between (1/8) and (1/7). Using the remaining 137 lipid profiles, MAD was 9.375 mg/dL for k=(1/8) and 10.25 mg/dL for k=(1/7) [p=NS] but MAD for k=(1/6), (1/5) and (1/4) [12.75, 19.7 and 35.0 mg/dL respectively] were significantly greater than MAD for k=(1/8)[p<.05] for TG values between 250 and 399 mg/dL. The outcome of this analysis suggests that best values of k in FTE may vary with TG and are not restricted to (1/5) or (1/6). It remains to be determined whether the best available FTE varies with analytical system. Data is currently being collected to determine if this approach can be used to generate an FTE with good properties for TG values =/>400 mg/dL.
B-109 Association of Glyoxalase I Gene Ala111Glu Polymorphism and Type 2 Diabetes Mellitus with Coronary Heart Disease in Han Chinese S. K. Yan1, X. Jin1, L. Y. Xia2, L. Zhang1, Y. H. Song2. 1Department of Laboratory Medicine, China-Japan Friendship Hospital, Beijing, China, 2Department of Laboratory Medicine, PUMC Hospital, Beijing, China
Objective Glyoxalase-I (GLO-I) is the important component of physiological detoxification enzyme systems of methylglyoxal (MG).As the first enzyme of glyoxalase enzyme system, it transforms glycosylation toxicity and the MG mutagenicity into non-toxic lactate discharging out of the body. In this study, we first investigated the association between GLO-I gene Ala111Glu polymorphism and type 2 diabetes mellitus (DM) with coronary heart disease (CHD) in Han Chinese. Methods We selected 161 healthy individuals, 99 patients with type 2 DM, and 71 patients with type 2 DM with CHD (DM+CHD) in Han Chinese from northern China. GLO-I gene Ala111Glu polymorphism was analyzed by the method of polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP). The levels of glycosylated hemoglobin (HbA1c) and serum lipids, lipoproteins, apolipoproteins were also measured. Results Neither the frequencies of genotypes nor frequencies of alleles of GLO-I gene Ala111Glu polymorphisms were statistically different among DM patients, DM+CHD patients and controls (P > 0. 05). We also found that there were no relationship between gender, family history of CHD, smoking, BMI and degree of coronary artery lesions and this polymorphism among different groups. Logistic regression analysis showed that age and HbA1c was the risk factor, but high-density lipoprotein
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cholesterol (HDL-C) was a protection factor [=-2.708,Exp()= 0.067,95%CI=0.009~0.488,P=0.008] for type 2 DM with CHD. The Ala111Glu polymorphisms of GLO-I gene might not the major genetic risk factors of type 2 DM with CHD. Conclusion GLO-I gene Ala111Glu polymorphism is not associated with changes in HbA1c and serum lipids, lipoproteins, apolipoproteins concentrations. We suggest that common variants in GLO-I gene are not significant susceptibility factors for type 2 DM with CHD in Han Chinese. These finding suggest that the genetic determinants of type 2 DM with CHD are complex and cannot be entirely explained through intermediate phonotypes. MG concerning its role in DM and in the development of diabetic complications may also be considered.

measured. Adiponectin, C-reactive protein, lipid profiles were measured in fasting blood samples. Results: Adiponectin levels were significantly lower (median [25th75th percentile]) (P=0.003) in diabetics (n=60) (11.1 [7.4-13.9] g/ml) than non diabetics (n=73) (12.1 [8.8-21.2] g/ml). No correlation between adiponectin and Creactive protein was found. When compared to controls the diabetic subjects showed reduced HDL and increased LDL cholesterol with less adiponectin. Adiponectin level was negatively correlated with LDL cholesterol. However, adiponectin levels were negatively correlated with BMI adjusting for age and diabetic status, and gender ( = -0.200, P=0.020; = -0.235, P=0.004). Conclusions: No correlation exists between adiponectin and C-reactive protein. However, this study showed that the adiponectin levels decrease with increasing adiposity, using BMI as a marker.
B-110 Characterization and validation of a novel homogeneous assay for small dense LDL cholesterol Y. Ito1, M. Fujimura1, M. Ohta1, T. Hirano2. 1Denka Seiken Co.,Ltd., Tokyo, Japan, 2Showa University, School of Medicine, Tokyo, Japan
Objectives: Many epidemiological and pathological studies have recently suggested the relationship between small, dense LDL-cholesterol (sd LDL-C) level and CHD occurrence. Several different methods have been employed for the determination of LDL particle size such as based on ultracentrifugation, electrophoresis, and nuclear magnetic resonance (NMR). All these methods require special equipment and very long assay time, making them too laborious for general clinical use. We recently developed a fully automated, homogeneous assay for sd LDL-C quantification. We describe the design of the new assay and report the verification results of the assay performance. Methods: The new assay for sd LDL-C quantification employs of two liquid, ready-touse reagents and consists of two steps. The reagent for the 1st step of the assay contains 3 key ingredients: 1) a polyoxyethylene benzylphenyl ether derivative that selectively decomposes chylomicron, VLDL and HDL, 2) a polyoxyethylene distyrenelphenyl ether derivative that selectively binds to sd LDL to protect sd LDL from the action of enzymes, and 3) a sphingomyelinase that has much more affinity to larger LDL particle than sd LDL. These ingredients work to decompose the other lipoproteins than sd LDL. Cholesterol released from such non-sd LDL lipoproteins are degraded to water and oxygen by the action of cholesterol esterase, cholesterol oxidase and catalase. At the 2nd step, cholesterol is released from sd LDL by the action of another detergent and lead to color development. Our new assay can be applied to both serum and plasma samples. It is completed in 10 minutes in a completely automated manner on routine chemistry analyzers without any off-line process. Results: The detection limit was found as 1 mg/dL and the assay linearity was observed up to 100 mg/dL. No significant interference was observed with 30 mg/dL bilirubin, 500 mg/dL hemoglobin, 50 mg/dL ascorbic acid and in all the cases the recoveries of sd LDL-C were in the range of 100% +/- 5%. The new assay always exhibited CVs well below 2% in within-run precision studies. The new assay was compared to the traditional ultracentrifugation method using 60 samples and the linear regression analysis showed: y = 0.99x - 3.4 , r = 0.954. The comparison study with our previous method using polyanion and a filter also showed excellent correlation (y = 0.99x - 1.5 , r = 0.904). Test samples were stable for the new assay at least for 7 days in a refrigerator. Consistent test results were also obtained when test samples were subject to repeated freezing and thawing at least for 5 cycles. In a case-control study employing CAD and non-CAD people, sd LDL-C measured by this new assay was significantly higher in CAD group compared to non-CAD group (p<0.05). Conclusions: The new assay was verified to possess robust and valid assay performance. This new assay may contribute to the wide spread use of sd LDL-C.
B-112 Measurement of Total Glyceride in Serum with Gas ChromatographyIsotope Dilution Mass Spectrometry S. H. Edwards1, S. D. Pyatt1, S. L. Stribling2, K. D. Dobbin2, M. K. Kimberly1, G. L. Myers1. 1Centers for Disease Control and Prevention, Atlanta, GA, 2Battelle Memorial Institute, Atlanta, GA
Background: CDC has used the chromotropic acid (CA) method for many years to standardize net triglyceride (or glycerol blanked triglyceride) measurements in the Lipid Standardization Program (LSP). However the chromotropic acid method has become increasingly troublesome with continuing technical problems; furthermore, it uses instrumentation that is outdated and extremely difficult to replace. CDC has developed an isotope dilution mass spectrometry (IDMS) method to measure total glyceride (the sum of monoglycerides, diglycerides, triglycerides, and endogenous free glycerol). We believe it is appropriate to implement an IDMS reference measurement procedure to generate target values for our reference materials used to standardize total glycerides (TG). The purpose of this project was to develop, validate and apply a rapid accurate and precise GC-IDMS method for the quantitation of TG in serum. Methods: We have developed a GC-MS method based on isotope dilution principles to separate and quantify total glycerides in serum. In brief, aliquots of diluted serum, calibration standards and reagent blanks that have been fortified with isotopically labeled 13C -glycerol were thoroughly mixed and hydrolyzed with alcoholic/KOH at 60 0C for 1 3 hr. After completion of the hydrolysis step the solvent was evaporated under nitrogen and the residue containing free and hydrolyzed glycerol was derivatized with acetic anhydride in the presence of pyridine. The derivatized product was then extracted with ethyl acetate and the organic layer was removed and dried under nitrogen at 60 0C. Subsequently the dried glycerol acetate derivative was reconstituted in methanol and analyzed by GC-MS. Results: The analytical recovery from a spiked serum matrix at three concentration levels ranged from 102 - 107% and averages at 104%. The calibration range was from 0.056 (5.0 mg/dL) to 1.13 mmol/L (100 mg/dL) and the LOD was determined to be 7 x 10-3 mmol/L. The linearity evaluation shows that the analyte response was linear across the range of the calibration curve. The method within run precision ranged from 0.3 to 1.5 % and the among run precision ranged from 0.8 to 2.0%. The accuracy of the assay was accessed through the analysis of three standard reference materials; SRM 1951a, SRM 1951b and SRM 909 and the bias from the certified reference value was determined. The relative biases were 1.3%, 1.6% and 1.5% respectively. Conclusion: This GC-IDMS method combined with liquid-liquid extraction sample preparation protocol provides a rapid accurate and precise mechanism for quantifying TG in serum. This method will replace the CA reference method procedure in the CDC LSP.
B-113 Withdrawn
B-111 Adiponectin and its relationship with anthropometric, C-reactive protein and metabolic variables in type 2 diabetic Trinidadians S. B. Nayak1, V. S. Bhogadi2. 1Faculty of Medical Sciences, University of West Indies, Champs Fleurs, Trinidad and Tobago, 2Diagnostic Laboratory services, North Central Regional Health Authority,, Champs Fleurs, Trinidad and Tobago
Objective: To determine and study the relationship of adiponectin with anthropometric, CRP and metabolic variables in type 2 diabetic Caribbean population. Method: It was a cross sectional study comprised 133 subjects Trinidadians aged 1865 years without known conditions or behaviors that alter adiponectin and Inflammatory Marker levels. Anthropometric indices and blood pressure were
B-114 Clinical Lipoproteomics Unveils Clusterin as a Novel Biomarker of Insulin Resistance. A. N. Hoofnagle1, M. Wu2, J. O. Becker1, J. D. Brunzell1, S. E. Kahn1, R. H. Knopp1, T. J. Lyons2, J. W. Heinecke1. 1University of Washington, Seattle, WA, 2Oklahoma University Health Sciences Center, Oklahoma City, OK
Background: Patients with obesity and insulin resistance are at increased risk for atherosclerosis. Insulin resistance lead to changes in plasma lipoproteins, however traditional Framingham Risk Factors cannot explain the increased risk. While low
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density lipoprotein cholesterol (LDL-C) has been implicated in atherogenesis, high density lipoprotein cholesterol (HDL-C) has been shown in numerous epidemiological studies to be a negative risk factor for atherosclerosis. The proteins associated with HDL particles likely confer atheroprotection. For example, apolipoprotein (Apo) A-I facilitates efflux of lipids out of macrophages, reducing the lipid burden within atherosclerotic plaques. Other proteins down-regulate the complement cascade and prevent neutrophil degranulation, making HDL antiinflammatory. Still other proteins reduce lipid peroxidation of lipoproteins giving HDL anti-oxidizing properties. The recent failed trial of the cholesteryl ester transfer protein inhibitor, torcetrapib, raises the possibility that not all HDL is equivalent and that it could become dysfunctional in vivo. We set out to determine if obesity and insulin resistance could lead to changes in HDL protein cargo, potentially explaining the increased risk of atherosclerosis by dysfunctional HDL. Methods: Using density gradient ultracentrifugation, we isolated HDL and non-HDL particles from 10 subjects in each of three groups: (1) lean, insulin sensitive, (2) lean, insulin resistant, and (3) obese, insulin resistant. We used targeted lipoproteomics by multiplexed isotope dilution-liquid chromatography-tandem mass spectrometry to quantify the concentration of ApoA-I, ApoM, ApoE, clusterin, and complement C3, proteins with recent interest as mediators of atherosclerosis. Results: By ANOVA, there was a significant difference between the three groups only for clusterin. By univariate analysis, HDL clusterin inversely associated with body mass index (p=0.006) and insulin resistance (p=0.004). HDL clusterin also strongly associated with a good serum lipid profile: a positive correlation with HDL-C (p<0.01) and a negative correlation with LDL-C, total cholesterol, and triglycerides (p<0.01 for each association). When controlling for lipids, there was still a significant inverse association of HDL clusterin with body mass index (p<0.04), but an association with insulin resistance was no longer significant. We then validated the strong inverse correlation between HDL clusterin and body mass index (p<0.001) and insulin resistance (p<0.001) in a second population from another institution. When we combined the populations (N=68), HDL clusterin significantly associated with insulin sensitivity, even when controlling for lipids and body mass index (p=0.02). On the other hand, non-HDL clusterin was directly associated with body mass index (p<0.001) and insulin resistance (p=0.006). There was also an inverse correlation between HDL clusterin and non-HDL clusterin (p<0.001), consistent with the hypothesis that clusterin transitions from HDL to non-HDL particles in obesity and insulin resistance. Conclusions: Clinical lipoproteomics has revealed that HDL clusterin associates with a good lipid profile and insulin sensitivity. Clusterin is known to be protective to the kidneys and protective from excessive damage de to cerebral ischemia. It is also known to be important in lipid metabolism and is regulated by stress. This is the first demonstration that clusterin provides a link between insulin resistance and lipoprotein metabolism and may finally provide a biomarker of dysfunctional HDL.
Lipids/Lipoproteins
B-116 Serum lipid analysis using biphasic electrophoresis S. P. Suh1, J. Park1, D. Cho1, S. Kee1, M. Shin1, J. Shin1, D. Ryang1, B. Park2. 1Chonnam National University Hospital, Gwangju, Republic of Korea, 2Mokpo National University, Muan, Chonnam, Republic of Korea
Background: Recently biphasic agarose gel electrophoresis method using Chol/Trig ComboTM for simultaneous detection of cholesterol and triglyceride on lipoprotein fractions has been developed to facilitate the classification and interpretation of abnormal lipoprotein patterns of patients with hyperlipidemia. The authors analyzed serum lipid profiles using Chol/Trig ComboTM in the patients of out-patient department of a university hospital. Methods: Measurement of serum cholesterol and triglyceride using enzymatic method was performed in the sera of 415 patients from August, 2007 to July, 2008. Simultaneously, we electrophoresed serum cholesterol and triglyceride using Chol/Trig ComboTM with analysis software (ED BANK, K. K Helena Kenkyujo, Sajitama, Japan). Results: According to ATP III guideline, we set up standard cholesterol as 200 mg/dL and triglyceride as 150 mg/dL, respectively, and the patients were classified into the control (C), hypercholesterolemic (HC), hypertriglyceridemic (HTG) and hypercholesterolemic/hypertriglyceridemic (HC/ HTG) groups, respectively. In HC group, LDL and HDL were significantly high and low (146 mg/dL in HC vs 87.5 mg/dL in C, P < 0.001; 42 mg/dL in HC vs 53 mg/dL in C, P < 0.05), respectively. In HTG group, VLDL and HDL were significantly high (44.5 mg/dL in HTG vs 24.5 mg/dL in C, P < 0.001; 59 mg/dL in HTG vs 53 mg/dL in C, P < 0.05, respectively). HC/HTG group had higher VLDL and LDL (56 mg/dL in HC/HTG vs 24.5 mg/dL in C, P < 0.001; 140 mg/dL in HC/HTG vs 87.5 mg/dL in C, P < 0.001, respectively). The positive modified LDL was detected at distinctly high frequencies in the HC/HTG and HTG groups (50.9% in HC/HTG vs 39.3% in HTG vs 19.8% in C, P < 0.01). However, there was no significant difference between the HC group (23.6%) and the control (19.8%). The most common Fredrickson types determined by using the biphasic electrophoresis were type IIa (58.4%) in HC, type IV (96.4%) in HTG, type IIb (55.4%) in HC/HTG, and indeterminate type (97%) in the control, respectively. Conclusions: This study demonstrates that the biphasic electrophoresis can be convenient and useful for the classification and interpretation of abnormal lipoprotein patterns of patients with hyperlipidemia.
B-117 Oxidized LDL, antibodies to oxidized LDL and its effect on survival of hemodialysis (HD) patients J. Racek1, M. Korotvicka1, J. Eiselt1, L. Malanova2, L. Trefil1, D. Rajdl1, M. Vostry1. 1Charles University, Faculty of Medicine, Pilsen, Czech Republic, 2B. Braun Avitum, Pilsen, Czech Republic
Background. Oxidative stress and lipid metabolism disorders belong among the factors participating in early atherosclerosis development in HD patients. Evidence exists that HD patients are exposed to enhanced oxidative stress. Increased oxidative stress combined with chronic inflammation may lead to an increased risk of CVD. Oxidized low density lipoproteins (oxLDL) are oxidatively modified lipoproteins associated with atherosclerosis. These modified particles serve as antigen for antibodies against oxidized LDL (IgoxLDL). Effect of oxLDL, IgoxLDL and routine lipid parameters on survival of HD patients was investigated. Methods. Blood samples were collected from 176 chronically haemodialysed patients (70 females and 106 males with mean age SD = 66.4 10.63 years) and 73 men with normal kidney function (mean age SD = 48.15 5.78 years) who served as controls. Mercodia OxLDL and Biomedica oLAb ELISA kits were used for the detection of serum levels of oxLDL and IgoxLDL, respectively. Lipid parameters were measured by routine photometric tests. Cox step wise model, Kaplan Meier and log rank test were used for results evaluation and expression. Results. The data were divided into tertiles. The first tertile of patients (oxLDL<47.24 U/L) had a tendency to worse prognosis of survival for 36 months (p=0.066) than the other two (second tertile boundaries 65.6 U/L). The second and third tertile showed no obvious difference between each other, generally said the patients with oxLDL levels higher than 47 U/L had almost the same survival rate. Expectedly, the values of oxidized LDL correlate with LDL-cholesterol as well as apoB values correlated with LDL-cholesterol and apoA-I with HDL-cholesterol, respectively. OxLDL antibodies showed significant correlation between oxLDL antibody titre and total cholesterol, LDL-cholesterol and triglycerides. The probability of survival in patients with high levels of IgoxLDL (more than 549.72 IU/L) was significantly lower (p<0.01). Conclusions. The examination of antibodies against oxLDL as a marker of oxidative stress in serum confirmed their negative effect on the survival in HD patients.
B-115 The comparison of plasma oxidation via Cu2+ and macrophage Y. Liu1, L. Ping2, J. Wu2. 1Renmin Hospital of Wuhan University, Wuhan, China, 2Basic Medical School of Wuhan University, Wuhan, China
Objectives: Compare plasma oxidation via Cu2+ and macrophage to found a more relevant model of the lipoprotein oxidation in the arterial wall than the in vitro oxidation of single isolated lipoproteins, e.g., low density lipoprotein (LDL). Methods: We measured the oxidation of whole plasma as an absorbance increase at 234 nm known to reflect the level of conjugated dienes in the samples. Plasma oxidation was induced by Cu2+ and macrophage. Results: Oxidation of human plasma measured in the presence of Cu2+ (10 mol/L) was completely inhibited by plasma concentration for 5% and 10%. This oxidation was partly inhibited by 2.5% and 1%, and the level of inhibition for 2.5% is higher than that of 1%, and its oxidation arrearage-time (240 min) longer than 1% plasma (160 min). The peak of the cellmediated plasma is taken place about 9 h. The arrearage-time of 5% and 2.5% plasma is equal, but the oxidative capability of the cell-mediated 2.5% plasma is higher than that of 5% plasma. The arrearage-time of the three patients is equal in cell-mediated oxidation. But along with the dose of LDL raises, the oxidation raises. The plasma oxidizability did not correlate with plasma antioxidant content in cell-mediated oxidation. Conclusion: Detecting the plasma oxidation can get messages of cellmediated LDL oxidized. It is more reliable model for studying the mechanism of LDL oxidized than the purified LDL.
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Oxidized LDL had positive effect on survival. Conceivable principle is that LDL also serves as a marker of nourishment status, and moreover, higher levels of LDL represent more substrate for oxidation.The study was supported by research project MSM 0021620819.

