case study [coagulation and hematology]

Laboratory Evaluation of Coagulation Inhibitors

Amy L. Adams, MD, Yara M. Audeh, BS, Regina de Luna, MD, Monette S. Baker, MD, Marisa B. Marques, MD Department of Pathology, Division of Laboratory Medicine, University of Alabama School of Medicine, Birmingham, AL
DOI: 10.1309/YLB7RF56KRNHTL94

Patient #: Age/Sex: Chief Complaint:

1 11-year-old female Epistaxis, fever, malaise, and anorexia

2 43-year-old male Ischemic stroke

Physical Exam Findings:

Afebrile, well-nourished child with cervical lymphadenopathy Unremarkable Hypertension

Past Medical History: Surgical History:

Tonsillectomy at age 9 years without increased bleeding No relatives with a bleeding disorder Aspirin for fever

None

11-year-old male 69-year-old female Epistaxis that occurred 1 week Easy bruising within the past prior to current evaluation; treated few weeks with fresh frozen plasma at another institution where a diagnosis of prothrombin deficiency was made Apparently healthy child in no acute Large hematoma at the site of an distress with no active bleeding IM injection; multiple ecchymoses in left arm with diffuse swelling Fever and lymphadenopathy approxi- Hypertension, diabetes mellitus, mately 6 months prior to presentation rheumatoid arthritis, and asthma Circumcision and tonsillectomy with- Cholecystectomy and out excessive bleeding hysterectomy without increased bleeding Negative for bleeding disorder None No relatives with a bleeding disorder Acetaminophen, atenolol, azithromycin, estradiol, glyburide, pravastatin sodium, prednisone, ramipril, and theophylline

Family History: Drug History:

Noncontributory Multivitamins

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Principal Laboratory Findings: [T1] Questions: 1. What are the most striking laboratory results for each of these patients?

2. What are the common causes of an abnormal PTT with a normal PT? 3. What clinical factors should be considered when evaluating patients with abnormal PTT and normal PT results? 4. What is your assessment of the most striking laboratory results for each of the four patients presented in these case

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studies? Include in your assessment the most likely diagnosis for each of the 4 patients presented, how each condition should be treated, and the role of the laboratory in managing these patients. Possible Answers: 1. One or more abnormal screening tests: PT (Patient #3), PTT (Patients #1-4), PTT-LA (Patients #1-4); abnormal PTT mixing study results (Patients #1-4); abnormal confirmatory test for the presence of an inhibitor, Staclot LA delta (Patients #1,3, and 4) and/or dRVVT ratio (Patients #2 and 3); low factor VIII activity with an increased factor VIII inhibitor level (Patient #4). 2. The most common reasons for prolongation of the PTT with a normal PT are heparin therapy, intrinsic factor deficiencies, presence of a lupus anticoagulant (LA), and/or an intrinsic factor inhibitor (eg, an antibody against factor VIII). 3. When assessing a patient with abnormal coagulation screening tests such as PT and/or PTT, several clinical factors are important to consider in guiding further evaluation in a cost-effective manner: gender, age, drug history, signs/symptoms, and concurrent medical conditions. 4. Patient #1: Since this patient is female, the probability of a factor deficiency is low, because the most common factor deficiencies, hemophilia A (deficiency of factor VIII) and hemophilia B (deficiency of factor IX), are X-linked disorders, and therefore only males are affected. Hemophilia carriers have decreased levels of factors VIII or IX, but these patients still have enough factor activity to provide a normal PTT result. Heparin is also an unlikely explanation for this patients prolonged PTT result

because she was not hospitalized and did not have an indwelling catheter. Finally, an antibody to factor VIII is very unlikely because the patient is a child, and specific factor inhibitors occur mainly in older individuals or in association with pregnancy. Thus, the most likely explanation for the prolonged PTT result in this patient is the presence of a LA. The term lupus anticoagulant refers to a heterogeneous group of autoantibodies that react with protein-binding phospholipids and thereby affect clotting tests that use a limited supply of phospholipid to form the clot (eg, PTT, PTT-LA, and dRVVT tests).1 Thus, LAs and anticardiolipin antibodies are called antiphospholipid antibodies. Anticardiolipin antibodies, however, do not prolong clot-based tests and are assayed by ELISA. The International Society on Thrombosis and Haemostasis (ISTH) has defined the following 4 criteria for the diagnosis of LA: (1) at least one abnormal screening test (PTT, PTT-LA, or dRVVT) result; (2) lack of correction of the prolonged screening test result after a mixing study has been performed; (3) shortening of an abnormal clotting time with the addition of excess phospholipid (eg, Staclot LA); and (4) exclusion of a specific factor inhibitor.2 Evaluation of the coagulation results for Patient #1 against these criteria indicated that she was likely to have an LA in her plasma [T2]. Screening tests such as PTT, PTT-LA, and dRVVT are very sensitive to interference by a phospholipid inhibitor and are typically prolonged in the presence of an LA. Because an abnormal screening test by itself is not specific for LA, additional testing must be done to confirm the diagnosis. A mixing study is helpful in this scenario and is performed by repeating the initial screening test with the patients plasma diluted 1:1 with normal plasma containing adequate levels of all clotting factors. This mixture would be expected to correct an abnormal screening test result that was due to a specific coagulation factor deficiency. In the case of an