ability to run many batches in a single day has made it possible to meet throughput requirements. One hundred samples were analyzed for LDL cholesterol using both ultracentrifuges resulting in a 1.31% mean difference between the two data sets, an R2 of 0.97, a slope of 0.97, and a y intercept of 3.53. Intra- and interassay precision on 10 replicates of two manufactured controls produced 3.5 % and 5.7% CVs at 58 mg/ dL and 5.3% and 4.1% CVs at 153 mg/dL, respectively. Conclusion: The Beckman Optima MAX ultracentrifuge has improved TAT for LDL determination by 47 % in our lab and, in most cases, results are delivered to physicians the same day of order. To date, about 4,400 patient samples have been analyzed using the new Optima-MAX centrifuge method.
B-118 Serum Apolipoprotein C-III in HDL: Key Diabetogenic Risk Factor among Turks G. Hergen1, A. Onat2, E. Ayhan3, M. Ugur3, H. Kaya4, M. Tuncer5, G. Can6. 1Yildiz TU, Istanbul, Turkey, 2Turkish Society of Cardiology, Istanbul, Turkey, 3Siyami Ersek Center for Cardiovascular Surgery, Istanbul, Turkey, 4Kartal Kosuyolu Heart Hospital, Istanbul, Turkey, 5Department of Cardiology, Centennial Universityf, Van, Turkey, 6Department of Public Healthg, Cerrahpaa Medical Faculty, Istanbul, Turkey
Aims Apolipoprotein C-III (apoC-III) is recognized as an indicator of prognosis for coronary risk in healthy subjects. We studied certain determinants of serum apoC-III and whether levels of it or its fractions predict metabolic syndrome (MetS), type-2 diabetes and coronary heart disease (CHD). Methods and Results In tracked 802 individuals of a general population residing in Central and Western Turkey in whom serum apoC-III had been measured by turbidimetric immunoassay in 2001, its predictive value in cardiometabolic risk was assessed, after exclusion of patients with the above stated diseases at baseline, over a mean follow-up of 4.4 1.2 years. Total apoC-III as well as both fractions were significantly, linearly and inversely related to smoking status and positively with alcohol usage; in women, total apoC-III was additionally associated with levels of complement C3, and HDLapoC-III was associated weakly and positively with apoA-I. Total or nonHDL apoC-III predicted incident MetS each with a hazard ratio (HR) of 1.8 (95%CI 1.35; 2.4), after adjustment for sex, age, smoking status, alcohol usage and lipid lowering drugs. After similar adjustment including systolic blood pressure, nonHDL apoC-III predicted incident CHD with a HR of 1.33 (95%CI 1.04; 1.70), which attenuated to borderline significance when additionally adjusted for nonHDLcholesterol. HDL- (or total) apoC-III predicted newly developed diabetes with a HR of 1.65 (95%CI 1.27; 2.11), after additional adjustment for waist circumference and HDL-cholesterol, and represented a better predictor than waist girth. Conclusions: Serum total apoC-III is linearly associated inversely with smoking and positively with certain inflammatory risk markers. HDL apoC-III is a stronger predictor of type-2 diabetes than waist girth among Turks. NonHDL apoC-III predicts independently and strongly the development of MetS and moderately incident CHD independent of non-lipid factors.
B-120 Association betwenn polymorphisms of the hepatic uptake transporter SLCO1B1 and reduced atorvastatin response. A. C. Rodrigues1, P. M. Perin1, S. Purim2, F. D. Genvigir1, M. V. Willrich1, S. S. Arazi1, M. H. Hirata1, E. L. Dorea3, M. M. Bernik3, R. D. Hirata1. 1Faculty of Pharmaceutical Sciences, University of Sao Paulo, Sao Paulo, Brazil, 2Applied Biosystems Ltda, Sao Paulo, Brazil, 3University Hospital, University of Sao Paulo, Sao Paulo, Brazil
Introduction: The statins or 3-hydroxy-3-methlyglutaryl-coenzyme A reductase inhibitors are considered one of the most effective classes of drugs for reducing low density lipoprotein (LDL) and total cholesterol (TC). Although, statin treatment has beneficial effects in the prevention of cardiovascular disease, considerable interindividual variation exists in response to statin therapy, as well as in the incidence of adverse effects. The organic anion transporting polypeptides-1B1, 2B1 and 1B3 encoded by, respectively, the SLCO1B1, SLCO2B1, and SLCO1B3 genes are implicated as major transporters in cellular uptake of statins. Objective: The aim of this study was to investigate the relationship between genetic variation in SLCO1B1, SLCO2B1, and SLCO1B3 genes and lipid-lowering response to atorvastatin. Material and Methods: Seventy-six unrelated individuals with hypercholesterolemia from Sao Paulo city, Brazil, were selected and treated with atorvastatin (10mg/day/4weeks). Blood samples were collected for serum lipids and apolipoproteins measurements and DNA extraction. Serum lipids and apolipoproteins were measured by standard methods using an ADVIA 1650 analyzer. Genomic DNA was extracted by a salting out method and SLCO1B1 (N130D/ 388A>G and V174A/521T>C), SLCO2B1 (71T>C and R312Q/935G>A), and SLCO1B3 (S112A/334T>G and A519A/1557A>G) gene polymorphisms were identified by TaqMan Real-time PCR, using TaqMan MGB probes (VICTM and FAM dye-labeled). The effect of each polymorphism on lipid and lipoprotein levels before and after atorvastatin administration was evaluated by Two-way repeated measures ANOVA followed by Holm-Sidak test. Results: The genotype frequencies of the SLCO1B1, SLCO1B3, and SLCO2B1 polymorphisms were in Hardy-Weinberg equilibrium. Linkage disequilibrium analysis revealed that S112A and A519A of SLCO1B3 were in complete disequilibrium (p< 0.001 value). For SLCO2B1, -71T>C was also consistently associated to R312Q polymorphism (2=12.35, p<0.001). Subjects carrying 388AA genotype exhibited significantly less total cholesterol (TC) and LDL-C reduction relative to AG (p=0.010) and GG genotypes carriers (p=0.013). For 521T>C polymorphism, 521TT genotype was associated to lower TC and apolipoprotein B (apoB) levels after atorvastatin treatment (TC: 197 26 mg/dL; apoB: 106 3 mg/dL) compared to TC genotype (TC: 177 21 mg/dL; apoB: 86 5 mg/dL). There was no significant relation between atorvastatin response and SLCO1B3 and SLCO2B1 polymorphisms (p>0.05). Conclusions: These results reveal that SLCO1B1 polymorphisms are associated with atorvastatin response and may be important markers for predicting efficacy of lipid-lowering therapy.
B-119 Turn Around Time Improvement for LDL Beta Quantitation A. J. Stelpflug, J. M. Hornseth, S. D. Hodel-Hanson, J. P. McConnell. Mayo Clinic, Rochester, MN
Background: Beta-quantitative LDL determination utilizes ultracentrifugation, selective precipitation, and enzymatic determination of cholesterol. In our lab, this time-consuming method required a 15 hour ultracentrifugation using a Beckman Optima LE-80K ultracentrifuge. To shorten assay turn around time, a new ultracentrifuge, a Beckman Optima MAX, was employed. The Optima MAX is a bench top ultracentrifuge capable of a much higher spin velocity ( 130,000 RPM or >1,000,000 x g). Method: Ultracentrifugation with beta-quantification of LDL-C and HDL-C determination by precipitation of the lower fraction with dextran sulfate and Ca++ was performed on serum samples using both types of ultracentrifuges. Ultracentrifugation was performed for 15 hours at 25,000 RPM (92,400 x g) at 10 C with the Optima LE-80K and for 70 minutes at 130,000 RPM (1,020,000 x g) at 4 C with the Optima-MAX. LDL-C and HDL-C were measured on a Roche Hitachi 912 using Roche cholesterol reagents and CFAS lipids calibrator. Accuracy and precision of the LDL results determined using the Optima MAX ultracentrifuge were assessed. Results: Ultracentrifugation time has been reduced from 15 hours with the Optima LE-80K to 70 minutes with the Optima MAX, resulting in a potential total process turn around time (TAT) savings of up to 83%. Actual laboratory TAT reduction dropped from 1.7 to 0.9 days, a 47 % savings. While maximum batch size has been reduced from a possible 100 samples on the Optima LE-80K model ultracentrifuge to 10 samples on the MAX, the purchase of two Optima MAX instruments and the
B-121 Effect of lifestyle modifications on a new oxidized low-density lipoprotein marker, serum amyloid A-LDL, in asymptomatic subjects with primary dyslipidemia K. Kotani1, T. Yamada1, N. Taniguchi1, M. Kawano2, I. Sakurabayashi3. 1Jichi Medical University, Dep Clin Lab Med, Shimotsuke-City, Tochigi, Japan, 2Jichi Medical University Saitama Medical Center, Saitama-City, Saitama, Japan, 3Saitama Memorial Hospital, Saitama-City, Saitama, Japan
[Background]: Dyslipidemia is a major health problem as a risk factor for cardiovascular disease in many countries. Lifestyle modifications are a cornerstone of the treatment for dyslipidemia. In dyslipidemia, oxidized low-density lipoprotein
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(oxLDL) is involved in atherogenesis. Among the several newly developed oxLDL markers, we have recently reported a significant relationship between serum amyloid A-LDL (SAA-LDL) and metabolic syndrome. SAA-LDL has also been shown to be closely correlate with vascular inflammatory markers, such as C-reactive protein. Using SAA-LDL, we may be able to elucidate the mechanism by which proper lifestyles can help prevent cardiovascular disease. We therefore have first investigated the effect of lifestyle changes on SAA-LDL in dylipidemic subjects. [Methods]: We performed a 6-month lifestyle modification program including diet and exercise for asymptomatic subjects with primary dyslipidemia (n=100, men=21%, mean age=59.4 years). All eligible subjects had more than 1 lipid abnormality, but were drug-free, within normal glucose levels and without any known history of cardiovascular, cerebrovascular, thyroid, kidney or liver diseases. None of the subjects were currently consuming alcohol or smoking. The prescribed diet consisted of 25 kcal/kg of ideal body weight per day and an appropriately balanced intake of fat, as well as exercise for at least 30 min at a moderate intensity at least 3 d/ week were recommended. The pre- and post-intervention fasting data on BMI, blood pressure (BP), blood glucose and lipid/lipoprotein, including SAA-LDL, were collected from each subject. Serum samples were incubated with the SAA-specific antibody coated on a micro-titer plate for 2 hours at room temperature and then an apo-B antibody was added (an ELISA system). The intraassay coefficients of variation at low and high concentrations were 2.6% and 4.7%, respectively. The interassay coefficients of variation at low and high concentrations were 5.0% and 6.7%, respectively. [Results]: During our intervention, the pre- and post-mean values of the measured variables were: follows: BMI 25.9 to 25.0 kg/m2, systolic BP 148.7 to 143.1 mmHg, diastolic BP 88.5 to 86.2 mmHg, glucose 5.65 to 5.48 mmol/L, LDL-cholesterol 3.47 to 3.43 mmol/L, HDL-cholesterol 1.59 to 1.62 mmHg, triglycerides 1.83 to 1.56 mmol/L, SAA-LDL 26.6 to 20.0 g/mL. A multiple regression analysis on the change in (log-)SAA-LDL revealed the degree of (log-)triglycerides (=0.27) and HDLcholesterol (=-0.23) to be independent and significant correlates. [Conclusions]: Presently, SAA-LDL is thought to be produced in the endothelium. Our data suggest that favorable changes of triglycerides and HDL-cholesterol levels by lifestyle modifications for dyslipidemia may prevent atherogenesis in part in relation to SAA-LDL. The measurement of SAA-LDL may be useful for the risk assessment of dyslipidemia.
Lipids/Lipoproteins
Conclusions. The results of this retrospective epidemiological investigation are consistent with the hypothesis that elderly patients have similar or even greater Lp(a) value than those observed in a general population aged <75 yrs. Therefore, elevated plasma Lp(a) concentrations may be compatible with longevity.
B-123 Research of hemorheological differences in age, gender and blood lipid level Y. Guo, H. Jiang, Y. P. Tian. Chinese PLA General Hospital, BeiJing, China
Objective: To investigate hemorheological changes in people with different age, gender and blood lipid level. Methods: Hemorheological level was tested by weissenberg rheogoniometer. There were 3 age groups in the study according to WHO:young group(44y), middle-age group (45~59y)and old group(60y).There were 4 groups in blood lipid level:control group, high TG group, high T-chol group, both high TG and T-chol group. Result: 1. Hemorheological changes in common people with different age and gender. Hemorheological level in male is higher than that in female. Middle age groups hemorheological level was highest in all age groups. There were differences in shear rate 1.00(1/S) and 5.00(1/S) in different age groups and gender. 2. Hemorheological changes in different blood lipid level. There are obvious differences among all groups of blood lipid level totally. There is no difference in hemorheological changes of gender groups and young group. In middle-aged/old group it was observed that the hemorheological level changed significantly with blood lipid level fluctuation and alteration trend was that hemorheological level became higher with lower T-chol level, but higher TG level on the contrast. There is a positive correlation between hemorheological and TG levels in each shear rate. Conclusion: Blood viscosity was significantly varied in gender, age groups and high blood lipid. A higher hemorheological level was easy to be observed in men and elderly people. There is an oppositive trend between high T-chol and TG levels in hemorheological changes .Higher TG level induced higher hemorheological level, especially in elderly people. Prompted social roles and sub-health state as well as decreased body fat metabolism may be influencing factors. Analysis of blood viscosity could help to provide a more suitable assessment for health and risks of diseases.
B-122 Lipoprotein(a) and ageing. G. Lippi1, G. L. Salvagno1, G. Targher2, M. Montagnana1, M. Franchini3, G. C. Guidi1. 1Laboratorio di Biochimica Clinica, University of Verona, Italy, 2Sezione di Endocrinologia e Malattie del Metabolismo, University of Verona, Italy, 3Servizio di Immunoematologia e Trasfusione, Azienda Ospedaliero-Universitaria di Parma, Italy
Background. Lipoprotein(a) (Lp(a)) is an emerging independent risk factor for a variety of thrombotic disorders, including myocardial infarction, stroke, peripheral artery disease, and venous thromboembolism. Although the role of Lp(a) as a cardiovascular pathogen is acknowledged, there are controversial data on its role on the overall mortality and, especially on its plasma levels in the elderly. Materials and Methods. We retrospectively retrieved data from our laboratory information system on 2128 medical outpatients who consecutively attended our laboratory between for Lp(a) and routine serum lipid assessment over the past 3 yrs. The cohort included 299 (88 men and 211 women) elderly outpatients aged 75 yrs and 1829 (828 men and 1001 women) patients aged <75 yrs. Results. Values are presented as means standard deviation(SD), except for Lp(a)(shown as median and 5-95 percentile distribution, table 1). Differences are assessed by the unpaired Students t-test or the Mann-Whitney test). Compared with those aged <75 yrs, elderly male patients had an overall more favourable traditional lipid profile, including lower values of cholesterol, triglycerides, LDL-C, total to HDL-C ratio, atherogenic index of plasma and higher values of HDL-C. Conversely, no significant differences could be observed between women aged <75 yrs and the elderly ones. In both genders, elderly patients had significantly higher Lp(a) median concentrations. However, after stratification of patients according to the 300 mg/L cardiovascular risk threshold for Lp(a), the percentages of those 75 yrs having Lp(a) levels above this threshold were marginally but not significantly higher in both genders (men: 28% versus 25%, p=0.499; women: 35% versus 30%, p=0.374).
B-124 Pilot Study: Effect of High Fat Intake and Carbohydrate Rich Meal on Serum Small Dense LDL-Cholesterol levels C. Tennert, R. Burkhardt, D. Teupser, J. Thiery. University Hospital Leipzig, Leipzig, Germany
Background: There is some pathophysiological and clinical evidence that small dense LDL (sdLDL-Cholesterol) can potentiate the risk for coronary artery disease. Serum sdLDL-Cholesterol (sdLDL-C) levels are frequently associated with high triglycerides and low HDL levels. However, little is known on the influence of postprandial hyperlipemia on sdLDL-C levels. The aim of the present pilot study was
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Lipids/Lipoproteins
to investigate the effects of high fat intake on sdLDL-C levels in healthy subjects. Methods: A carbohydrate rich meal (medium size pizza) was given healthy male and female volunteers (n=10, aged 20-29 years). One group (n=5) was given additional 250ml whipped cream (20% fat). Blood samples were taken before, 3 hours and 5 hours after the meal. During this time the volunteers were only allowed to drink water. The following metabolic parameters were analyzed at each time point: glucose, insulin, high sensitive CRP, triglycerides, total cholesterol, LDL-cholesterol, HDLcholesterol, ApoA-I, ApoB, Lp(a), sdLDL-C and free fatty acids. The analyses were performed on a Roche Modular System except insulin (DiaSorin). For sdLDL-C determination a novel precipitation assay (Denka Seiken) was used. Results: Additional fat intake caused an increase of triglyceride levels 3 hours after meal compared to baseline (1.13 mmol/L0.34mmol/L and 1.84 mmol/L0.48mmol/ L, respectively, p=0.029), no significant increase was observed in the carbohydrate control group. Postprandial hypertrigyceridemia did not change the levels of sdLDLC in the control and the additional fat intake group (0.280.12mmol/L and 0.260.10mmol/L, respectively). Free fatty acids decreased significantly in the control group at 3 hours and increased 5 hours after the meal. In the group with additional fat intake a decrease of free fatty acids was observed, but did not reach statistical significance (p=0.0501). No significant influence of a carbohydrate rich meal was noted on postprandial total cholesterol, LDL-C or HDL-C. For both groups a significant increase in insulin was seen 3 hours after the meal but without difference between the high fat and carbohydrate group. In the high fat group a significant increase of glucose levels was observed 3 hours after the meal. No postprandial effects were observed on CRP in both groups. Conclusions: Our preliminary results demonstrate that postprandial hyperlipidemia does not change sdLDL-Cholesterol levels in healthy young subjects.

B-126 The effect of apolipoprotein A-I cross-linking caused by glycation on ATP binding cassette transporter A-I-mediated cholesterol release Y. Kurosaki1, S. Endo1, H. Ikeya2, Z. Ogawa1. 1Kitasato University, Kanagawa, Japan, 2Kanagawa Rehabilitation Hospital, Kanagawa, Japan
[Introduction] High density lipoprotein (HDL) in plasma has critical roles in the process that cholesterol in peripheral tissues was transported to liver , which is called reverse cholesterol transport (RCT), and are inversely related to atherosclerosis susceptibility. Apolipoprotein A-I (apo A-I), the HDL major apolipoprotein, is one of the most important proteins, including playing a critical role in RCT with binding lecithin cholesterol acyltransferase (LCAT). Recently, it has been reported that apo AI is glycated by methylglyoxal, which is generated in chronic hyperglycemia state, and formed cross-linking. The change of apo A-I conformation caused by glycation effect a change in the ability of LCAT activation. Apo A-I also has related with ATP binding cassette transporter A1 (ABCA1)-mediated cholesterol release from cells. This is an important step to transport cholesterol to liver accumulated in peripheral tissues. In this study, MRC-5, one of the fibroblast containing ABCA1, was used to clarify how effect the change of apo A-I construction by glycation on apo A-I/ ABCA1-induced cholesterol efflux from cells. [Methods] Apo A-I was purified with DEAE column from HDL obtained with ultracentrifugation. Purified apo A-I was adjusted to 1 mg/ml and incubated at 37C for 048 h with 3 mmol/l methylglyoxal. The cross-linking of glycated apo A-I was confirmed with immunoblotting. MRC-5, which is one of fibroblast, was cultured to 1105 cells/well, approximately. Normal apo A-I or glycated apo A-I was added to the medium (final concentration of 0-20 g/ml) after filter sterilization and cultured for 24 h. Cholesterol in the medium was extracted with chloroform/methanol (2/1) and that concentration was determined. [Results] When the purified apo A-I was incubated with methylglyoxal, apo A-I was confirmed to be cross-linking. Then MRC-5 was cultured with normal apo A-I or glycated apo A-I, and cholesterol in the medium was determined. In the medium added normal apo A-I, cholesterol concentration was increased depending on apo A-I concentration within 0-10 g/ml. On the other hand, cholesterol concentration in the medium added glycated apo A-I was not increased. [Discussion] When the normal apo A-I was added to MRC-5, cholesterol concentration in the medium was increased. It was supposed that apo A-I interacted with ABCA1 in MRC-5 and induced cholesterol efflux from MRC-5. In contract, glycated apo A-I induced no cholesterol efflux. It was suggested cross-linked apo A-I with glycation decrease the interaction between apo A-I and ABCA1 and thereby decrease the ABCA1-mediated cholesterol efflux from peripheral cells.
B-125 Homocysteine vis--vis lipid profile and Lp(a) as a marker of vascular disease in north Indian urban population S. Bhargava1, A. Manocha1, M. Kankra1, S. Das1, E. K. Bhargava2, L. M. Srivastava1, A. Ali3. 1Sir Ganga Ram Hospital, New Delhi, India, 2University College of Medical Sciences and associated Guru Tegh Bahadur Hospital, New Delhi, India, 3Jamia Millia Islamia University, New Delhi, India
Background: The parameters of the lipid profile - total cholesterol [TC], HDLcholesterol [HDL-C], LDL-cholesterol [LDL-C] and triglycerides [TG] - and lipoprotein (a) [Lp(a)] have been implicated in the pathogenesis of vascular disease which shows a higher prevalence in the Asian population. In recent studies, homocysteine [HCY] has also been implicated in the pathogenesis of vascular disease, but opinions differ on its importance vis--vis the conventional risk factors. In this study, we have compared the levels of HCY, the lipid profile and Lp(a) in North Indian urban patients of vascular disease. Materials and methods: TC, HDL-C, LDL-C, TG, Lp(a) and HCY were estimated in the serum of 300 patients of vascular disease [confirmed by angiography, CAT scan and Duplex Sonography] and 200 controls. HCY was estimated quantitatively by ELISA [chemiluminescence]. All the parameters of lipid profile and Lp(a) were estimated by immunoturbidimetry on random access fully automated chemistry analysers. The 300 patients included 100 patients of coronary artery disease [CAD], 100 of cerebrovascular disease [CVD] and 100 of peripheral vascular disease [PVD]. ANOVA and posthoc test were used for statistical analysis. Results: The mean levels of TC [182.51+2.26 mg/dL], LDL-C [111.69+1.96 mg/dL], TG [146.38+5.15 mg/dL], Lp(a) [27.89+1.43 mg/dL] and HCY [10.71+0.22] in the controls were all in the high normal range, corroborating evidence that people of Asian origin have a higher risk for vascular disease. Amongst the lipid profile parameters, only TG was significantly higher [p<0.05] in CAD [208.13+15.45] and CVD [206.11+16.63] as compared to the controls, while Lp(a) [29.60+2.75 mg/dL in CAD, 28.93+1.99 mg/dL in CVD, and 28.27+2.14 mg/dL in PVD] was not significantly different from that in the controls. HCY was significantly higher [p<0.0001] in all three disease groups [CAD=20.77+1.89, CVD=27.84+2.75, PVD=28.41+2.81]. Also, the mean HCY concentrations in CAD patients was significantly lower than that in CVD and PVD patients (p<0.05 and p<0.02, respectively). Conclusions: Amongst the parameters of the lipid profile and Lp(a), only TG is a significant marker and that, too, only in CAD and CVD. On the other hand HCY emerges as a marker of much greater significance in all types of vascular disease, even more so in CVD and PVD. Thus, it is recommended that HCY be estimated along with other conventional risk factors, especially since it is easily modifiable by the administration of water-soluble vitamins of the B-group.
B-127 The Association of Serum Small Dense Low Density Lipoprotein (sdLDL) ,ApolipoproteinB and ApolipoproteinB/ApolipoproteinA1 Ratio with Coronary Artery Stenosis L. Hosseini Gohari1, R. Karimzadeh Gassab2, M. Firoozray3, A. Zavarei4, H. Bassiri4. 1Iran University of Medical sciences Faculty of Allied Medicine& Cellular and Molecular Center, Tehran, Iran, Islamic Republic of, 2Iran University of Medical Sciences, Faculty of Medicine , Biochemistry Depatment, Tehran, Iran, Islamic Republic of, 3Iran University of Medical Sciences ,Faculty of Medicine,Biochemistry Department, Tehran, Iran, Islamic Republic of, 4Shahid Rajaii Heart Center, Tehran, Iran, Islamic Republic of
Background:Recently, small dense low density lipoprotein(sdLDL) has been highlighted as a new risk factor for the coronary artery disease(CAD).Small dense LDL are believed to be atherogenic since these particles are taken up more easily by arterial wall.They are readily oxidized and have reduced affinity for LDL receptor and increased affinity for arterial proteoglicans. Apolipoprotein B(apoB) and apolipoprotein A1(apoA1) are the major proteins of atherogenic lipoproteins and high density lipoproteins(HDL),respectively. LDL cholesetrol is only a measure of the cholesterol level in the LDL whereas apolipoprotein B(apo B) is a measure of the cholesterol levels of all the atherogenic particles,including very low density lipoprotein ,intermediate density,and low density lipoproteins.Therefore,it might be a better marker than other lipids. The aim of the present study was to evaluate the association of serum small dense LDL , apoB,apoA1 and apoB/apoA1 ratio with the coronary artery stenosis. Methods: 86 patients with coronary stenosis , 35 patients without coronary stenosis
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identified by angiography , and 30 healthy individuals were studied. SdLDL was measured by a direct homogenous LDL-C assay in the supernatant of serum which remained after heparin-magnesium precipitation. Serum apolipoprotein A1 and apolipoprotein B were measured by using immunoturbidimetric method . The levels of plasma total cholesterol and triglyceride were determined using enzymatic method.HDL and LDL cholesterol were measured directly with HDL-C and LDL-C diagnostic kits . All values are expressed as the meanSD.One-way analysis of variance (ANOVA) was used to compare mean values among groups.A statistically significant difference was defined as p<0.05.Linear regression analysis and Pearson correlation coefficient were used to access correlation between two parameters. Results: The results showed that the sdLDL levels were higher in patients with coronary stenosis than patients without coronary stenosis and healthy individuals(21.547.1, 16.884.4 and 15.455mg/dl, p=0.001, respectively). In addition the level of apoB and apoB/apoA1 ratio were significantly higher in patients with coronary stenosis than patients without stenosis and healthy individuals (113.7121.8 mg/dl and 1.1000.24, 100.8818.7 mg/dl and 0.5890.26, 102.309.6 mg/dl and 0.7510.16, p=0.001, respectively).SdLDL levels were positively correlated with the level of apoB (r=0.589), apoB/apoA1 ratio(r=0.416), triglyceride (r=0.494) ,LDL-C(r=0.749), total cholesterol (r=0.354) and were inversely correlated with the level of HDL-C(r = -0.586)(p<0.01). Conclusion:The elevated levels of small dense LDL,apoB and apoB/apoA1 ratio were associated with coronary artery stenosis.Therefore, determination of serum sdLDL for the prognosis of CAD is recommended.
Lipids/Lipoproteins
calculated LDL (cLDL) cholesterol and elevated C-reactive protein (CRP). Forty one percent of patients in the Jupiter trial had the metabolic syndrome and the median body mass index was 28.3, indicating that greater than 50% of subjects enrolled were overweight or obese. CRP concentration is known to be increased in obese patients and in patients with the metabolic syndrome. It is also this group of patients where discordant results have been observed between cLDL cholesterol and LDL particle concentration or apolipoprotein B (apoB). Objective We investigated whether patients with an elevated CRP (>2.0 mg/L) and normal cLDL cholesterol (<130 mg/ dL) exhibited elevated LDL particle concentrations. Method Fasting EDTA plasma samples were analyzed for traditional lipids, CRP, and LDL particle concentration in 277 consecutive patients undergoing lipoprotein analysis in our laboratory. Mean patient age was 57 years (range, 19-88 years). CRP (high sensitive assay kit), total cholesterol, and triglyceride levels were measured on a Roche Cobas c501 using the Roche reagents and Roche CFAS lipids calibrator. HDL cholesterol was measured using dextran sulfate/calcium chloride precipitation and subsequent analysis for cholesterol as described above. cLDL cholesterol values were calculated using the Friedewald equation. LDL particle concentrations were analyzed in our lab utilizing the LipoScience NMR LipoProfile method. Results Of the 276 samples with calculable LDL cholesterol, 74 (27%) exhibited cLDL cholesterol <130 mg/dL and CRP > 2.0 mg/L, while 113 (41%) had cLDL cholesterol <130 mg/dL and CRP < 2.0 mg/L. Forty-six percent of patients with normal cLDL cholesterol and increased CRP showed increased LDL particle concentrations (>1300 nmol/L) and 16% of that same population had LDL particle concentrations >1600 nmol/L, as compared with 28% and 5%, respectively, in the subset with cLDL cholesterol <130 mg/dL and CRP < 2.0 mg/L. There was no correlation between CRP concentration and cLDL cholesterol (r = 0.022) in patients with cLDL cholesterol < 130 mg/dL (n = 197), but in this group a significant (P < 0.05), although weak positive correlation between CRP level and LDL particle concentration was observed (r= 0.288). Conclusion Elevated CRP in patients with normal cLDL cholesterol, at least in part, identifies patients with elevated LDL particle concentration. This effect may explain some of the benefits observed in response to statins in the Jupiter trial and could be confirmed by performing similar LDL discordance analyses in the Jupiter population.
B-128 Elevation of osteopontin is associated with smooth muscle phenotype changes in thoracic ascending aortic aneurysm R. Majumdar, L. Savastano, J. P. McConnell, T. M. Sundt. Mayo Clinic and Foundation, Rochester, MN
OBJECTIVE: Thoracic ascending aortic aneurysm (TAA) is a highly lethal condition, the pathobiology of which remains poorly understood. Following DNAmicroarray analysis, we showed that, among several genes, OPN (osteopontin) consistently highly over expressed in aneurysms associated with the tricuspid aortic valve (TAV). Furthermore, over expression of OPN is correlated with the TAA showing moderate to severe idiopathic cystic medial degeneration (CMD) histologically and is independent of hypertension, valve pathology or gender of the patients. The over expression of OPN in TAA might be associated with phenotypic changes in vascular smooth muscle cells (vSMC) leading to dilatation of the aorta. The purpose of the investigation was to determine whether OPN over expression correlates with vSMC phenotype changes in human TAA. METHODS: Patterns of expression of the selected genes OPN, MHC-SM2 (smooth muscle myosin heavy chain -type2, marker for contractile form of vSMC), MMP9 and MMP2 (matrix metalloproteinases) were assessed by real time RT-PCR, Western blotting and gel zymography from human aneurismal aortas with CMD (n = 6) and control (nonaneurysmal) aortas (n = 6). RESULTS: Real time RT-PCR analysis showed OPN mRNA level was significantly increased (21.74 8.62 vs. 1.64 0.88, p<0.002), whereas MHC-SM2 mRNA level was significantly decreased (1.98 0.83 vs. 10.60 5.36, p<0.002) in TAA samples as compared with control samples. This was confirmed by Western blotting, which demonstrated an increase in OPN protein whereas there was substantial decrease in MHC-SM2 protein in TAA compared to control samples. Furthermore, while there is significant over expression of MMP9 at the mRNA level (41.34 43.37 vs. 1.76 1.11, p<0.002) accompanied by increase in enzyme activities in TAA vs. the controls, there was no difference in MMP2 mRNAs (3.22 4.57 vs. 1.76 0.36, P = 0.930) or enzyme activities. CONCLUSIONS: Over expression of OPN has an inverse relationship with the MHC-SM2 in the formation of TAA. Since OPN expression is an early event in aneurysm formation, OPN and MMP9 may be directly involved in the SMCs phenotype changes that lead to decreased vessel elastic recoil, reduced wall strength, and eventual dilatation.
B-130 Fenofibrate treatment up-regulates BCL6 and reduces serum VCAM levels A. K. Tsai1, D. K. Arnett2, M. Y. Tsai1. 1University of Minnesota, Minneapolis, MN, 2University of Alabama, Birmingham, AL
Background: Proinflammatory cytokines are thought to play important roles in the progression of atherosclerosis. They have been previously implicated in plaque formation, stability, and rupture as well as in coronary artery disease (CAD). The cytokines are known to up-regulate several vascular adhesion molecules such as soluble vascular cell adhesion molecule 1 (sVCAM-1). Fenofibrate is widely used for the management of combined hyperlipedemia. Its pharmacologically active metabolite fenofibric acid is a potent agonist for the nuclear transcription factor peroxisome proliferator-activated receptor- (PPAR-). Activation of PPAR- results in increased lipolysis and plasma clearance of atherogenic triglyceride (TG) rich lipoproteins and reduced TG synthesis. Fenofibrate also has other beneficial effects such as enhancement of cholesterol efflux, and reduction of lipoprotein oxidation. PPAR- independent actions of fenofibrate have also been reported. Our laboratory has previously demonstrated that fenofibrate treatment up-regulates the expression of BCL6 in peripheral white blood cells. In vitro studies have demonstrated that BCL6 may have an anti-inflammatory effect, and thus may influence sVCAM-1 levels. While several studies have identified sVCAM-1 as a marker of atherosclerosis, only limited data is available on the effect of lipid-lowering drugs on sVCAM-1 levels. Methods: In the current study, we selected 95 participants with hypertriglyceridemia (baseline plasma triglyceride levels >200 mg/dL) from the Genetics of Lipid Lowering Drugs and Diet Network (GOLDN) study, and compared their plasma sVCAM-1, triglyceride, LDL-cholesterol (LDL-C) and HDL-C levels before and after treatment with 3-week open-label fenofibrate (160 mg/d). Plasma triglyceride (TG), HDL-C, and LDL-C were measured on the Roche/Hitachi 911 Automatic Analyzer (Roche, NJ). sVCAM-1 was measured using an ELISA assay (Quantikine, distributed by R&D Systems). The analytical coefficients of variation were 2.6%, 3.9%, 1.8%, 6.2% for TG, HDL-C, LDL-C, and sVCAM-1 respectively. Data that were nonGaussian in distribution (triglyceride) were log-transformed. Between-group differences were examined by paired t tests. Single-factor ANOVA was used for multiple comparisons. Significance was defined as p<0.05. Results: After treatment, plasma levels of sVCAM-1 and triglyceride decreased significantly (p<0.03 & p<0.001 respectively), and HDL-C levels increased significantly (p<0.001). While there was a significant decrease in the sVCAM-1
B-129 Elevated C-Reactive Protein is Associated with Elevated LDL Particle Concentration in Patients with Normal Calculated LDL Cholesterol J. P. McConnell, S. J. Hartman, J. M. Hornseth, A. S. Jaffe. Mayo Clinic, Rochester, MN
Background The recently published Jupiter trial showed that statin therapy reduced the number of cardiovascular events in an apparently healthy population with normal
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concentrations across all quartiles, patients with higher baseline sVCAM-1 showed a more dramatic decrease. Across the quartiles of baseline TG, sVCAM-1 showed a trend towards significance (p<0.08). Overall, neither baseline HDL-C nor LDL-C was associated with a change in sVCAM-1. Similarly, no associations with changes in sVCAM-1 levels were identified between reduction in TG, reduction in LDL-C, or increase in HDL-C. Conclusion: Based on these results, we conclude that fenofibrate treatment decreases the level of sVCAM-1. These findings are consistent with our previous studies that show that treatment with fenofibrate increases the expression of BCL6. The increase in BCL-6 may in turn reduce the expression of sVCAM-1 and other markers of inflammation and oxidation.

Methods: Protein fractions of whole serum were isolated by Fast Liquid Protein Chromatography (FPLC) using a superpose 6 column resulting in sixty-six 0.5 ml fractions. Each fraction (50l) was incubated with 25l (320mM) of an oxygen radical generator (2,2-azobis(2-amidinopropane) dihydrochloride (AAPH) (WAKO)), 50l (8.16 x 10-5 mM) fluorescein disodium (Sigma), and 100l PBS. Fluorescein was gradually quenched by AAPH and measured every minute for 120 min (fluorescent plate reader (Biotek)). The loss of fluorescence correlates to the net amount of oxidation and extent of damage that occurs due to competition between AAPH and the sample examined. A calibration standard curve was established for each run using increasing Trolox (Sigma) concentrations (6.25M, 12.5 M, 25M, 50M, 100M). HDL isolated by ultracentrifugation and oxidized by cupper was used as a negative control. The net area under curve was graphed and the samples AOC was calculated using the regression equation between Trolox concentration and the net AUC expressed as micromole Trolox equivalents per liter sample (mol/L). The Trolox concentration and cholesterol content of each FPLC fraction was superimposed on the FPLC chromatograph to identify fractions corresponding to HDL. The AOC specific to HDL was calculated by only including fractions corresponding to the HDL particle. All samples were kept frozen at -80 C before being run for the ORAC assay and were run within the first 24 hours of collection. General chemistry test profile was in addition performed for all samples. Results: The intraday and interday coefficient of variation in 10 samples examined over 5 days in triplicates was <10% with SD of 2.24. The AOC of HDL isolated by ultracentrifugation was 6.2M compared with 5.9M and 1.6M for HDL oxidized with cupper for 2 and 6 hours, respectively. The median and interquartile range of the total AOC of whole serum was 785.2M (649.2M -1011.5M) as compared with the HDL specific AOC of 105.1M (72.8M -125.1M). The average HDL AOC of whole serum was 13.1% ranging from 4.5% to 21.7%. There was no linear relationship between the AOC of HDL and HDL-C levels (r2=0.01; p=0.56), albumin levels (r2=0.03; p=0.39), or total protein in whole serum (r2=0.06; p=0.25). Conclusions: We found no correlation between HDL-C levels and the AOC associated with the HDL particle, suggesting that the cholesterol concentration of the HDL particle does not reflect the function of the particle. The ORAC method is reliable and quick for assessing HDL specific AOC in whole serum.
B-131 Comparative study of Serum and Biliary Lipid Profile of Libyan Gallstone patients J. R. Peela1, A. M. Jarari1, S. E. Saiety2, T. N. Patil3. 1Department of Biochemistry,Faculty of Medicine,Al-Arab Medical University, Benghazi, Libyan Arab Jamahiriya, 2Department of Surgery,7th October Hospital,AlArab Medical University, Benghazi, Libyan Arab Jamahiriya, 3Al-Arab Medical University, Benghazi, Libyan Arab Jamahiriya
The prevalence of cholesterol gallstones in Libya is very high depending on dietary patterns and ethnic background. It is now widely accepted that the primary event in the pathogenesis of cholesterol gallstones is an altered lipid metabolism giving rise to a greater proportion of cholesterol relative to other bile lipids being secreted from the liver into bile. An abnormality in lipid metabolism may arise from a combination of a number of different factors such as excess dietary cholesterol/fat, obesity, diabetes and genetic factors. The association of cholesterol super saturation of bile with cholesterol gallstones paved the way to a physical-chemical basis for gallstone formation. It however, soon became clear that other factors including nucleation of cholesterol crystal, binding together of these crystals with mucin, and hypomotility of the gallbladder played an equally important role in gallstone formation. In the present study bile and serum retrieved from 46 patients of Cholelithiasis were collected during cholecystectomy at local hospital i.e., 7th October Hospital of AlArab Medical University, Benghazi, Libya. In all the 46 bile collected from 6males and 40 females with a male to female ratio of 1:7.67. Between age groups 19 to 88, peak incidence of age is at 40. Serum and biliary lipid profile was done by authentic enzymatic methods. Serum lipid profile was done for 19 healthy controls of both sexes between 25 to 55. Results had shown there no statistical significance of serum and biliary triglycerides. There is significant raise of both serum HDL and LDL when compared with controls ( p < 0.01). Biliary cholesterol ( p< 0.01), HDL( p < 0.01) and LDL( p < 0.01) were raised significantly when compared with serum samples. In males, only biliary total cholesterol (p < 0.05) and HDL ( p < 0.001) cholesterol were showing significance. In female gallstone patients of 40 years and below were showing highly significant levels of HDL( p < 0.001) and LDL( p < 0.001) in serum and total cholesterol( p < 0.001) ,HDL( p < 0.001) and LDL( p < 0.001) in bile. Serum HDL( p < 0.001) , Biliary cholesterol( p < 0.001) and HDL( 0.001) were showing highly statistical significance in females above 40 years where as biliary LDL( p < 0.05) was showing little significance in this group. As per the present study there is correlation of serum and biliary lipid profiles in causing gallstones in Libyan population. Both serum and biliary HDL is consistently showing higher values Libyan patients unlike other studies.
B-132 A Modified Oxygen Radical Absorbance Capacity Method for the Assessment of the Anti-oxidant Capacity of High Density Lipoproteins A. A. Sethi, M. Sampson, E. Vazquez, C. Pham, A. T. Remaley. National Institutes of Health, Bethesda, MD
Objective: No reliable, reproducible or high-throughput method is available to asses the antioxidant capacity of High Density Lipoproteins (HDL) in human serum. The oxygen radical absorbance capacity (ORAC) has previously been used to asses the antioxidant activity in foods. We evaluated the ORAC method and hypothesized that the Anti-Oxidative Capacity (AOC) of the HDL particle measured by ORAC does not reflect the HDL-C levels. Design: Serum from 25 patients at the National Institutes of Health was randomly selected based on their HDL-C levels (median: 64 mg/dL; range 14 mg/dL-204 mg/dL). Selection was random and blinded to patient diagnosis.
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interactive difference plots and a novel procedure to optimize the diagnostic performance by adjusting reference values. The Clark Error (EG) grid and Consensus Error grid (CE) are applied to the verification of POCT glucose measurement procedures. The FDA requires error grids with 360 observations for CLIA waiver applications. Provided limits are defined, the program can be used for other analytes (CLSI EP27). Software. The EXCEL workbook contains four worksheets. The input sheet allows duplicates, useful in estimating an uncertainty profile. The error grid is defined in another worksheet; the Allowable Total Error and the Limits for Erroneous Results and up to twelve lines delineating special areas. For each tier, third or tertile of data the ordinal linear regression, principal and Deming regressions. The correlation coefficients are calculated and regression lines optionally displayed. Deviating results from the equal line or regression lines can be counted. The Bland-Altman graph mirrors the partitioning of the data. The tolerance limits are calculated. Four situations are identified in the evaluation of reference intervals. In each case the sensitivity and specificity and likelihood ratio are calculated and prevalence-sensitive indicators e.g. post-test probability. The Goal seeking function facilitates appraisal of the reference interval. Results. In this study only single measurements were made. The tertiles contain a third each of the observations and the thirds the results in each third of the measuring interval, in this case 90, 8.9 and 1.1 % in the low, mid and high thirds, respectively. This is an unfavorable distribution of results and the tertiles would cut off the dataset at 4.8 and 5.3 mmol/L, also unfavorable. Therefore the tiers up 5 mmol/ L and from 6,5 mmol/L were chosen to better represent hypo- and hyper- and normoglycemia which gives 168, 157 and 34 results in each tier, sufficient for a regression analysis. The OLR gives the slopes and (intercepts) 0.95 (0.14), 0.93 (0.54), 0.94 (0.50), 0.64 (2.09) and 0.97 (0.21); with =10 the Deming characteristics were 1.05 (0.0), 0.91 (0.64), 0.94 (0.07) and 0.99 (0.17) of the total, low, mid and high partitions, respectively. If the ATE is defined as a 20 % deviation from the equal line it includes 94 % and if 10 %, 71 % of the observations. In the EG, seven results were in the observation Zone C whereas in the CG only one result was found in the corresponding zone. To optimize the diagnostic agreement between methods the reference interval was changed to 5.3-6.2 mmol/L. This may also depend on the use of the new procedure and diagnostic needs. Conclusion. An interactive regression analysis adopting the principles of the error grid and a feature to optimize reference intervals improves the evaluation and understanding of the performance of new instruments also for clinical risk assessment.
Tuesday PM, July 21