Principal Laboratory Findings

Test 1 Platelet count PT, sec PT reference interval, sec INR Prothrombin activity PTT PTT mixing study PTT-LA Staclot LA delta Thrombin time dRVVT: Check Mix Sure ratioa Factor VIII activity Factor VIII inhibitor 13.8 11.3-14.6 82 55.0 43.0 108.0 20.4 43.1 2 12.5 11.3-14.6 78.0 90.0 16.0 78.6 56.0 37.5 2.1 200 N/A Results for Patient # 3 287 25.0 10.7-13.0 2.3 6 62.0 42.0 113.0 61.5 17.0 99.2 70.2 1.4 N/A <1% 64 BU 4 324 11.2 10.7-13.0 99.5 60.4 131.4 19.9 15.3 42.2 130-400 x 103/L Reference Range

T1
1.00 50-150% 25.0-34.0 sec Correction to 25.0-34.0 sec 36.1-50.1 sec <8.0 sec <18.0 sec 29.6-42.9 sec Correction to 29.6-42.9 sec N/A <1.3 50-186% Undetectable

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135 N/A

adRVVT ratio = (dRVVT check)/(dRVVT sure) PT, prothrombin time; INR, International Normalized Ratio; PTT, partial thromboplastin time; LA, lupus anticoagulant; dRVVT, dilute Russell viper venom time; BU, Bethesda units; N/A, not applicable.

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Evaluation of Coagulation Test Results for Patients 1 Through 4 Against the ISTH Criteria for the Diagnosis of a Lupus Anticoagulant
Criterion 1 Prolonged coagulation screening tests (PTT, PTT-LA, or dRVVT) Uncorrectable screening test by mixing study Shortening of abnormal clotting time with addition of excess PL Exclusion of a specific coagulation factor inhibitor + + + + 2 + + + + Patient # 3 + + + -

T2
4 + + + -

+, criterion met; -, criterion not met; ISTH, International Society on Thrombosis and Haemostasis; PTT, partial thromboplastin time; PTT-LA, partial thromboplastin time-lupus anticoagulant; dRVVT, dilute Russell viper venom time; PL, phospholipids.

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abnormal screening test due to the presence of an inhibitor such as an LA, the test result obtained on the mixture will still be abnormal because the inhibitor will interfere with the clotting factors from both plasma sources. The Staclot LA (Diagnostica Stago M, Parsippany, NJ) test is a commonly used confirmatory test in the workup of a possible LA. Using the Staclot LA procedure, 2 PTT-based clotting times are performed, 1 with a limited amount of phospholipid (tube 1) and the other with an excess of hexagonal phospholipids (tube 2). If the difference (or delta) between the clotting time values from tubes 1 (low phospholipid) and 2 (excess phospholipid) is greater than 8 seconds, a phospholipid-binding antibody such as an LA may be present. However, a decrease in the clotting time after the addition of excess phospholipid is not specific for an LA because this can happen in other conditions with a prolonged PTT, such as the presence of factor VIII antibodies. Although an LA is more common than any specific coagulation factor inhibitor, factor VIII antibodies are the second most common inhibitors, and their presence must be ruled out in order to establish the presence of an LA. Factor VIII inhibitors are measured with the Bethesda assay, which quantifies the amount of inhibitor based on the ability of the patients plasma to inhibit the activity of factor VIII present in normal plasma. One Bethesda unit is the amount of antibody capable of decreasing the activity of normal factor VIII by 50%. A positive Bethesda assay in conjunction with a low factor VIII activity confirms the presence of a factor VIII inhibitor. The factor VIII activity for this patient was normal, however, thus ruling out the presence of a factor VIII inhibitor. In this case the patients prolonged PTT, PTT-LA, and diluted Russell viper venom time (dRVVT) [T1], along with a mixing study that did not correct the PTT and a high Staclot LA delta, confirm the presence of a LA. Lupus anticoagulants are only rarely associated with bleeding. More often they are asymptomatic or are associated with thrombosis. This patients PT and prothrombin activity were normal. Therefore, this patients epistaxis was a red herring and may have been due to thrombocytopenia from a viral illness or from the effect of aspirin therapy on her platelets. Most LAs in children are associated with viral infections, are transient, and are not expected to cause thrombosis under these clinical conditions.3 Patient #2: Similar to Patient #1, the coagulation results for Patient #2 are consistent with the presence of an LA. The