Poster Session: 2:00 pm - 4:30 pm Point-of-Care Testing
B-133 A Comparison of the DCA Vantage Analyzer and Afinion AS100 Analyzer Using HbA1c Reagent J. Cox, E. Cowden, G. Krauth, J. Li, D. Ledden. Siemens Healthcare Diagnostics Inc., Norwood, MA
The DCA Vantage Analyzer from Siemens Healthcare Diagnostics is a quantitative, point-of-care (POC) immunoassay analyzer for use in patients with diabetes. It offers two parameters: an HbA1c test for glycemic control in whole blood and a microalbumin/creatinine ratio test for early detection of kidney disease. The DCA Vantage Analyzer is the successor of the DCA 2000+ Analyzer. The Axis-Shield Afinion AS100 Analyzer is also a quantitative POC instrument that uses boronate affinity to test the percentage of HbA1c in whole blood. A study was conducted comparing the HbA1c tests of the DCA Vantage Analyzer and the Afinion Analyzer on commercially available controls and patient samples. The precision study obtained acceptable total precision (CV) for both analyzers: 2.65% for the Afinion and 3.62% for the DCA Vantage Analyzers. Out of 117 Afinion cartridges tested, 39 failed to provide results due to system errors. A correlation study comparing both analyzers to the Tosoh analyzer as a reference method found an Afinion median percent bias to reference of -3.6% (1.4% CV); that of the DCA Vantage Analyzer was 0.3% (1.5% CV). Linearity testing showed 91.2% mean percent recovery for the Afinion and 94.6% for the DCA Vantage Analyzers. Testing commercial controls was unsuccessful on the Afinion system; values could be generated only by using the recommended Afinion proprietary controls. Scipac, Canterbury, BioRad, and Bionostics controls all yielded results on the DCA Vantage Analyzer, with an overall CV of 2.6%. Sample time in the capillary was not an issue for either instrument, for samples treated with anticoagulants. When removing reagent cartridges from the protective foil packaging for different intervals of time up to 60 minutes, the Afinion and DCA Vantage Analyzers had comparable total CVs: 1.3% and 3.7%, respectively. When stored at 2C-8C, Afinion cartridges could produce no results until warmed for 20 minutes. The Afinion package insert states that cartridges should be warmed for at least 15 minutes before use. The DCA package insert advises the same warm-up period following storage at 2C-8C. DCA cartridges were able to produce results after 15 minutes. Lot-to-lot variability was 2.2% for the Afinion and 3.6% for the DCA Vantage Analyzers. Uric acid interference was tested at 5.0 and 10 mg/dL. The Afinion Analyzer had 97.7% and 94.2% recoveries, respectively; the DCA Vantage Analyzer had 96.0% and 96.3% recoveries, respectively. The data show that both systems have acceptable performance for total precision, reproducibility, linearity and comparison to reference method. The Afinion system exhibited an error rate of 11%. The Afinion Analyzer, with its sample limitations, cannot process commercial controls and may generate errors with proficiency samples that are not compatible. In addition, the Afinion controls recommended for use are difficult to resuspend. The DCA Vantage Analyzer is a more flexible system with many more options, such as flexibility with commercial controls and the ability to adjust slope and intercept, which allows correlation to reference methods; and a large capacity for onboard data storage.
B-135 Verification procedures of concentration measurements POCT instruments for glucose
O. V. Chernichuk1, L. A. Khorovskaya2, V. L. Emanuel2. 1KhantyMansyjsk Clinical Regional hospital, Khanty-Mansyjsk, Russian Federation, 2Pavlov's State Medical University, St.-Petersburg, Russian Federation
Objective. There is a wide variety of POCT instruments for B-glucose concentration measurements on the market. Measurement procedures should be robust enough to allow patients and non-laboratory staff to use the instruments. Manufacturers do a thorough verification of the instruments and laboratories verify and compare alternate instruments in a purchasing process and before commissioning the instruments. Important criteria includes verifying within- and between instrument variations. Also, pre- and post-analytical aspects are important e.g. operator skills and computer connectivity. Users need to verify that they can reach the specifications declared by manufactures. There are different statistical approaches to estimate the performance of measuring systems. In this report we use a protocol from CLSI EP15 and an interinstrument split-sample technique. Pre-and post analytical sources of variation are not considered. Method. Six different glucometers from the same manufacturer (Johnson and Johnson, USA) were compared. The CLSI 15 protocol was expanded to include five measurements in each series. One series was measured each day for five days. The instruments were calibrated according to the manufacturers instructions. One batch of reagents were used for each instrument. Reference material of two concentrations from Bio- Rad was used. Inter-instrument variation was estimated by split sample. Data from the EP15 design were used; thus, data of the fourth day was transferred to a two-point regression study that also allows estimation of the withinand between laboratory variances and the combined uncertainty Results. The outcome can be interpreted in different ways. On a group level, Students dependent ttest showed that there was no difference between the claimed and found repeatability and reproducibility except for reproducibility of low concentrations where the instruments performed better than specified. For individual instruments, the information from manufacturers (performance interval) did not allow a direct comparison. Inter-instrument comparison demonstrated a combined uncertainty of 3,9% for low and 2,1% for high concentrations. Conclusion. A simplified and
B-134 Comparison of measurement performance using an interactive procedure and application of Clark and Consensus Error Grids L. A. Khorovskaya1, O. V. Chernichuk2, A. Kallner3. 1Pavlov's State medical University, St Petersburg, Russian Federation, 2Khanty-Mansiysk Regional Clinical Hospital, Khanty-Mansiysk, Russian Federation, 3Karolinska university hospital, StockholmSt Petersburg, Sweden
Background. New measurement procedures are often compared with previous in verification procedures aiming at establishing the uncertainty in terms of precision and trueness (CLSI EP15). We describe an EXCEL application that allows establishing an error grid, continuous changes of partitioning for analysis of data,
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recognized verification procedure for measuring systems was shown to also be applicable for POCT measurement of glucose. An inter-instrument comparison procedure indicated that a harmonization of measurements might improve the monitoring and treatment of diabetes mellitus.

the first set of POC replicates was compared to the values from the RL. The absolute difference between replicates was =/> .3 INR units in 6/125 (4.8%), 21/180 (11.67%) and 13/34 (38.2%) of patients in groups 1, 2 and 3 respectively. Chi-square statistics with Bonferroni correction indicated that all pair wise comparisons were statistically significant (p<.005). Regression of the POC value (Y) on the RL value (X) using Table Curve 2D software (Systat; San Jose, CA) revealed a best fit relationship given by: Y = -6.14 + 6.48 exp (.12 X) [r-sq = .868] Expansion of the exponential term in a Taylor series around X = 0 suggests a linear relationship between X and Y at smaller values of INR becoming increasingly nonlinear as INR increased. Based on the regression estimate a POC value of 4.0 would correspond to an RL value of 3.8. The latter value is modestly elevated relative to the upper limit of the therapeutic range for INR at EBNHC and BMC (3.5). On the basis of this data the INR threshold for CL confirmation was set at 4.0 instead of what appeared to be an arbitrarily established threshold of 5.0. This was felt to give the clinician additional information to assess possible modification of therapy while resulting in only a modest increase in the number of specimens tested in the CL (less than 5 per week). The collected data suggested increasing imprecision of POC data and an increasingly non-linear relationship between POC and RL values with increasing INR. Derivation of a threshold INR value for confirming POC results should be based on data analysis and not defined arbitrarily.
B-136 When is a Point-of-Care Test (POCT) really a POCT? C. Schromm1, L. Casey2, D. Lantz3, E. Pearlman4. 1Codman Square Health Center (CS), Boston, MA, 2Dorchester House Multiservice Center (DH), Boston, MA, 3East Boston Neighborhood Helath Center (EB), Boston, MA, 4Boston Medical Center, Boston, MA
In January 2008 multiple providers at EB claimed an increased frequency of negative group A streptococcus (GAS) rapid antigen tests (RATs) followed by GAS (+) throat cultures. This occurred when a recommendation from the Infectious Disease Society of America (IDSA) to eliminate reflex cultures from patients =/> 16 years old with negative RATs was under consideration. As part of the inquiry into the EB complaint the authors compared RAT performance at three community health centers (CHCs). Sensitivity was defined as: [(# of positive RATs)] / [(# positive RATs) + (# of GAS positive throat culture specimens)] This definition assumed that every throat culture positive for GAS was associated with a preceding negative RAT. The numbers generated would therefore underestimate true sensitivity. The lab managers at the three CHCs however, thought that this assumption was close to the truth and as the same definition was used at all three CHCs the resulting data would remain informative. A sensitivity of .926 was suggested by the manufacturer (Genzyme; Framingham, MA) in the package insert. In November of 2006 prior to any provider complaints, sensitivity at EB was .74 compared to a composite value of .926 at CS/DH. In the last quarter of 2007 the sensitivity at EB was .675 compared to .921 at CS/DH for the calendar year 2007 [z score = 20.09; p<.001]. In January, 2008 (when the complaints were received) the sensitivity was .70 at EB and .917 at CS/DH. RAT was standardized at CS and DH with both CHCs using transport media from a common source (Fisher Healthcare; Ontario, Canada). EB however used transport media from another vendor [BectonDickinson (BD); Sparks, MD]. In February the RAT manufacturer claimed that BD transport medium had been reformulated [an allegation denied by BD] and was no longer compatible with its RAT. EB switched to a dry swab transport system in March, 2008 with some improvement in sensitivity to .78 in April, 2008 compared to .929 at CS/DH [z score = 6.84; p<.001]. In May, 2008 it was decided to retrain staff at EB and standardize according to the CS/DH protocol. Simultaneously IDSA recommendations were implemented at CS/DH. Sensitivity data at EB in June (.70) and July (.69) remained disappointing. In attempting to explain this continuing performance discrepancy it was noted that the RAT at CS/DH was performed in the central clinical laboratory by lab personnel but at EB was used as a true CLIA waived POCT and performed by individuals without clinical laboratory training. The authors and eventually the RAT manufacturer concluded that the continuing poor sensitivity at EB was technique driven. Review of the package insert revealed that the RAT involved 11 steps two of which were timed. Although the data is retrospective and the conclusions can only be viewed as tentative, disturbing questions are raised about the meaning of waived testing under actual field conditions.
B-138 Evaluations of the Clinical Application of the CoaguChek-XS INR Detection Analyser Z. Zhang, J. Yang. Wuhan Asia Heart Hospital, Wuhan, China
Objective: Comparison of the INR results of relation, bias and consistency by the two automation analysis systems, we evaluated the application value of the Roche CoaguChek XS system(POCT). Methods: We tested INR results of 169 patients who accept oral anticoagulant therapy by Roche CoaguChek XS analysis system and the Beckmam ACLTOP analysis system. We handle the results with correlated regression analysis to evaluate the relation and regression coefficient. We observe the bias extent of the tested results with the Bland-Altman analysis. The results analyzed by subgroup T test. Results: The relation of the CoaguChek XS system and the ACL TOP system is good(r=0.9539 y=1.0748X-0.1452) .The mean bias of the two metheds is -0.06. The results of the CoaguChek XS system are slightly larger than that of the ACL TOP system. When INR>3.51, the results of the CoaguChek XS system are significiantly larger than that of the ACL TOP system, and there has significiant difference between the two group results (P<0.05). When INR<3.50, the two results has no significiant difference (P>0.05). Conclusion: The CoaguChek XS INR detection analyser (POCT) can completely monitor INR of the patients who accept oral anticoagulant therapy,we can get the accurate result quickly ,and the operation is simple and convenient.
B-139 Performance and practicability of a new ambulatory blood glucose monitoring system (Accu-Chek Mobile) under everyday conditions in diabetic subjects B. M. Gambke1, M. Brndle2, N. Hermanns3, U. Keller4, B. Teupe5. 1Roche Diagnostics, Design Verification and Validation, Mannheim, Germany, 2Kantonsspital, St. Gallen, Switzerland, 3Forschungsinstitut der Diabetesklinik, Bad Mergentheim, Germany, 4Universittsspital, Basel, Switzerland, 5Diabetes-Dorf Althausen, Bad Mergentheim, Germany
Objective/Relevance: The Accu-Chek Mobile system uses a new concept providing the reagent on a tape in a cassette. Objectives of the study were an examination of the relative accuracy using capillary blood, reproducibility with control solutions, and an evaluation under everyday conditions. Methodology: Intended users were asked to use the Accu-Chek Mobile system in parallel to their own system. They had a minimum of 20 days to complete daily controls and 50 glucose measurements from the same pricking using both systems. Experiences with Accu-Chek Mobile system were assessed by a questionnaire. People rated statements on a scale from 1 strongly agree to 6 strongly disagree. Data were recorded by the participants themselves. Results: 46 subjects with diabetes type 1 or 2, 29 from Germany, 17 from Switzerland, age 19 to 71 years, with a broad range of experiences in the use of blood glucose self monitoring, agreed to test the Accu-Chek Mobile system. Pooled measurements with control solutions yielded a SD of 6 mg/dl for the low control and
B-137 When Should a Point-of-Care (POC) INR be Confirmed? D. Lantz1, E. Pearlman2. 1East Boston Neighborhood Helath Center, East Boston, MA, 2Boston Medical Center, Boston, MA
In November, 2006 the EBNHC introduced POC INR testing in its coumadin clinic. At that time a decision was made to confirm POC INR values =/> 5.0 at a central laboratory facility. At a Joint Commission laboratory inspection in June 2008, the surveyor thought the value of 5.0 was too high and noted that no explanation was given for this particular choice of threshold. This led the new laboratory medical director (ESP) to review the original validation data. Specimens from 339 patients were tested on two POC instruments (Coagacheck; Roche Diagnostics: Indianapolis, IN). Citrated plasma specimens (3.8 percent) from 254 patients were also sent to a referral laboratory [RL] (Quest Laboratories; Cambridge MA) where INR was determined on a CA-7000 (Siemens; Deerfield IL) using thromboplastin reagent with ISI near 1.0. The 339 replicates were divided into three groups depending on the mean POC INR with 125, 180 and 34 patients in Groups 1 (INR <2.0), 2 (INR between 2.0 and 3.5) and 3 (INR >3.5) respectively. For purposes of correlation the INR values in
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CVs of 7.5% and 6.0% for the high controls. The Accu-Chek Mobile system is calibrated to provide IFCC-plasma-like glucose values. Simultaneously measured blood glucose values (Accu-Chek Mobile and comparison system) were converted to plasma-like values if appropriate. Regression analysis for several systems can be found in the table below. Data from the questionnaire showed high acceptance with ratings between 1 and 3 in the range from 84.4% to 100%. Data of the regression analysis according to Passing/Bablok for the comparison of the Accu-Chek Mobile system with Roche, Bayer, Lifescan, and Abbott systems N Median Comparison Plasma glucose Correlation measurement deviation systems range [mg/dL] coefficient [r] s (Y-X)/X [%] 978 33 - 486 0.949 0.377 Roche (1) 585 47 - 343 0.925 5.298 Bayer (2) 538 34 - 466 0.969 5.958 Lifescan (3) 616 34 - 384 0.922 4.293 Abbott (4) (1): Accu-Chek Aviva, Accu-Chek Compact Plus, Accu-Chek Sensor (2): Ascensia Contour, Ascensia Contour Link, Ascensia Dex 2 (3): One Touch Ultra, One Touch Ultra 2, One Touch Ultra Smart, One Touch Ultra Easy, Euroflash (4): FreeStyle, FreeStyle Mini, FreeStyle Lite, FreeStyle Freedom, Precision Xceed Conclusion: The new blood glucose monitoring system (Accu-Chek Mobile) tested in ambulatory diabetic subjects demonstrated excellent agreement to other systems on the market. Handling caused no problems and the ease of use was highly appreciated. The new Accu-Chek Mobile system thus shows very good performance and practicability in the hand of the intended users. Accu-Chek and Accu-Chek Mobile are trademarks of Roche.
B-141 A Rapid Lateral Flow Dot-Blot Assay for Broad Spectrum Bacterial Detection J. Fleming, C. Norton, M. Dykstra. GenPrime, Inc., Spokane, WA
Objective: We are developing a broad spectrum bacterial test that is inexpensive, simple to use, and requires minimal sample preparation. The device is sensitive and designed to detect a wide variety of bacteria including both gram positive and gram negative organisms. It utilizes a novel dot-blot method in which the sample is applied directly onto a lateral flow membrane. Detection is achieved when gold nanoparticles conjugated to pattern recognition peptides bind to peptidoglycan in bacterial membranes. This test is being developed for use in platelet concentrates, but should prove useful in other clinical applications where bacterial contamination tests are required. Methods: A human recombinant peptidoglycan recognition protein (rhPGRP-s) was conjugated to 40nM gold nanoparticles using a method that yields highly concentrated particles. After conjugation, the nanoparticles were coated, blocked, and stabilized with BSA. These protein-coated gold nanoparticles were used for detection of peptidoglycan on lateral flow membranes. Pure peptidoglycan was dotted onto a lateral flow membrane as a positive control. Negative controls consisted of dotting various HCl digested and non-digested proteins and glycoproteins on the membrane. The rhPGRP-s-gold nanoparticles were diluted into a blocking buffer and allowed to wick up the membrane by capillary action. A positive result was determined by the development of a visible red dot at the site of sample application. Bacterial cultures were grown in appropriate growth media and concentrations were determined by the standard plate count method. One mL samples of the bacterial cultures were centrifuged for 3 minutes at 7,500 x g and the supernatant was removed. The bacterial pellets were digested in 5M HCl, heated for one minute at 100C, and 5 uL was dotted directly onto the membrane prior to wicking with the rhPGRP-s-gold. For bacterial detection in platelets, apheresis concentrates were spiked with known concentrations of bacteria. Spiked platelets were then treated according to the following protocol. One mL samples were lysed by adding 100 uL of a 16% Triton X-100 solution. The samples were centrifuged for 3 minutes at 7,500 x g to pellet the bacteria and the supernatant was removed. The resulting pellets were digested in 5M HCL, heated for one minute, and 5 uL samples were dotted onto the membrane as before. Results: The assay was both specific and sensitive for peptidoglycan. None of the proteins evaluated as negative controls cross reacted with the PGRP-gold and the sensitivity of peptidoglycan detection was shown to be less than 2.5 ng/mL. A titration of Escherichia coli showed detection at 5 x 104 CFU/mL. Furthermore, the assay was able to detect both gram positive and gram negative organisms of clinical significance when spiked into platelets. These included gram negative aerobes such as Escherichia coli, Serratia marcescens, Klebsiella oxytoca, Enterobacter cloacae, and Salmonella enterica and gram positive aerobes, Bacillus cereus, Bacillus subtilis and Staphylococcus epidermidis. Conclusions: These results indicate that our dot blot assay can be used to detect bacteria in clinical samples such as platelet concentrates. The assay is specific, sensitive, simple and requires minimal sample preparation.
B-140 Multicenter Performance Evaluation of the Alfa S40 Clinical Analyzer: A New Physicians Office Chemistry Analyzer M. Rosenthal, T. Harrigan, A. Nair, L. Parisi. Alfa Wassermann Diagnostic Technologies, West Caldwell, NJ
INTRODUCTION: The Alfa S40 Clinical Analyzer, made by Hitachi, is a fully automated, stand alone, bench top, random access analyzer intended for use in clinical and physician office laboratories (POL). Each reagent is contained in a sealed cartridge, which also includes the reaction chamber/cuvette. A 2D barcode on each cartridge contains all ID, parameters and calibration information for each chemistry. Our goal was to evaluate the bias and precision of this analyzer in several POL labs. METHODS: We evaluated 21 different routine chemistries both in-house and in 3-4 POL sites using patient serum specimens using a protocol based on CLSI EP9A2 (Method Comparison and Bias Estimation Using Patient Samples, 2nd Edition). Each site measured these analytes in singlicate on at least 223 specimens with values distributed over the reportable range of each analyte on both the S40 and the Alfa Wassermann ACE Clinical Chemistry System (19 chemistries) or the Olympus AU640 (2 chemistries). Precision studies were conducted at the same POL sites using a protocol based on CLSI EP10-A3 (Preliminary Evaluation of Quantitative Laboratory Measurement Procedures, 3rd Edition). Three samples with increasing levels of each analyte were tested three times a day for at least five days at each lab. RESULTS: The method comparison (Deming regression) and precision range for all labs and samples (WR = within-run precision) for the 21 chemistries are shown in the table. Chem ALB ALP ALT AMY AST BIL* BUN CA CHO CK CO2 Bias Slope Correl 0.985 0.9839 0.984 0.9966 0.977 0.9986 0.943 0.9969 1.062 0.9972 0.964 0.9966 0.969 0.9940 0.957 0.9546 0.955 0.9768 1.038 0.9991 0.976 0.9595 Precision WR Total 0.6-2.5 0.8-2.8 1.5-4.4 1.5-4.9 0.6-3.7 0.7-3.8 1.0-6.4 1.0-6.6 0.8-4.8 0.9-7.0 0.0-10.4 0.0-10.4 0.7-2.5 0.9-2.5 0.4-3.9 0.7-4.3 0.3-2.0 0.7-2.0 0.5-3.7 0.8-3.7 1.3-6.6 2.0-6.6 Chem CRE CRP* GGT GLU HDL IP LD TG TP UA Bias Precision Slope Correl WR Total 1.031 0.9978 1.0-10.1 1.3-10.8 0.969 0.9962 1.0-7.5 1.5-7.5 1.014 0.9994 0.6-12.5 0.9-13.0 1.074 0.9947 1.1-2.9 1.3-3.4 0.997 0.9666 1.2-2.8 1.2-3.4 1.070 0.9874 0.9-3.4 1.2-3.4 0.970 0.9943 0.9-4.3 0.9-5.1 1.021 0.9931 1.3-3.9 1.3-3.9 0.914 0.9692 0.6-3.1 0.7-3.1 1.038 0.9754 0.3-2.2 0.7-2.3 *AU640 comparison method (x)
B-142 Evaluation of Glucose on the EPOC Point of Care Blood Analysis System J. Nichols, M. Rodriguez. Baystate Health, Springfield, MA
Objective: To evaluate the analytical performance of a new glucose sensor on the EPOC blood analysis system (Epocal Inc., Ottawa, ON, Canada) at a tertiary care health system. Methods: The EPOC blood analysis system offers a fast, 35-second biosensor analysis using a Smartcard technology that includes integrated calibration and quality checks. Signals during analysis are detected by a card reader that wirelessly transmits results to a handheld, portable PDA or other computerized data management system. Within-run precision was estimated from analysis of a heparinized, venous blood sample on two card readers conducted by staff at the hematology/oncology center and core laboratory. Precision was also estimated from reproducibility of duplicate analysis of patient samples during method correlation. The EPOC blood analysis system was compared to the i-STAT 1 analyzer (Abbott Laboratories, Abbott Park, IL). Leftover samples from laboratory analysis were used to perform duplicate analysis in a randomized order on both the EPOC and i-STAT methods according to CLSI EP9-A2 document. This study qualified for expedited review by our institutional review board.
CONCLUSION: The S-Test reagents showed minimal bias and good precision in all the laboratories.
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Results: A venous patient specimen collected from a volunteer was analyzed (N=10 12 replicates) by four laboratory staff at two locations, an outpatient hematology/ oncology center and at the inpatient hospital core laboratory, using two EPOC card readers. Within run precision varied from 2.2 - 4.4% CV at an average glucose of 40 50 mg/dL (2.22-2.78 mmol/L). Precision estimated from duplicate analysis of leftover patient specimens (N=80) varied from 1.8 - 2.1% CV over the range of 20 - 606 mg/ dL (1.11 - 33.7 mmol/L). Method comparison was performed using 4 lots of EPOC cards. Correlation statistics were estimated from the first replicate of each sample: y = 1.02X - 0.9 (standard error of the estimate, Syx = 6.7 mg/dL or 0.37 mmol/L), correlation coefficient = 0.998. Average bias at the clinical decision levels was: 45 mg/dL (-0.86 +/-1.16) 2.50 mmol/L (0.05+/-0.06) 120 mg/dL (1.44+/-0.90) 6.67 mmol/L (0.08+/-0.05) 180 mg/dL (3.28+/-0.93) 10.0 mmol/L (0.18+/-0.05) Conclusions: The glucose sensor demonstrated excellent precision and comparability of patient results to the i-STAT analyzer. No differences were noted between operators. Overall, the operators found the EPOC system easy-to-use with a technical performance that would meet patient needs. The portability, room temperature storage of test cards, and wireless connectivity offer several operational advantages over current POCT devices. An expanded menu of analytes is currently under development and will include other metabolites, cardiac markers and hemostasis.

B-144 Basic performance of fertility assays on PATHFAST K. Igami, Y. Okamura, S. Tashiro, Y. Tazaki, H. Kohta, K. Shoji, N. Arai, Y. Sugie, H. Yokoi. Mitsubishi chemical medience corporation, Yachiyoshi, Japan
Background/Purpose: There is a growing awareness of infertility and the requirement for its medical treatments are enormously expanded today. In this study, we suggest the clinical usability of rapid measurement of gonadal hormone in pointof-care testing (POCT) settings and evaluate the fertility marker panel for the quantitative determination of FSH, LH, Prolactin, Estradiol, Progesterone and Testosterone with PATHFAST, a bench top chemiluminescent enzyme immunoassay analyzer. Principles/Methods: All tests are performed with PATHFAST analyzer device with the appropriate PATHFAST reagent kits. In the PATHFAST fertility marker assays, we use all a one-step immunoassay based upon CLEIA-MAGTRATION technology. We utilize the sandwich assay for FSH, LH and Prolactin with two monoclonal antibodies recognizing specific antigens, and the competitive assays for Estradiol, with polyclonal antibody recognizing specific steroid hormone and for Progesterone and Testosterone with monoclonal antibody. Since all in one reagent cartridge is used in PATHFAST system, no water supply, drain system, washing buffer and substrate bottles are required. The assay data for all fertility markers can be obtained within 26 minutes. Both whole blood and plasma samples were tested on the PATHFAST analyzer, since PATHFAST accepts whole blood as well as plasma samples. Results: In all fertility markers, accuracy and precision within-device CVs were below 15%. Method comparison with other commercial fertility tests yielded excellent correlation. Good correlation was obtained between whole blood and plasma samples. Cross-reactivity between gonadal hormones was scarcely detected. The calibration frequency was every 4 weeks for any one lot. Conclusions: The PATHFAST fertility marker assays showed favorable basic performance. Application of whole blood samples also supports easy operation and rapid measurement. The simultaneous measurement of multiple markers in one batch (up to 6 tests) provides a set of measurements of different markers briefly. On the basis of the present evaluation, we conclude that the analytical performances and the main technical features of PATHFAST fertility marker assays can be a suitable system in various clinical sites during infertility treatment.
B-143 Assessment of point of care and laboratory capillary, venous whole blood, plasma and serum glucose analysis in patients receiving glucose self-monitoring H. Shao, A. Bao, Y. Li. Department of Clinical Laboratory, Renmin Hospital of Wuhan University, Wuhan ,Hubei, China
Objective Self-monitoring of blood glucose has been increasingly used in patients with diabetes mellitus. Both the laboratory in hospital and point of care glucose meter can meet the requirement of patients who need frequent self-monitoring of blood glucose. Although point of care glucose meter is widely used in diabetes patients, much is unknown about the accuracy of point of care glucose meter for measuring glucose in self-monitoring population, and little is elucidatory about the influence of sample source on blood glucose analysis. The aim of this study is to assess the accuracy of Roche advantageII glucose meter, explore the comparability and bias of glucose meter and laboratory measurement with hexokinase method. Also, the study intends to observe the impact of different sample source (capillary, venous whole blood, plasma and serum) on glucose analysis. Methods In 58 patients with diabetes mellitus, capillary blood was obtained from fingerstick for point of care glucose testing using Roche advantageII glucose meter, simultaneously, whole blood was drawn from ulnar vein for both point of care and laboratory measurement of glucose. Plasma and serum separated from the whole blood was tested with hexokinase method in Olympus AU2700 chemical analyzer in core laboratory. A t test was used to determine differences in whole blood glucose values between point of care meter and laboratory testing. As the plasma glucose measurement with hexokinase method was considered to be the reference method, the t test was applied to observe differences in glucose values between each sample source (capillary in point of care testing, serum in core laboratory testing) and plasma value resulted in core laboratory. Bland and Altman bias plot was used to indicate differences between glucose values resulted in point of care device and laboratory testing, and differences between glucose values of each sample type and referenced plasma testing. Results Glucose values for point of care capillary blood and laboratory plasma test did not differ significantly (P>0.05). Glucose value for point of care venous whole blood and laboratory plasma test did not show significant difference (P>0.05), though mean value of whole blood glucose from point of care meter was lower than that of laboratory plasma value. No significant difference was found in serum and plasma glucose values (P>0.05). Besides, fingerstick blood and venous whole blood did not show difference significantly in glucose value measured in the same point of care meter (P>0.05). Conclusions Glucose values for point of care samples did not differ significantly from core laboratory values. Both capillary and whole blood are suitable sample for point of care glucose self-monitoring. Whole blood, serum and plasma did not induce unacceptable bias when measuring glucose value in laboratory.
B-145 Analytical and clinical performance of the PATHFAST HCG assay K. Igami, Y. Sugie, S. Tashiro, Y. Okamura, H. Yokoi. Mitsubishi chemical medience corporation, Yachiyo-shi, Japan
Background/Purpose: Human chorionic gonadotropin (HCG) is one of the most common markers in the gynecologic health care. Its measurement is useful not only for pregnancy test and prognostication but also for diagnosis of abnormal pregnancy or trophoblastic disease, and follow-up study of the clinical treatment. In this study, we assessed the analytical performance of a wide-ranged HCG quantitative assay on the rapid and easy handling system, the PATHAFAST. We also showed clinical data from the females at different stage of pregnancy. Principles/Methods: HCG was determined with PATHFAST HCG-preg reagent kits, those reference Standards were calibrated against the World Health Organizations Forth International STD of HCG for Immunoassay, (WHO 4th IS 75/589). Functional sensitivity was studied by limiting-dilution analysis using specimens with low concentrations of HCG. Within-run and between-day imprecision were evaluated using pools of human specimens at low, mid and high concentrations. Over 100 specimens distributed across the analytical range were analyzed with two comparison methods: PATHFAST HCG (Mitsubishi Chemical Medience Corporation) and VIDASHCG (Biomerieux). Results: The PATHFAST HCG-preg has an analytical assay range from 1 to 5,000 mIU/mL. Repeatability and within-device C.Vs were ranged from 3.0% to 5.9% and 2.2% to 5.4%, respectively. The calibration frequency was every 4 weeks for any one lot. Good correlation was obtained between whole blood and plasma samples; n =48, r = 0.994, y =0.998 x + 0.192. The samples from 5,000 to 2,000,000 mIU/mL were reported as > 5,000 mIU/mL without pre-dilution. Assay correlation with other commercial HCG tests were determined: n = 106, r = 0.991, y =1.002 x - 0.364 (PATHFAST HCG-preg vs. PATHFAST HCG), n = 102, r = 0.979, y =1.015 x + 9.18 (PATHFAST HCG-preg vs. Biomerieux VIDAS HCG). The histogram analysis of the data from 250 pregnant females showed exponential increase of the HCG values
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triggered by gestation, and it elevated with increasing gestational age. The peak of mean value was at 8 to 9 weeks of gestation. Conclusions: The PATHFAST HCG-preg system was shown to have a suitable analytical performance for the rapid analysis of HCG. Because of its easy handling which is adapted to whole blood test, PATHFAST HCG-preg is considered to be useful system for the pregnancy test and the diagnosis of ectopic pregnancy or abortions in the small clinic such as physician office laboratory.
representative, proposed a protocol driven pathway which included early blood sampling, enhanced POCT and result availability for senior clinical review. It was decided to use Point of Care testing (POCT) equipment in A&E to provide core Biochemistry results in an attempt to reduce the TAT for remaining laboratory tests. Processes were changed such that all A&E patients had a venous sample taken for analysis on a Roche Omni b221 blood gas analyser. TAT data was downloaded from the pathology computer (Meditech) for the following: Receipt in laboratory to clinical availability of results Mean time of processing and % reported within one hour. The graph below shows the changes before and after introducing the changes in June 2006.
B-146 Evaluation of accuracy and precision of multiple point of care glucose meters for glucose monitoring in a medium comprehensive hospital in China A. Y. BAO. Renmin Hospital of Wuhan University, Wuhan, China
Objective: Glucose meter was widely used in hospital and home for patients selfmonitoring of blood glucose. Since rapid analysis by glucose meter is especially useful for glucose measurement in critically ill patients, multiple glucose meters were equipped in some clinical wards or ICUs of a comprehensive hospital. However, little is known about the accuracy and precision of glucose meters in clinical wards. This study aims to evaluate the accuracy and precision of different glucose meters in a comprehensive hospital, and provide specific suggestions to different clinical wards using glucose meter in order to improve the quality of point of care testing. Methods: 20 Repeated whole blood and capillary blood glucose analysis were performed in each point of care glucose meter (Roche Advantage II, J&J Lifescan, Bayer Elite XL) to obtain data of precision, expressed in coefficient of variation% (CV%). Venous whole blood samples obtained from 16 patients were measured almost simultaneously in 19 point of care glucose meters and core laboratory for glucose. A small drop of whole blood was used for glucose meter to get glucose value, and separated plasma from the same blood sample was analyzed in Olympus AU2700 chemical analyzer with referenced method (hexokinase method) for glucose value. All samples used in this study had normal Hct levels (range 36%-48%). Correlation coefficient r and lineal regression equation were obtained by SPSS13.0 software. Calculated relative bias% of each glucose meter was compared with an acceptable criteria, less than 15% of bias%, recommended by American Diabetes Association. ANOVA was used to explore the difference among three different kinds of glucose meter, a P value <0.05 was considered statistically significant. Results: All of 19 point of care glucose meters evaluated in this study had a total coefficient variance of less than 5% (range 2.8%-4.9%) for both capillary and whole blood samples. Within-run precision of glucose meters ranged from 2% to 5%. Of the 19 evaluated glucose meters, 9 (47.4%) of them completely fulfilled the acceptable criteria at 3 medically decisive levels (2.8 mmol/l, 7.0 mmol/l and 11.1 mmol/l), 3 (15.8%) of them failed to fulfill the above criteria at 3 levels, while the others (36.8%) fulfilled the criteria at one or two levels. The bias% of all glucose meters compared with laboratory values ranged from 0.34% to 25.2% (absolute value). Additionally, the glucose value among three different kinds of glucose meter did not differ significantly (P>0.05). Conclusions: Not all of the glucose meters used in clinical ward or ICU of this comprehensive hospital were able to meet the acceptable criteria of accuracy of glucose measurement. The existed positive or negative bias between point of care glucose meter and laboratory reference method may cause minor to moderate mistakes in glucose analysis, which is perilous for a doctors decision making. Measures of continuous quality improvement, including personnel training, improved internal quality control and external quality assessment, appropriate storage of testing strips, should be taken to enhance the performance of point of care glucose meter.
Discussion: Improvements in TAT were linked to the increased analysis of samples in A&E using POCT equipment for the determination of urea, sodium, potassium, ionised calcium, glucose, and lactate. Analysis of Creatinine and Troponin I was not available in A&E resulting in samples being sent to the main laboratory for these and other requested analytes. This information was presented at the daily facilitated A&E meetings for the first twelve months of the project. Conclusion: The introduction of extended POCT reduced laboratory TAT which continues to be monitored on a weekly basis as a service performance indicator and is now reported to the Pathology Board and Trust Information Management on a monthly basis.
B-148 Visual revelation of quality control outlying observations with parallel box plots. A practical example for SureStep Flexx glucose meters. V. M. Genta1, L. Wyer2, Y. Shen1, D. Graubner1, J. M. Shea3, W. Tang1. 1Sentara Virginia Beach General Hospital, Virginia Beach, VA, 2Sentara Healthcare, Norfolk, VA, 3Sentara Norfolk General Hospital, Norfolk, VA
Introduction. Recent studies indicate that a strict control of glycemia is crucial in assuring favorable outcomes for hospitalized diabetic patients. Consequently, speed and accuracy of the analytical method play an important role. While portable glucose meters assure a rapid turnaround time, the variability of reagent strips, meters and operators' technique has to be controlled. Materials and Methods. In Sentara Healthcare system the performance of 350 SureStep Flexx (Johnson and Johnson) meters used in 8 hospitals by potentially 5000 nurses with different lots of reagent strips (Johnson and Johnson) is centrally monitored by electronically collecting the observations obtained with low and high quality control material (QCM) (Johnson and Johnson) in Lifescan DataLink (Johnson and Johnson) and then transferring them to Minitab (Version 15, Minitab, Inc) statistical software. For each concentration of QCM the performance by Hospital, nurse, strip lot and QCM lot is monitored with descriptive and exploratory data analysis, LevyJennings and Shewart charts, histogram, six sigma capability indexes, general linear model (GLM) and Bonferroni's multiple comparisons of means statistical techniques. This practical example uses the observations collected for a period of a month. Results. While the traditional QC descriptive statistics and productivity indexes indicated that the overall performance was acceptable (Low QCM : mean= 48 mg/dL, CV= 6.4 %, CpK= 1.01, PpK= 0.97. High QCM : mean= 353 mg/dL, CV= 5.7%, Cpk= 0.93, PpK= 0.92), the histogram showed a quasi-normal distribution and the Levy-Jennings and Shewart charts did not reveal unnatural patterns. However, the parallel box plot for the low QCM revealed that for a number of hospitals, strip lot and QCM lot there were clusters of outlying observations. The parallel boxplot showed homogeneity of variance by hospital, strip and QCM lots; this visual impression was confirmed with the Bonferroni's multiple comparisons of variances (P> 0.05).The outlying observations were easily extracted with Minitab from the database and they were attributable to a number of nurses. Bonferroni's multiple comparisons confirmed that the mean performance of these nurses was statistically significantly different from the other nurses (P < 0.001). Direct observation of the technique employed by these nurses revealed that they were overloading the
B-147 Changes in Workflow for Accident and Emergency department involved near patient testing to reduce turnaround times B. A. Bewley, A. A. Gidman. Addenbrookes NHS Trust, Cambridge, United Kingdom
Background:Amalgamation of all acute admission units at Addenbrookes Hospital Cambridge into a single Accident and Emergency (A&E) Department in 2006 virtually doubled the number of patient episodes subject to the Department of Health (DoH) 4 hour A&E target. Prior to the amalgamation process re-engineering identified several improvements, including faster turn around times (TAT) for laboratory results, as necessary to achieve DoH targets. Method: The multi-disciplinary re-engineering group, including a Pathology
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reagent strip with control material. Since this faulty technique also affects patient testing, these nurses were retrained. Conclusions. This practical example clearly shows that the parallel boxplots display is a powerful graphical tool for revealing location, magnitude of variance, skewness, tail heaviness and outlying observations which may not be detected with classical descriptive QC techniques. These visual impressions can be confirmed with inferential statistical techniques for comparisons of multiple means and variances. Furthermore, Minitab statistical software has functions which facilitate data mining and extraction of observations attributable to individual locations, operators, reagent strips and QCM lots. Since Minitab graphical displays can be automatically updated upon acquisition of new observations in the database, the immediate electronic transfer of observations from Lifescan DataLink to Minitab would facilitate the detection of arising QC problems in real time.

570). Testing for multiple disease markers at the point-of-care, however, often requires prohibitively expensive combinations of different tests and formats. Advances in microfluidic, optical sensor, and microarray technologies hold promise for new, small sample volume, multiple pathogen assays. But despite impressive proof-of-concept demonstrations, relatively few integrated micro-optical diagnostic devices have achieved significant market penetration. This is in part due to manufacturing complexity underlying microfluidic device configurations and the relatively expensive optical systems required for high quality, high sensitivity imaging. A robust, sensitive assay system is described here that combines a disposable plastic cartridge with extremely low cost (estimated instrument manufactured cost ~$100) illumination and fluorescence imaging. Based on a novel, multi-mode planar waveguide configuration, the system capitalizes on advances in volume-manufactured consumer electronic components to deliver an imaging system with minimal moving parts and low power requirements (the unit operates off a laptop USB port). The cartridge has been designed for manufacture, with feature sizes and tolerances compatible with conventional injection molding. The single-use fluidic cartridges have no valves or active pumping, and offer workflow similar to conventional lateral flow based devices. By integrating low density microarrays in the cartridge, multiple targets can be simultaneously assayed even with small (e.g., 20 microliter) sample volumes. Two low cost systems are demonstrated. Data are presented for a multiplexed HIV and hepatitis C virus (HCV) serology assay that includes multiple pathogen-specific antigens as well as in-array sample and procedural positive and negative controls. Results for 90 clinical serum and plasma samples representing HIV and HCV positives, co-infections, and negative controls are presented, showing excellent correlation with known disease status. A second dataset presents nucleic acid testing results, specifically focusing on multiplexed RNA sandwich assays for influenza virus subtyping. Demonstration data based on synthetic RNA targets and H1N1 and H3N2 probe pairs designed using the NCBI Influenza resource are presented, including standard curves and hybridization specificity measurements. Limit of detection in this model system is approximately 10 femtomolar RNA target. A second system is a two-color cell imager, with magnification optimized for enumeration of immunostained human T cells. By incorporating a second diode laser and an emission filter changer, the waveguide system is demonstrated as a very low cost, two-color fluorescence microscope. To demonstrate the system, peripheral blood mononuclear cells were stained with anti-CD4-Alexa647 and anti-CD45-Alexa790 antibodies. Registered images were used to generate fractional CD4/CD45 staining results that show excellent correlation with flow cytometry. The cell imager is under development as a very low cost CD4+ T cell counter for HIV disease management in limited resource settings.
B-149 A rapid and compact on-site molecular analyzer S. S. Wong. Lynntech, Inc., College Station, TX
Current molecular analysis is limited to use in central laboratory locations because it is bulky, costly, time-consuming, and requires users with considerable expertise in molecular biology to execute. This poster will describe an innovative nucleic acid analyzer and include data to demonstrate its utility in enabling an operator with minimal or no expertise in molecular biology to perform molecular diagnostics in both laboratory and non laboratory environment with improved speed and decreased instrumentation cost. We will also briefly describe our efforts in building a simple and low-cost automated sample preparation module. We are developing an incredibly simple, closed loop convective flow real-time PCR thermal cycler. The thermal cycler uses RayleighBnard convection to drive the liquid through three different temperature zones that enable denaturing, annealing, and extension steps commonly used in a PCR protocol. In our design, because the PCR mix is moved between fixed temperature zones, the need for dynamic thermal control is therefore eliminated. This significantly reduces the complexity of the level of hardware needed to run the device. In addition, the fixed temperature zones allow random-access and eliminate the need for sample batching. More importantly, rapid cycling times are achievable because reagents quickly attain thermal equilibrium with their surroundings as they are transported through successive temperature zones. This design is particularly attractive because unidirectional flow through the loop allows residence times within specific temperature zones to be precisely controlled for a particle assay condition. Moreover, the use of PCR tubing materials allows the user to monitor the PCR process in real-time with fluorescent dyes or probes. The versatility of the convective flow thermal cycling system was demonstrated by using it to amplify multiple targets (5 different respiratory viruses) with a wide product size range (1.3 kb to 191 bp). Our result also demonstrated that, given the same amount of PCR amplification duration, our cycler produced significantly more PCR products than a commercial cycler. We have also successfully demonstrated convective PCR amplification and detection of three species of Gram negative and Gram positive bacteria spiked in non-leukoreduced human blood platelets. Gel electrophoresis images of PCR products (16S rRNA gene) clearly demonstrated that the device is capable of detecting 100 CFU/mL bacteria (1 CFU/mL per PCR reaction) in the presence of excess human genomic materials from the platelets. We expect the sensitivity will be further improved when fluorescent detection set up is used to carry out real-time PCR reactions. In summary, we expect that our molecular analyzer can be adapted to run virtually any existing and future PCR or real-time PCR based clinical diagnostic assays. The ability to carry out rapid, near-patient molecular diagnostics will improve patient outcome and reduce the spread of highly infectious diseases. The device is specially designed for scenarios where the number of samples is relatively low and rapid turnaround is needed.
B-151 Evaluation of the Analytical Performance of the Nova StatSensor Whole Blood Creatinine Meter and Reagent Strip for Chronic Kidney Disease Patients K. L. Schnabl1, S. Bagherpoor2, J. Dubois3, P. Yip2. 1Dept. of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada, 2University Health Network, Toronto General Hospital, Toronto, ON, Canada, 3Nova Biomedical Canada LTD, Mississauga, ON, Canada
Introduction: Chronic kidney disease is a growing health problem worldwide. Whole blood creatinine measurement at the point-of-care may assess risk for kidney injury prior to administration of nephrotoxic agents or monitor renal function. To address the need for point-of-care testing creatinine analytical performance data, the StatSensor creatinine meter was evaluated for imprecision, linearity, comparability to laboratory results, and interference. Methodology: Experimental design followed CLSI guidelines. Total, between-day, and within-day imprecision were evaluated over 20 days with three levels of control material performed twice per day with 2 meters across 2 reagent strip lots. Manufacturer linearity material and whole blood from healthy volunteers spiked with increasing amounts of interferent were used to assess linearity and interference, respectively. Venous whole blood patient samples were analyzed to compare whole blood creatinine levels measured on the creatinine meter against plasma values obtained on the Abbott Architect c8000 chemistry system. Data were analyzed using Microsoft Excel and EP Evaluator. Results: Creatinine meter imprecision data are shown in the table. An independent means t-test showed no significant meter-to-meter variation at all control levels. Manufacturer linearity material verified the measurable range from 93-863 mol/L (1.0-9.8 mg/dL).
B-150 Robust, low-cost fluidic cartridge and fluorescence imaging system for point-of-care, multiplexed protein, nucleic acid, and cellular assays M. J. Lochhead1, K. M. Todorof1, J. T. Ives1, J. R. Heil1, M. R. Givens1, M. J. Delaney1, K. D. Moll1, K. R. Vogel1, R. T. Schooley2, C. J. Myatt1. 1Precision Photonics Corporation, Boulder, CO, 2University of California, San Diego, CA
Timely infectious disease diagnosis remains a major public health challenge, particularly in limited-resource settings. For example, best practice guidelines in HIV management call for diagnosis of opportunistic infections at the time of HIV screening or prior to initiation of antiretroviral therapies (JAMA 2008, 300(5), 555-
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Creatinine Meter Imprecision Low Control (n=80) Mean (mol/L) 92.5 Total Imprecision (%CV) 6.10 Between Day Imprecision (%CV) 3.10 Within Day Imprecision (%CV) 5.29 Comparability of Architect (x) Correlation (R2) vs Creatinine Meter (y) All Subjects 0.9658 Renal 0.9559 Pre-Dialysis 0.9117 Post-Dialysis 0.9668 Other 0.9814 Medium Control 180 7.49 4.22 6.24 Equation y = 0.731x + 12 y = 0.725x + 13 y = 0.719x + 13 y = 0.865x - 14 y = 0.902x - 6.3 High Control 558 6.11 3.63 4.96 N 191 154 97 57 31
B-153 Analytical and Clinical Evaluation of Cardiac Troponin I and hCG on the Radiometer AQT90 Flex POC Testing, Random Access Analyzer B. J. Kilburn, K. M. Schulz, M. M. Murakami, F. S. Apple. Hennepin County Medical Center, Minneapolis, MN
The goal of this study was to evaluate the analytical performance of Radiometers whole blood AQT90 Flex POC testing, random access analyzer for cardiac troponin I (cTnI) and beta hCG (hCG). For cTnI the LoB, LoD, and LoQ were 0.0043 g/L, 0.0095 g/ L, and 0.0095 g/L, respectively. The linear range was 0 to 50 g/L. Day to day imprecision (%CV) was 10% at 0.039 ug/L and 20% at 0.020 g/L. 8% of the normal subjects (n=425 healthy blood donors) had a measurable concentration, with a 99th percentile value of 0.023 g/L. There were no significant differences between whole blood and plasma, or between men and women. Correlation of 373 patient specimens with AQT cTnI concentrations < 5 g/L between the AQT and the Siemens Stratus CS demonstrated the following regression: AQT cTnI = 0.258 Stratus CS + 0.001; r = 0.92. 382 patients that presented with symptoms suggestive of ACS were evaluated, of which 85 (18.9%) had an MI. The ROC curve area under the curve for the baseline (0 to 2 hours after presentation) sample was 0.91. The clinical sensitivity and specificity using the 99th percentile value were 64.7% and 93.3%, respectively. For hCG, the assay was linear between 2 to 5000 IU/L, with no hook effect up to 250,000 IU/L. No matrix effect was found between EDTA whole blood, EDTA plasma, lithium heparin whole blood or lithium heparin plasma. Based on 505 normal subjects, the reference intervals were: male (n=255) < 2 IU/L; female (n=250, all) <6 IU/L; postmenopausal (n=38) <6 IU/L; premenopausal (n=87) <2 IU/L. Imprecision (%CV) was 10 % at 8 IU/L. Correlation of 184 patient specimens with AQT90 hCG concentrations in the range of 2-5,000 IU/L (with the AQT90 FLEX hCG assay) demonstrated the following regression: AQT90 hCG = 1.291 Stratus CS - 7.2; r = 0.99. In conclusion, we have shown that the Radiometer AQT90 Flex random access analyzer provides diagnostically accurate rapid, whole blood, POC testing for both cTnI and hCG; 2 critical tests that need to be available in emergency settings on a 24/7 basis.
Patient comparison data across an analytical measurement range of 41-923 mol/L (0.5-10.4 mg/dL) is shown in the table. Interference studies showed significant bias in the presence of creatine above 0.25 mmol/L and urea above 20 mmol/L. Conclusions: The StatSensor creatinine meter provides reliable creatinine measurement across a broad clinically important range and should prove useful in urgent situations. Calibration difference and matrix effects in renal patients may contribute to the observed bias. Slope or offset adjustment of results to agree with the central laboratory should be considered upon implementation.
B-152 Reliability of Critical Values Obtained By Point Of Care Glucose Testing R. Schifman, S. Renner, E. Cooper, E. Douros. Southern AZ VA Healthcare System, Tucson, AZ
Point of care glucose (POCG) testing is central to glycemic control, insulin treatment and managing risk of hypoglycemia. The objective of this study was to evaluate the reliability of critical POCG results in a VA healthcare facility in which laboratory personnel oversee quality of POCG testing and maintain a comprehensive competency program for over 750 operators, mostly nurses. POCG was measured by Roche AccuChek Inform that has a lock-out capability to prevent unauthorized testing by untrained or uncertified operators. After initial training, certification is maintained by quality control performance, annual competency exam, direct observation and proficiency testing. A total of 279 critical POCG values (<40 or >600 mg/dl) were observed over a 7month period within healthcare areas including; acute care (44%), critical care (25%), geriatrics (18%), ED (9%), and psychiatry (3%). Of all critical values, 65% were repeated in <5 min, and 12% in 6-15 min. Testing was not repeated within 15 min in the remaining 23% of which 59% were critical low and 31% critical high results. The highest frequency of non-repeats within 15 min was from ED (50%), critical care (22%) and acute care (20%). A significant clinical discrepancy was defined as a repeat value that was shifted in opposite direction within or outside the reference range, e.g. critical low value upon retesting was within or above reference range. The overall frequency of significant clinical discrepancies was 35% and 50%, for critical low values and 6% and 14% for critical high values at <5 min, and 6-15 min retesting periods respectively. For critical low POCG values the frequency of significant clinical discrepancies when repeated within 5 min was 100% for ED, 50% for critical care 35% for geriatrics 26% for acute care and 25% for psychiatry. For critical high POCG values the frequency of significant clinical discrepancies when repeated within 5 min was 18% for geriatrics, 10% for critical care and 6% for psychiatry. Of 12 POCG values below linearity (<10 mg/dl), 75% had significant clinical discrepancies upon repeat testing at <5 min. This study shows that critical low POCG results are frequently not reproducible and probably erroneous, while critical high values are more reliable as demonstrated by repeat testing within 5 minutes. In spite of an ongoing comprehensive competency and training program overseen by the laboratory, operators, especially in critical care and ED, did not comply with repeat testing policy for critical POCG results. Furthermore, these areas showed the highest frequency of clinically discrepant results upon retesting. These findings, if confirmed, indicate that special attention should be directed at critical POCG results, especially with suspected hypoglycemia since a relatively high proportion may be due to measurement errors. Therefore, immediate (<5 min) retesting for confirmation of critical POCG results should always be performed. Finally, compliance with repeat testing and frequency with which critical hypoglycemia is verified may be useful ongoing quality indicators of individual operator performance and competency.
B-154 Influence of Methemoglobin on the accuracy of Whole Blood Total Bilirubin Measurements on CO-Oximetry Analyzers P. Pamidi, M. DeAbreu, D. Kim, S. Mansouri. Instrumentation Laboratory, Lexington, MA
Total bilirubin measurements are traditionally performed on plasma or serum samples and such assays suffer from interference from hemolysis or sample turbidity from lipids. Most modern blood gas and CO-Oximetry analyzers started to offer total bilirubin assay on whole blood samples thereby eliminating need for the separation of red blood cells while offering no interference from hemolysis and turbidity within clinically significant levels. However, the blood based bilirubin assays have to appropriately correct for the significant spectral background of 1000 times more concentrated hemoglobin. CO-Oximetry based total bilirubin measurements are generally optimized for samples containing functional hemoglobins and the results correlate well with plasma based analyzers. Accuracy of measuring whole blood total bilirubin in the presence of methemoglobin (metHb) on two CO-Oximetry based systems is compared in this study. Radiometer ABL 735, Instrumentation Laboratory GEM Premier 4000 (total bilirubin assay currently under development) and Reichert Unistat (plasma or serum assay) analyzers were compared in this study. Fully oxygenated blood samples were spiked with metHb stock solution and bilirubin samples from two different sources (NIST SRM 916 and pooled clinical samples from Lahey Clinic, Burlington MA) for this interference study. Sample Oxyhemoglobin + Cinical Serum metHb Control metHb +NIST bili MetHb + Clinical Serum Plasma from MetHb + Clinical Serum 1 month old Cord Blood Sample tHb, g/dL MetHb, % 15 16 16 16 N/A 13 0.5 25 25 25 0 9 Total Bilirubin, mg/dL GEM ABL 735 Unistat Premier 4000 25.9 2.8 23.7 18.1 14.0 2.4 24.6 4.1 23.1 18.8 14.4 4 25.3 0.4 13.9 14.7 14.7 0
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The data shown above highlights the impact of methemoglobin on whole blood based bilirubin measurements as compared to plasma measurements from the same sample. In conclusion, bilirubin measurements on samples containing clinically significant levels of methemoglobin require appropriate bias corrections or sample interference flags. Bilirubin measurements on GEM Premier 4000 will attempt to correct or appropriately flag the results for samples containing methemoglobin.

intervals: 0-2 hours, 2-4 hours, 4-6 hours, 20-22 hours and 22-24 hours. The SDDs were then regressed against the time intervals of 2 to 14 hours; extrapolation to zero time represents the sum of sb and short term analytic variation (sa). Results: Substitution of experimentally derived analytic error permitted the calculation of CVb: pH: 0.3%; pCO2: 5.7%; pO2: 13%; Na+: 0.6%; K+: 4.8%; Cl-: 0.8%; HCO3-: 3.2%; iCa++: 2.4%; and glucose: 10.3%. Except for iCa++ where the CVb is 50% higher than for healthy individuals, the CVb of the other electrolytes very closely matches the lowest estimates. Conclusions: Derivation of the ratio of biologic to analytic variation indicates that the ABL800 is extremely suitable for ICU testing. This analysis should be extended to other point of care instrument systems.
B-155 Withdrawn
B-156 A Point-of-Care Test for Diagnosis of Early Acute Myocardial Infarction K. W. IP. Hong Kong University of Science and Technology, Hong Kong SAR, Hong Kong
Background: Heart-type fatty acid-binding protein (H-FABP), a small protein (15 kD), holds promise for early diagnosis of acute myocardial infarction (AMI). H-FABP appears earlier in blood than large proteins after cell damage due to its small molecular size. Its characteristics of high concentration heart tissue contents and low normal plasma values give the possibility of a rapid increase above the respective reference values, thus providing an early indication of the appearance of myocardial injury. Objectives: To take full advantage of the characteristics of the early marker H-FABP, rapid analysis is a crucial parameter. We evaluated the diagnostic performance of a one-step FABP immunotest and compared with the gold standard marker, cardiac troponin I (cTnI), and the traditional marker, myoglobin at the early stage. Methods: Consecutive patients presenting to the Emergency Department suspected of AMI within 6 h of symptom onset were included. The diagnostic performances of a one-step H-FABP immunotest and its combination with the gold standard troponins at admission and 2 h after admission were compared with different cardiac markers. Results: Two hundreds patients presenting to hospital with a median symptom onset of 2.3 h (IQR 1.7 - 4.0 h) were enrolled in this study and 107 (53.5%) had AMI. The H-FABP immunotest was found to have a better sensitivity (76.6%) than the other cardiac markers and a better specificity (88.2%) than myoglobin at admission and 2 h after admission. Both sensitivity and negative predictive value (NPV) increased to over 90.0% at 2 h after admission, which were sufficient to safely allow ED discharge non-AMI patients. The areas under the receiver operator characteristic (ROC) curves to distinguish AMI from non-AMI for H-FABP were greater than those of the other cardiac markers at admission and 2 h after admission. The area under the curve for the combination of H-FABP with cTnI was the greatest at admission [0.834 (95% CI: 0.774 - 0.894)]. Conclusion: The developed one-step H-FABP immunotest improves the detection of myocardial damage at an early stage in patients suspected of AMI. With our outlook to combine H-FABP with the gold standard marker, troponins, this one-step immunotest with duo markers is able to provide a wider range of diagnostic window and allow more accurate targeting of appropriate therapy.
B-158 Microalbuminuria: a bedside tool to predict outcome in critically ill patients S. Basu1, M. Bhattacharya1, A. Majumdar1, S. Chaudhuri1, T. Chatterjee2, S. Todi1. 1Advanced Medicare and Research Institute Hospitals, Kolkata, India, 2Jadavpur University, Kolkata, India
Introduction: Prognostication in critically ill patients is of vital importance. We have tried to show the role of microalbuminuria in this regard. Methods: Design: Prospective, observational, cohort study. Microalbuminuria estimated as urine albumin-creatinine ratio (ACR) measured on Intensive Care Unit (ICU) admission (ACR1) and after 24 hrs (ACR2) and expressed as mg/g. Setting: A 20 bedded mixed medical-surgical ICU in a tertiary care hospital. Patients: 277 were recruited for the study between January 2007 and May 2008. Patients with ICU stay of less than 24 hours, pregnancy, menstruation, anuria, hematuria, urinary tract infection, proteinuria due to renal and post- renal structural diseases, were excluded. Procedures: Spot urine samples were collected within 6 hours and at 24 hours of ICU admission. Patients were classified as sepsis and no sepsis group as per the ACCP/ SCCM guidelines on admission. Demographics and APACHE II scores were obtained from the ITU data-base. The difference of ACR2 from ACR1 was calculated and expressed as delta () ACR. Results: The predictive values of APACHE II, ACR1, ACR2 and ACR for ICU mortality by receiver operating characteristics (ROC) curve analysis revealed that ACR2 could predict mortality nearly as well as the time tested APACHE II scoring system.{Area under curve (AUC) : 0.775} and a comparable ACR2 (AUC: 0.701) (p = 0.147) (Img1). ACR2 at a cut-off concentration of 101.1 mg/g had sensitivity of 68.9% and specificity of 65.9% with PPV of 28.1% and NPV of 91.6 % to predict ICU mortality. Conclusions: Degree of microalbuminuria at 24 hrs of ICU admission is a predictor of ICU survival.
B-159 Clinical concordance of eGFR measurement between laboratory plasma and whole blood point-of-care creatinine methods N. L. Korpi-Steiner, A. M. Wockenfus, J. M. Lakin, C. D. Koch, B. S. Karon. Mayo Clinic Rochester, Rochester, MN
Background: Patients with estimated glomerular filtration rate (eGFR) less than 60 ml/min/1.73 m2 are at high risk of contrast induced nephropathy; and accordingly eGFR measurement from serum or plasma creatinine is recommended prior to contrast administration for certain at-risk patients. Whole blood creatinine assays employed at the point-of-care would be ideal for this use, though bias and interferences in these assays may limit their utility. The aim of this study was to compare the clinical concordance of eGFR calculation using three whole blood creatinine methods compared to a reference plasma creatinine assay. Methods: Excess lithium heparin whole blood samples (n=266) submitted for stat evaluation of creatinine/eGFR prior to contrast administration for CT procedures were used. Creatinine values, patient age, gender and ethnicity were recorded for each sample. The eGFR was calculated using the MDRD equation and creatinine values obtained from iSTAT (Abbott Laboratories, Abbott Park IL) and StatSensor (Nova Biomedical, Waltham MA); whereas the MDRD adjusted for IDMS traceability was used to calculate the eGFR for the Radiometer ABL800 Flex (Radiometer A/S, Bronshoj Denmark) and reference method. Whole blood was centrifuged to produce plasma for measurement by the Roche Cobas Integra 400 (Roche Diagnostics, Indianapolis IN) used as the reference assay. Overall concordance was calculated as
B-157 The Use of Serial Patient Blood Gas, Electrolyte and Glucose Results to Derive Biologic Variation: a New Tool to Gauge the Acceptability of ICU Testing G. S. Cembrowski1, S. Redel1, D. V. Tran1, T. N. Higgins2. 1University of Alberta Hospital, Edmonton, AB, Canada, 2DynaLIFEDX Laboratories, Edmonton, AB, Canada
Background: Most estimates of biologic variation (sb) are based on periodically acquiring and storing specimens, followed by analysis within a single analytic run. We demonstrate for many intensive care unit (ICU) tests, only patient results need be statistically analyzed to provide reliable estimates of sb. Methods: Over eleven months, approximately 28,000 blood gas measurements (including electrolyte panels and glucose) were performed on one of two Radiometer ABL800 FLEX analyzers (Radiometer, Copenhagen, Denmark) on 1,676 ICU patients. We tabulated the measurements of paired intra-patient blood samples drawn within 24 hours of each other. After outlier removal, we calculated the standard deviations of duplicates (SDD) of the intra-patient pairs grouped in two-hour
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the percent of whole blood eGFR values falling into the correct plasma eGFR category (i.e. below or above 60 ml/min/1.73 m2). Results: The mean ( standard deviation) bias between StatSensor and Integra 400 creatinine was -0.23 0.18 mg/dL. Mean bias between i-STAT and Integra 400 creatinine was 0.13 0.08 mg/dL; while the Radiometer demonstrated a mean bias of -0.05 0.09 mg/dL. The Radiometer ABL800 Flex displayed the best overall concordance with plasma eGFR (93%). The i-STAT demonstrated slightly lower overall concordance (87%) but the best sensitivity (97%) for detection of plasma eGFR < 60 ml/min/1.73 m2; due to systematic overestimation of plasma creatinine by this method. The StatSensor demonstrated lower concordance (75%) with plasma eGFR < 60 ml/min/1.73 m2 compared to the other two whole blood methods. However, the StatSensor offers a slope and intercept offset to match whole blood to plasma creatinine, and use of the offset improved concordance significantly. Conclusions: The Radiometer ABL800 Flex demonstrated the best correlation to plasma creatinine and the best clinical concordance when creatinine values were used to calculate eGFR. The i-STAT device systematically overestimates plasma creatinine resulting in increased sensitivity but decreased specificity for determination of eGFR < 60 ml/min/1.73 m2. The StatSensor significantly underestimates plasma creatinine but offers a slope and intercept offset that partially compensates for this effect. The optimal device for use in rapid determination of eGFR from whole blood creatinine may depend upon whether it is more important in a given practice to optimize sensitivity, specificity, or overall concordance for determining plasma eGFR < 60 ml/ min/1.73m2.
Centaur plasma results (lithium heparin) with values greater than 4.6 mIU/mL being classified as numerically positive for hypothyroidism. When both devices were compared to the Centaur method, the ThyroChek and ThyroTest were in concordance with the Centaur for 61% and 88% of patient samples, respectively. However, the ThyroChek and ThyroTest concurred with each other in only 45% of the patient samples. The ThyroChek had a 38% false negatives on comparison with values >4.6 while the ThyroTest device had only 2% FN suggesting an apparent sensitivity of the ThyroCheck of 33% as compared to the ThyroTest device of 96%. Surprisingly, nine samples out of 32 apparent false negatives with ThyroCheck had values >10mIU/mL, particularly four with very high values: 28mIU/mL, 39.2mIU/mL, 54.4mIU/mL, 77.2mIU/mL. In addition, our study also revealed that the ThyroTest had a 8 apparent false positives (FP) in comparison to the ThyroChek device which hand only 1. Many of the ThyroTest FP occurred in the 4-5 mIU/mL range however. This lower detection threshold for TSH might prove useful for clinicians who elect to treat patients with borderline/subclinical hypothyroid values. The results of this study indicate that the ThyroTest rapid testing device for TSH is more accurate than the ThyroChek device based on comparison with the Siemens Centaur method using >4.6 mIU/mL as a cutoff for hypothyroidism. Centaur >4.6 mIU/mL <=4.6 mIU/mL Thyrochek Positive 16 1 Thyrochek Negative 32 36 Thyrochek Positive 44 8 Thyrochek Negative 2 31
B-160 A New Rapid Immunochromatography Test To Evaluate Exposure To Second Hand Tobacco Smoke J. M. Gonzalez, M. W. Foley, N. M. Bieber, P. A. Bourdelle, R. S. Niedbala. Lehigh University, Bethlehem, PA
Passive exposure to tobacco smoke causes a variety of illnesses ranging from allergic responses to some forms of cancer. Assessment of second hand tobacco smoke (SHS) exposure, particularly among vulnerable populations, such as children may allow intervention and/or prevention of future disease. A minimally invasive saliva-based onsite test to detect such exposure would constitute a valuable tool for researchers and clinicians. In our efforts to develop such a test, we found that detecting low levels of cotinine, a metabolite of nicotine, in a visual format is difficult; therefore, we report the use of an inexpensive reader that utilizes a CMOS image sensor to quantify a reporter signal generated in the visible range. This reader from Alverix Inc. allowed development of a rapid and sensitive test for cotinine. The rapid test was designed as follows; Protein-A was conjugated to colloidal gold nanoparticles. Cotinine derivatives were conjugated to BSA and striped onto a nitrocellulose strip. A control strip of Goat anti-human IgG was also striped above the test line. While developing the test, a series of anti-cotinine antibodies were obtained and purified. Derivatives to cotinine were also synthesized and matched to the purified antibodies for binding and displacement. During this process the test strips were evaluated using the Alverix reader. Levels of cotinine were detectable to 2.0ng/ mL and clearly distinguished using the instrument to calculate the ratio between the reflectance of the test and control lines. Conversely the same strips could not be easily interpreted by visual observation and resulted in a 10x improvement in sensitivity. Additionally the reader allowed for kinetic monitoring of the test which further aided the development process. The use of the reader accelerated the selection and optimization of reagents and suggests that such readers may improve the use and/or development of onsite rapid tests.
B-162 Use of Samples from Indwelling Venous Catheters for Glucose Meter Testing C. D. Koch, A. M. Wockenfus, T. M. Wangen, B. A. Sievers, J. K. Brown, B. S. Karon. Mayo Clinic, Rochester, MN
Background: Recent studies have found that drawing samples from indwelling venous catheters (IVC) for point-of-care (POC) glucose testing in critically ill patients may result in unacceptable levels of positive bias and imprecision. Studies that have examined IVC POC glucose measurement in non-critically ill patient populations have had mixed results. It is unclear whether positive bias and increased imprecision from IVC sampling is due to exogenous glucose contamination of IVC blood, inherent bias and imprecision of devices used, or properties of IVC blood that interfere with POC glucose meter analysis. Method: To determine how whole blood POC and laboratory plasma glucose measurement from IVC samples differs from the same measurements drawn by venipuncture, we compared glucose results from IVC samples to those obtained nearly simultaneously by venipuncture in 18 non-critically ill hospitalized patients. Each patient had a venipuncture performed for analysis of whole blood POC glucose on the Roche AccuChek Inform and analysis of plasma glucose, as the reference value, on the Roche Cobas Integra 400 Plus (Roche Diagnostics, Indianapolis IN). These results were compared to whole blood POC and laboratory plasma glucose analyzed from the same IVC sample, after a 10 mL flush and 5 mL discard volume according to institutional procedure. Results: The mean ( standard deviation) bias between IVC glucose meter and venous plasma (reference) glucose was 9 17 mg/dL, while the mean bias between venous meter and venous plasma (reference) was -1 9 mg/dL. The close agreement between venous meter and venous plasma (reference) glucose indicates that inherent bias and imprecision of the meter is not responsible for bias and imprecision observed during IVC meter analysis. The mean bias between IVC plasma and venous plasma (reference) was 1 6 mg/dL. The close agreement between IVC plasma and venous plasma (reference) glucose indicates that the positive bias and imprecision of IVC samples is not due to contamination of IVC blood with exogenous glucose, as the same sample was used for IVC meter and IVC plasma glucose analysis. 1 of 18 IVC glucose meter values was more than 20% higher than the venous plasma reference value, while all 18 venous meter and IVC plasma results were within 15% of the venous plasma reference value. Conclusions: The positive bias and greater imprecision of glucose meter results, when sampled from an IVC, is not limited to critically ill patients. The cause of this bias and imprecision does not appear to be related to contamination of IVC samples with exogenous glucose, but rather properties of IVC blood samples that are not optimal for glucose meter analysis. Care should be taken when interpreting glucose meter values obtained from IVC samples.
B-161 Evaluation of Two Point-of-Care (POC) Whole Blood Tests for Thyroid Stimulating Hormone (TSH) K. E. Blick, C. T. Beavers, B. E. Blick, H. D. Fry. Un of OK Health Sci Ctr, Oklahoma City, OK
We evaluate two point-of-care immunoassay devices, ThyroChek and ThyroTest, for the measurement of TSH on whole blood (EDTA) by comparing results on paired samples with numerical values obtained on the Siemens Centaur. A total of 85 patients were included in the study ranging as follows: 2 patients; <3mIU/mL;55 patients;37mIU/mL;19 patients;7-20mIU/mL; and 9 patients;>20mIU/mL. The detection cutoff level for each POC device was set at 5 mIU/mL with values >5 indicative of a positive screen for primary hypothyroidism. Each POC result was compared with
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B-163 Disinfectant wipes containing hydrogen peroxide induce overestimation of glucose results obtained with Lifescan Surestep Flexx glucose meter P. Desmeules, J. thier, P. Allard. CHU Ste-Justine, Montreal, QC, Canada
Introduction and objective. Sanitizing of glucose meter between successive measurements is a frequent procedure performed in hospital. In our center, ready-touse wipes containing 0,5 % hydrogen peroxide (Virox wipes) have been used to clean glucose meters LifeScan Sure Step Flexx. This glucose meter uses strips based on glucose oxidase reaction which generates hydrogen peroxide that reacts with the horseradish peroxidase. The hydrogen peroxide from the wipes can interact with this reaction by increasing the end-point of the detection reaction. We have demonstrated and investigated this interference. Methodology. Two scenarios of cleaning have been studied. Firstly, when the exceeding cleaning product (Virox) is wiped off with a sterile cloth from the glucose meter surface before the analysis and secondly without wiping. In both scenarios, an initial glucose measurement of the sample was done before cleaning the glucose meter. Then, the device was cleaned and glucose measurements were performed every 30 minutes. Simultaneous measurements were performed at the core lab (using ABL800) for comparison. Results. Typically, we observed a maximal increase of 60, 30 and 25% for samples containing low ( 3 mmol/L), normal or high level of glucose respectively. Then, the glucose value of the sample gradually decreases (many hours) until reach the initial glucose values. Moreover, if the Virox product was not wiped off, an overestimation of more than 100% for low and normal glucose values have been observed. Conclusions. It is clear that erroneous results can be obtained for many hours after this cleaning procedure. This interference cannot be detected by the operator since no error code appeared on the glucose meter. This cleaning procedure may induce risky results in many situations such hypoglycemic monitoring of newborn. The utilization of this widely used disinfectant wipes for sanitizing of glucose oxidase based glucose meter must be forbidden to avoid hazardous overestimation of glucose results.

analysis on the Cholestech LDX. Conclusions: In general capillary sampling can be expected to increase intra-assay precision (decrease reproducibility) of lipid measurement; however the magnitude and clinical significance of this effect varies by analyte. Total cholesterol measurement by both capillary whole blood and serum sampling on the Cholestech LDX results in relatively precise and accurate estimation of laboratory serum cholesterol. HDL cholesterol analysis on the Cholestech LDX is more accurate with capillary whole blood compared to serum, with a trade-off of increased intra-assay precision when capillary sampling is used. Both intra-assay CV and the standard deviation around the mean bias were twice as great with capillary sampling of triglycerides compared to serum sample analysis on the Cholestech LDX. Capillary sampling significantly increases imprecision in the measurement of triglycerides; such that use of capillary sampling to follow changes in triglyceride levels among individuals or populations may not be advisable.
B-165 Improvement of glycemic control of diabetic patients from primary care by an educational intervention together with an on-line quality control system from the central laboratory of Clinical Biochemistry M. Rodriguez-Oliva, C. Sanchez-Mora, R. Goberna, V. SANCHEZMARGALET. VIRGEN MACARENA UNIVERSITY HOSPITAL, SEVILLE, Spain
Objectives: To improve the glycemic control of diabetic patients from primary care by the implantation of an on-line quality control system from the central laboratory of Clinical Biochemistry together with an educatinal intervention. Material and methods: 7 portable glucose meters (PCx, Abbott) were set in 2 different primary care centers and connected to the central laboratory for quality control. The educational intervention was carried out both on the nurse personnel and diabetic patients. The meters from patients were controled by the PCx in every visit (every 3 months). 206 consecutive diabetic patients were recruited for the intervention and 12 months follow-up. Informed consent form was signed by the patients included and the local Ethical Commitee aproved the protocol. 135 patients completed the intervention and the 12 months follow-up. HbA1c and glucose levels were determined. Results: The on-line connected meters in the primary care centers fullfilled the analytical goals of a total error <7.9. The diabetic patients studied were 53% women and 47% men, and averaged 68 years-old. 43% of the patients meters were changed because of bad performance (>10% total error) comparing glucose determination with the on-line connected meter in the primary care center, which is controlled by the central laboratory of Clinical Biochemistry. After 1 year of follow-up, HbA1c decreased in average from 7.5% down to 7%. The differences in the mean HbA1c values were statistically significant (p<0.05). 66% of the patients improved the glucose control decreasing the HbA1c value. Conclusions: Glucose meters used by diabetic patients for the glycemic autocontrol can be indirectly controlled by the implantation of on-line connected glucose meters in the the primary care center together with an educational intervention, both on the nurse personel and the diabetic patints. This kind of intervention from the central laboratory of Clinical Biochemistry may help to improve the glucose self-monitoring of diabetic patients.
B-164 Effect of capillary sampling technique on the precision and accuracy of point of care lipid measurement N. K. Myhre, G. R. Deobald, B. S. Karon. Mayo Clinic Rochester, Rochester, MN
Background: Little information is available regarding the influence of sample type (capillary whole blood vs. venous serum) and sampling technique on the precision and accuracy of lipid measurement on the Cholestech LDX device. We estimated intraassay coefficient of variation (CV) and mean bias for lipid measurement based on replicate capillary sampling and replicate analysis of venous serum samples obtained by venipuncture; compared to a laboratory serum reference method for cholesterol, HDL cholesterol, and triglycerides. Methods: Two capillary punctures were performed on 40 healthy volunteers for duplicate capillary whole blood analysis of cholesterol, HDL cholesterol, and triglycerides on the Cholestech LDX (Cholestech Corporation, Hayward CA). Venipuncture was then performed within 2 minutes on each volunteer for duplicate analysis of venous serum cholesterol, HDL cholesterol and triglycerides on the Cholestech LDX; and for serum analysis of lipids on a Roche Hitachi P Modular Analytics Systems (Roche Diagnostics, Indianapolis IN) used as the reference method. Intra-assay coefficient of variation (CV) was estimated from the standard deviation of duplicate measurements divided by the population mean; while mean ( standard deviation) bias was calculated by comparing average (of the duplicate) Cholestech LDX value to the reference value for each individual. Results: For total cholesterol measurement, intra-assay CV estimated from duplicate capillary sampling was 2.3%, compared to an intra-assay CV of 1.4% when serum samples were analyzed in duplicate from the same patients (n=38). Mean bias between Cholestech LDX capillary whole blood and laboratory serum cholesterol was -4.7 3.7%; compared to a mean bias between Cholestech LDX serum and laboratory serum cholesterol of - 4.6 2.1%. For HDL cholesterol intra-assay CV was 4.7% with replicate capillary whole blood sampling vs. 2.8% for serum on the Cholestech LDX (n=36). Surprisingly, mean bias vs. laboratory serum HDL was 0.4 9.9% for capillary whole blood and 3.9 9.2% for serum sampling on the Cholestech LDX. For triglyceride measurement intra-assay CV was 5.0% for capillary sampling and 2.2% with serum on the Cholestech LDX (n=32). Mean bias vs. laboratory serum triglyceride was -1.4 11.6% with capillary whole blood and -4.1 6.1% for serum
B-166 Haemoglobin A1c: Comparison of three point of care analysers with the central laboratory assay C. Sanchez-Mora, M. Rodriguez-Oliva, V. Sanchez-Margalet, R. Goberna. Virgen Macarena University Hospital, Seville, Spain
Background: HbA1c measurement is the most important parameter to assess the glycemic control of diabetic patients. HPLC is the reference method for HbA1c determination. However, POCT devices for the measurement of HbA1c are available in medical offices, and they are specially being used in endocrinology and pediatric medical offices Objectives: To evaluate three POCT analysers by comparing the determinations with the automated HPLC analyser in the Clinical Biochemistry laboratory. Methods: HbA1c was determined in 53 blood samples sent to the central laboratory from diabetic patients. Every sample was determined in duplicate by every POCT analyser (DCA Vantage Siemens, Int2it Bio-Rad, and Afinion Izasa). Results were compared with those obtained by the reference method in the laboratory: HA
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8160 Menarini. Student's' t Test for paired samples and Pearson coefficient of lineal regression were employed for statistical significance using SPSS v.13.0 software. For imprecission studies SD and CV were determined by 20 consecutive measurements of a sample. Results: The means of the differences of the measurement performed by each POCT analyser ad the referente method (95% confidence interval) were: -0.278 for DCA, 0.627 for Int2it, and -0.348 for Afinion. Pearson coefficient of correlation were: r =0.974 for DCA, r = 0.980 for Int2it, and r = 0.991 for Afinion. Imprecission results were: CV = 3.35% - SD = 0.31 for DCA, CV = 5.37% - SD = 0.57 for In2it, CV = 1.95% - SD = 0.17 for Afinion. Turnaround time (TAT) of Afinion was 3 min, 6 min for DCA, and 11 min for Int2it. Conclusions: The three POCT analysers of HbA1c have a good correlation with the reference method, aceptable precission and they have short TAT.
B-168 Novel Humidity Check with MULTISTIX 10SG Urine Strips on the CLINITEK Status Analyzer from Siemens Healthcare Diagnostics L. S. Schulman, C. T. Zimmerle, M. J. Pugia. Siemens Healthcare Diagnostics Inc., Elkhart, IN
Urine strips require proper storage conditions at all times. Using strips that were not properly stored or handled can cause false-positive results. Improper storage conditions occur when bottles are uncapped for excessive periods of time, bottles are improperly sealed, or the user leaves the test strip out of the bottle for extended periods before use. Exposure to high humidity for as little as 10 minutes degrades performance. To prevent false results, we devised an algorithm on the CLINITEK Status analyzer, which will alert the user to a strip that has been compromised. When urine strips were exposed to high humidity for 20 minutes, 100 percent of them produced false-positive leukocyte results with normal urine. (A positive leukocyte result is an indication of urinary tract infection, for which a course of antibiotics is usually prescribed.) Using the new algorithm, all false-positive results for MULTISTIX 10SG urine strips exposed to air/moisture for 20 minutes were prevented. The new method reduces false-positive results on the CLINITEK Status analyzer due to improper storage or incorrect use of MULTISTIX 10SG urine strips.
B-167 Creatinine levels in patients with renal disease: Discordance between POC meter and automated enzymatic methods. J. Straseski, L. Phelan, W. Clarke. Johns Hopkins Medical Institutions, Baltimore, MD
Objective: Point of Care (POC) creatinine and eGFR measurements are commonly used to confirm renal function before administering MRI contrast agents. Monthly correlation analyses at Johns Hopkins Hospital have revealed a subgroup of patients whose results do not correlate between the EZ CHEM Creatinine Meter and an enzymatic central laboratory method. This study aimed to determine the frequency of such discrepancies and explore possible causes by further investigating the patient population in question. Methods: The EZ CHEM Creatinine Meter (Nova Biomedical, Waltham, MA) is a POC instrument that measures creatinine in whole blood based on the current generated when creatinine in the specimen mixes with reagent on the test strip. Fifty heparinized whole blood samples from hospital inpatients were tested with four different EZ CHEM Creatinine Meters. Results were compared to an endpoint creatininase enzymatic method (Roche Hitachi Modular) using plasma from each specimen. Patient age, gender, race, hematocrit, blood pH, pO2, currently prescribed medications and diagnosis were all documented and compared between groups. Results: Using the automated (enzymatic) method, creatinine values from the fifty patient specimens ranged from 0.20 to 12.90 mg/dL (median 1.30; adult male reference range 0.6 - 1.3 mg/dL). Fourteen samples (from 10 individuals) had creatinine results that differed between the two methods by > 0.5 mg/dL (28% of all samples). The mean EZ CHEM creatinine value in this group of patients was 4.62 mg/ dL, compared to 5.32 mg/dL obtained using the automated method. Of the discrepant creatinine values, 71% of results reported by the POC instruments were lower than the automated method values, representing a mean bias of -0.69 mg/dL. To investigate the discordant results, a group of control patients (n=10) were selected from the original group of samples. Controls were age, gender and race-matched to the patients with discordant results. The mean creatinine value for the control group was 1.41 mg/dL reported by EZ CHEM meters and 1.26 mg/dL using the automated method (mean bias 0.14 mg/dL). No differences in hematocrit or currently prescribed drugs were noted between the two groups. Comparisons of blood pH and pO2 could not be made, as most patients in the discrepant group did not have blood gas values available. However, 9 of 10 patients whose results did not correlate between the POC instrument and the automated method had some form of renal insufficiency. Diagnoses of these patients included end stage renal disease, chronic kidney disease and acute renal failure. In comparison, 3 of 10 patients in the control group had varying degrees of renal insufficiency. Conclusions: Overall, the EZ CHEM Creatinine Meter reported creatinine results that were statistically significantly lower than those obtained using the automated enzymatic method. Result discrepancies were observed in patients with elevated creatinine values (above 2.0 mg/dL). Patients with creatinine values in this range commonly have renal insufficiency, which may play a role in the lack of agreement between these two instruments. Assessing renal function in these patients with a POC instrument such as the EZ CHEM Creatinine Meter may produce falsely low values.
B-169 CLINITEK Status Analyzer Detects Urine Sample Interferences with the Use of MULTISTIX 10SG Urine Strips L. S. Schulman, C. T. Zimmerle, M. J. Pugia. Siemens Healthcare Diagnostics Inc., Elkhart, IN
Urine sample interferences can cause falsely elevated or falsely lowered urine strip results. These interferences include high specific gravity, which may cause falsely lowered glucose results as well as falsely lowered leukocyte results; elevated glucose, which may be another cause of falsely lowered leukocyte results; and high pH and visibly bloody urine, each of which may cause falsely elevated protein results. Algorithms using MULTISTIX 10SG urine strips from Siemens Healthcare Diagnostics were devised which will alert the user of these occurrences on the CLINITEK Status analyzer, occurrences that may otherwise cause false-negative or false-positive results with strips lacking this technology. MULTISTIX 10SG urine strips with and without these algorithms were compared to Teco URS-10, Clarity Urocheck 10 SG, and ARJ Medical Uritest urine strips. Effects of the various sample interferences on the MULTISTIX 10SG urine strips were reduced by as much as 100%. The new algorithms significantly reduce false-positive and false-negative results with MULTISTIX urine strips that may occur from urine sample interferences.
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B-170 Probing the Influence of Critical Care Variables on Glucose Measurements by the Nova StatStripTM Point-Of-Care Glucose Meter. P. Chan1, M. Rozmanc2, A. Wong2, Z. Gong2. 1Sunnybrook Health Sciences Center and University of Toronto, Toronto, ON, Canada, 2Sunnybrook Health Sciences Center, Toronto, ON, Canada
Introduction: Critical care variables such as hematocrit (Hct), electrolytes, pCO2, pO2, etc, have been reported to influence certain types of point-of-care glucose meters. However, their effects are sometimes difficult to assess because of their endogenous origin. Objective: To probe the influence of pH, pCO2, pO2, HCO3-, Na, K, Cl-, Base excess, iCa, lactate, total hemoglobin (THb) and Hct on glucose measurements by the StatStripTM handheld glucose meter (Nova Biomedicals, MA). Methods: 163 arterial and 47 cord blood samples were collected in heparinized syringes. Whole blood samples were analyzed for glucose and 12 critical care parameters using the StatStripTM glucose meter and the ABL800 Flex critical care analyzer from Radiometer respectively. Plasma glucose concentration was measured on the same samples after a 10-min centrifugation, using a glucose-oxidase method on the Roche Modular Analyzer. To identify critical care variables that may have an effect on the StatStripTM glucose measurement, paired differences of glucose measurements using the glucose meter and the laboratory analyzer were fitted into a stepwise multiple linear regression model together with all the critical care variables. Paired differences of glucose measurements were also organized into quartiles according to the levels of each critical care parameter. One-way ANOVA and trend analysis were performed on the means of the quartiles. Correlation between paired difference in glucose measurements and critical care variables were assessed and the Pearson's correlation coefficients were calculated. All statistical analyses were performed using the SigmaStat 3.01 from SPSS. A p-value <0.05 is considered significant. Results: Forward stepwise multiple linear regression analyses identified that iCa (p<0.001) and lactate (P<0.05) contributed significantly to the prediction of the paired difference in glucose measurements. One-way ANOVA of the latter, however, found significant differences among the quartiles of iCa, THb and Hct (but not lactate). Significant trend across quartiles were also observed in these three variables. Linear correlations were significant (p<0.001) between the paired difference of glucose measurements and iCa (Pearson's Correlation Coefficient, r=0.282), THb (r=0.232) or Hct (r=0.231). Comparing between the highest and the lowest quartiles, the means of paired glucose difference were 0.257 (95% CI: 0.171 to 0.342) and -0.302 (-0.459 to -0.146) mmol/L for iCa, 0.227 (0.138 to 0.316) and 0.175 (-0.337 to -0.013) mmol/L for THb, and 0.232 (0.143 to 0.32) and -0.194 (0.359 to -0.029) mmol/L for Hct, respectively. Discussion & Conclusion: Although iCa, and THb (collinear with Hct) demonstrated a statistically significant influence on the glucose measurement by the StatStripTM glucose meter as compared to the reference laboratory measurement, the effects (as shown by the difference between the highest and the lowest quartiles) were only 0.56 and 0.40 mmol/L for iCa and THb respectively. These small differences, together with weak correlations (r<0.3), are unlikely to have any clinical significance. In conclusion, the StatStripTM glucose meter is relatively free from the confounding effects of endogenous critical care parameters and should be a good candidate for use in critical care areas.

beyond 5 to 8 weeks of gestation. Reports have demonstrated that hospital POC qualitative hCG devices vary in the detection of different hCG variants. Objective: To investigate whether an excess of an hCG variant was the cause of three false negative qualitative hCG results on the Osom (Genzyme Diagnostics) qualitative POC device. Methods: Three patient urine specimens with false negative qualitative hCG results on the Osom device were quantified by variant-specific hCG immunoassays for intact hCG, hCG, and hCGcf. To investigate which hCG variants affect qualitative hCG device performance, purified hCG variants (hCGcf, hCG, hCGn and hCGn) were added to an hCG positive urine sample from a pregnant woman and tested on the Osom, hCG Combo (Cardinal Health) and ICON25 (Beckman Coulter) devices. Results: Three urine samples from patients with false negative qualitative hCG results on the Osom device were selected for further analysis. These three urine samples had hCG concentrations from 89,195 to 176,498 IU/L (Siemens Centaur); concentrations significantly below our previously established concentration necessary to produce a hook effect on the Osom device (~1,600,000 IU/L). However, dilution of these patient samples produced positive results on the Osom device, suggesting that a high dose hook effect may be responsible for the false negative results. Analysis of these three patient urine samples by variant-specific immunoassays demonstrated that hCGcf accounted for 64 - 78% of total hCG immunoreactivity. Addition of an excess of purified hCGcf to hCG positive urine (17,800 IU/L, Siemens Centaur) produced false negative results for both the Osom and ICON 25, but not the hCG Combo device. Addition of an excess of purified hCG to hCG positive urine also produced false negative results on the Osom brand while purified hCGn and hCGn diminished the positive signal intensity. We propose that an excess of an hCG variant produces false negative results on these devices by binding only one of the two antibodies, preventing an antibody-antigen-antibody sandwich from forming. Significantly, the hCG Combo device was not subject to interference by any of the variants tested. Conclusions: Elevated concentrations of hCGcf can cause false negative results on the Osom and ICON 25 brands, while the hCG Combo device was not subject to this interference.
B-172 Assessment of the Accuracy and Imprecision of the StatSensor Ketones POC Monitoring Device Compared to Central Lab Methods D. L. Meany1, W. Clarke1, L. Soto1, K. Reilly1, F. W. Chu2, J. Pei3, J. DuBois3. 1Johns Hopkins Medical Institutions, Baltimore, MD, 2Far Eastern Memorial Hospital, Taipei, Taiwan, 3Nova Biomedical, Waltham, MA
Introduction: Proper, early, and accurate assessment of ketosis by the diabetic patient or health care professional is important in diagnosis and prevention of an acute condition known as diabetic ketoacidosis (DKA). An accurate quantitative easy to use method for measurement of ketones in whole blood has not been available in the Point of Care (POC) until recently. The traditional methods were qualitative nitroprusside methods that primarily detect acetone and acetoacetic acid. A new POC device StatSensor ketones was developed to measure hydroxybutyrate (HB). HB represents ~80% of the ketone bodies present in the blood, which is the metabolite of greatest interest clinically. We evaluated concurrently the performance of the StatSensor device, in two different medical center settings, by comparing the StatSensor results to a photometric method adapted to a clinical chemistry analyzer. Methods: Following an IRB approved protocol, a capillary and venous blood were collected from consented patients with the ketone level simultaneously measured on the new Nova StatSensor ketone meter and the Stanbio ketone reagent method adapted to the respective chemistry analyzer at each medial center. The results were analyzed statistically for slope, intercept, correlation coefficient (r), and bias. Imprecision data was also collected and analyzed for the duration of the study. Results: A total of 169 paired patient analyses were randomly performed on capillary and venous whole blood prior to analysis on the central lab analyzer. The correlation of the StatSensor ketones method versus the Stanbio method adapted to the Hitachi 707 is Y=1.05X-.06 and the correlation coefficient is 0.978. StatSensor imprecision was observed as follows: at 0.1 mg/dl standard deviation = 0.006 mg/dl, at 1.4 mg/dl CV- 3.7%, at 3.2 mg/dl CV = 4.4%, and at 6.0 mg/dl CV = 4.0% and was comparable to the central lab method. Conclusions: The StatSensor meter and reagent strips were easy to use, provided rapid test results (10 sec) with comparable results to a central lab method. The StatSensor ketones device is well suited for self-monitoring, for use in emergency settings and at the patients bedside. This will be a valuable new diagnostic tool for managing diabetes patients and in rapidly diagnosing DKA in an emergency setting.
B-171 False Negative Results in Point-of-care Qualitative hCG Devices Due to Excess hCG Beta Core Fragment M. Cervinski1, A. M. Gronowski1, U. H. Stenman2, A. Woodworth3, L. Ashby4, M. G. Scott1. 1Washington University School of Medicine, St. Louis, MO, 2Helsinki University Central Hospital, Helsinki, Finland, 3Vanderbilt University, Nashville, TN, 4Barnes-Jewish Hospital, St. Louis, MO
Background: Qualitative point-of-care (POC) hCG devices are ubiquitously used in hospitals and other health-care settings. While robust, these devices are subject to rare false positive and negative test results due to incorrect reading time, insufficient or dilute urine, mislabeled specimens and biochemical pregnancy (i.e., early pregnancy loss). The high dose "hook effect" has been reported to cause false negative results in qualitative hCG devices. Testing is also complicated by the heterogeneity of hCG in both serum and urine. In addition to intact hCG (intact and subunits), variants include: hyperglycosylated hCG (hCG-h), nicked intact hCG (hCGn), hCG missing the -subunit C-terminal peptide, free -subunit (hCG), hyperglycosylated free -subunit and nicked hCG (hCGn). Another variant, the beta core fragment (hCGcf) is predominantly detected in urine and is the most prevalent form in urine
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B-173 Comparison of a Point-of-Care Whole Blood Beta-Hydroxybutyrate System with a Clinical Laboratory Reference Method A. C. Chin, D. D. Koch. Emory University, Atlanta, GA
OBJECTIVE: The primary intent of our study was to demonstrate performance of a point-of-care beta-hydroxybutyrate (-HB) meter in a real-world setting. We compared capillary blood obtained by skin puncture with venous blood. Patients were seen in two locations of Grady Hospital. The test strip assays were conducted by healthcare professionals who perform point-of-care testing. The results were compared to -HB measurements using plasma from the venous specimen on a laboratory -HB method. RELEVANCE: A need exists for a rapid -HB test to offer immediate diagnosis of ketosis and guide therapeutic decisions. The -HB molecule represents 80% of the ketones in blood, especially when large concentrations of NADH are present. The semi-quantitative nitroprusside methods are frequently employed in clinical settings currently; however, these methods do not react with the most clinically significant ketone body, -HB. Hence, a negative nitroprusside test does not rule out ketoacidosis. Several authors demonstrate that -HB determination is a useful tool that allows differentiation between simple hyperglycemia and potentially lifethreatening ketosis (1-3). A quantitative, whole blood method for -HB has been developed that is specific for the quantitative measurement of -HB. METHODOLOGY: The meter (StatSensor) and strip are products of Nova Biomedical Corporation (Waltham, MA). The central laboratory assay is the Pointe Scientific, Inc. (Canton, MI) -HB Reagent as developed for the Beckman Coulter, Inc. DxC (Brea, CA). The point-of-care method was judged using the following performance standards: EA = 0.56 @ 2.8 mg/dL, EA = 1.0 @ 5.0 mg/dL. These allowable errors are similar to analytical quality requirements used for comparable POC analytes. The decision levels are at the upper limit of the reference range and an elevated concentration of -HB, respectively. All of the participants were already having finger sticks and/or blood drawn for clinical purposes. IRB approval was obtained for the study. Precision was tested using patient samples at several -HB concentrations, and by within-run and day-to-day experiments using whole blood control materials. Approximately 270 patient samples were selected in method comparison experiments. Four meters designed specifically for -HB were tested through this study; two were larger, hospital-based meters that have full connectivity capabilities, and two were small meters for self-monitoring of ketones, similar to selfmonitoring glucose meters. Two lot numbers of strips were evaluated in these experiments. VALIDATION: The StatSensor meter and -HB reagent strips demonstrated excellent precision; example data are: SDs of 0.031 at 0.81 & 0.044 at 1.32 mg/dL. In every case, the random and systematic errors were less than the allowable error. CONCLUSION: The StatSensor meter and reagent strips are easy to use, produce the result rapidly (10 sec), and perform well in emergency settings. The meter met EA at each of the two decision levels. Medical assessment will be facilitated in these locations and patients will benefit from this whole blood -HB technology. REFERENCES: (1) Charles, R.A. et al. Singapore Med. J. 48(11): 986-9 (2007). (2) Naunheim, R. et al. Acad. Emerg. Med. 13(6): 683-5 (2006). (3) Guerci, B. et al. Diabetes Metab. 31: 401-6 (2005).
concurrent glucose testing on the three POC instruments. A sample was also collected in a pediatric lithium heparin container. Hematocrit was obtained using a Clay-Adams MHCT II manual hematocrit centrifuge. The sample was centrifuged within 5 minutes of collection and then analyzed for glucose and triglycerides on the Vitros 350 analyzer. Results: The Vitros 350 reference glucose measurements ranged from 10-251mg/dL (median of 76mg/dL). Relative to the Vitros 350 glucose determination (denoted as x), a least squares regression fit analysis of the measured glucose data yielded the following; Nova StatStrip= 1.13x - 2.6 (R2 of 0.97), Abbott PCx=1.22x + 8.8 (R2 of 0.95), LifeScan SureStep=1.11x + 4.0 (R2 of 0.96). The hematocrit within this patient set ranged from 25-66% (median of 45%) and the measured triglycerides ranged from 10-211 mg/dL (median of 51 mg/dL). The median percent bias of all measured glucose values relative to the Vitros 350 was +10% for the Nova StatStrip, +32 % Abbott PCx, and +16% for the LifeScan SureStep. Differing degrees of relative measured glucose percent bias was observed in samples with low hematocrit. The median relative bias for samples with <40% hematocrit (n= 41) was +12% for the Nova StatStrip, +39% for the Abbott PCx, and +20% for the LifeScan SureStep. Elevated levels of triglycerides (greater than 150 mg/dL, n=10) did not appear to substantially affect the percent bias relative to the reference method for all POC glucometers. At our institution, there is a strong desire to not miss any hypoglycemic infants with a potentially hazardous reference glucose concentration of 45mg/dL. Samples that exhibit potentially low glucose values by POC screening can be further tested by the reference method. In this data set, 21 samples exhibited glucose concentration of 45mg/dL by reference method. For these samples, the highest observed Nova StatStrip concentration was 50mg/dL, the highest observed Abbott PCx concentration was 65mg/dL, and the highest observed LifeScan SureStep concentration was 55mg/dL. Conclusions: Accuracy of POC glucose meters and the effects of common potential interferences, particularly hematocrit, should be investigated prior to adoption in the neonatal clinical setting. Institutions considering implementing POC meters should undertake studies to establish guidelines for repeating low glucose concentrations by their reference method.
B-174 Comparative Evaluation of Three Point-of-Care Glucose Meters with Neonatal Patient Samples Exhibiting Varied Hematocrit and Triglyceride Concentrations S. LeSourd, L. Fortune, K. Sanderson, B. Wood, R. W. Hall, J. A. Bornhorst. University of Arkansas for Medical Sciences, Little Rock, AR
Background: Waived point-of-care (POC) glucose meters are potentially attractive for use as a screening tool in neonatal populations. However, neonatal populations exhibit challenges for POC devices such as wide variation of hematocrit levels and increased lipid levels. The goal of this study was to comparatively evaluate the performance of Nova StatStrip, the Abbott Precision PCx, and the Johnson & Johnson LifeScan SureStepFlexx POC glucometers in a neonatal population exhibiting a wide range of hematocrit and triglyceride concentrations. Measured glucose concentrations were compared to the Ortho Diagnostics Vitros 350 glucose oxidase plasma reference assay, which is regarded as relatively insensitive to variations in hematocrit and lipemia. Methods: A total of 140 neonates (<1 month old) that had a pre-existing order for a plasma glucose level were identified. Whole blood was collected at the bedside for
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Comparative Study of Serum and Biliary Lipid Profile in Libyan Gallstone Patients

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Comparative Study of Serum and Biliary Lipid Profile in Libyan Gallstone Patients

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Cardiac Markers

Tuesday, July 21, 2:00 pm - 4:30 pm

Tuesday PM, July 21

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What is the True INR?

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B-79 INCIDENCE OF DEFICIENCY OF G6PD IN SCREENING IN BRASILIA, CAPITAL OF BRAZIL NEWBORN

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R. J. Molinaro, D. Bolliger, F. Szlam, J. Levy, K. Tanaka. Emory University, Atlanta, GA

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