PTT, PTT-LA, and dRVVT results were abnormal, and the dRVVT mixing study did not correct the dRVVT check results [T1]. In the case of Patient #1, the presence of a LA was confirmed using the Staclot LA test. For Patient #2, the confirmatory test was the dRVVT ratio. The dRVVT is a clotting test that utilizes the ability of Russell viper venom to activate factor X. As a screening test for the presence of a LA, the test is performed with a low amount of phospholipid (dRVVT check). If abnormal, the dRVVT may be repeated using a 1:1 mixture of the patients plasma with normal plasma (ie, plasma known to contain all clotting factors in adequate amounts with no substances that will interfere with any of the components of the coagulation cascade). If an inhibitor is identified by lack of correction in the mixing study, another dRVVT with concentrated phospholipid is performed (dRVVT sure). If an LA is present, it will essentially be neutralized by the excess phospholipid, allowing the dRVVT to correct. A dRVVT check to dRVVT sure ratio greater than 1.3 is consistent with the presence of a phospholipid inhibitor such as an LA. Moreover, the presence of a factor VIII inhibitor in this patients plasma was ruled out by his high factor VIII activity level, probably due to an acute phase reaction. Because this patient exhibited a thrombotic manifestation (stroke) in conjunction with an LA, this patient can be diagnosed as having the antiphospholipid syndrome (APS). Phospholipid antibodies are found in 18% of ischemic stroke patients younger than 44 years of age. In addition, neurological symptoms are more likely to occur in such patients in the presence of other risk factors, such as hypertension and smoking.4 Lastly, since this patients baseline PTT was prolonged due to the presence of a LA, the PTT could not be used to monitor heparin therapy. The anti-Xa (activated factor X) assay is helpful in this situation because it is not affected by the presence of an LA and it provides a very reliable measurement of anticoagulation in a patient receiving heparin. The laboratory should alert the patients physician to request the anti-Xa assay in this clinical setting. Patient #3: This patients prolongation of both the PTT and PT results suggests either the presence of an inhibitor or the deficiency of a specific coagulation factor within the common pathway of the coagulation cascade. However, the abnormal screening tests, the lack of correction with a mixing study, and the abnormal confirmatory test (dRVVT ratio and Staclot

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LA delta) results indicate an inhibitor, most likely LA, as the probable culprit [T1]. In patients with an LA and bleeding, it is important to check for hypoprothrombinemia. Occasionally, an LA is directed against prothrombin (factor II), leading to immune clearance of this factor from the plasma. In such cases, the prothrombin level is low, and the PT is prolonged. This scenario should be suspected if the INR is greater than 1.4. Because the INR for Patient #3 was 2.3, while the prothrombin activity was low [T1], and all of the ISTH criteria for the presence of an LA were met [T2], these findings are highly suggestive of an LA with specificity for prothrombin. A history of a preceding viral illness is common in patients with an LA, but an episode of bleeding, as seen in Patient #3, is not. Lupus anticoagulants are more often associated with thrombosis. If the LA is directed at prothrombin, however, a hypoprothrombinemia can develop resulting in epistaxis, easy bruising, hematoma formation, menorrhagia, and prolonged bleeding after trauma or surgery. As noted in T1, the prothrombin activity level was low (6%). This type of acquired prothrombin deficiency is more likely than a congenital form of prothrombin deficiency, as this patient had previously undergone both a tonsillectomy and circumcision without excessive bleeding. Fortunately, even with acquired prothrombin deficiency, the prognosis of LAs in children is good, as most are transient and resolve without specific treatment.3 Supportive therapy with management of bleeding episodes as they occur is the appropriate treatment for this patient. When bleeding episodes occur, fresh frozen plasma (FFP) is used to control bleeding. Fresh frozen plasma contains all of the clotting factors at an activity level of 100%. In vivo, a prothrombin activity level of 20% to 40% is generally adequate for achieving hemostasis. This level is often reached with the amount of prothrombin in FFP, in which case no other treatment is needed. While there is not a specific prothrombin concentrate available, a prothrombin complex concentrate exists which contains, in addition to prothrombin, factors VII, IX, and X. The only currently available formulation on the market is Bebulin, which contains a high level of prothrombin activity. This prothrombin complex concentrate has 2 main advantages over FFP for the treatment of prothrombin deficiency. First, it is safer because it undergoes a viral inactivation process to prevent the transmission of HIV, hepatitis B, and hepatitis C viruses. Second, the concentrate contains a higher level of prothrombin activity in a smaller volume, so less volume needs to be given to the patient to achieve hemostasis. This is particularly important in children or other patients who cannot tolerate excessive fluid. Also, for patients with more severe bleeding or for those being prepared for a surgical procedure, a hemostatic level of prothrombin can be achieved more quickly and with less overall volume. The loading dose of concentrate may be calculated by first determining the patients blood volume (BV), which is based upon the patients weight in kilograms. For children and slender adults, blood volume is presumed to be 70 mL per kilogram of body

weight. For obese adults, the blood volume (BV) is estimated using 60 mL per kilogram. Next, the plasma volume (PV) is determined by the following formula: PV = BV x (1 - Hematocrit) Finally, the dose of concentrate to be given is calculated as follows: Dose, IU = (Desired % Activity Current % Activity) x PV, where 100% activity equals 1 IU/mL. The amount of each clotting factor present in 1 vial of prothrombin complex concentrate is based upon the amount of factor IX present, measured in international units (IU). For example, a vial of Bebulin should have 120 IU for every 100 IU of factor IX. The package insert for each formulation of prothrombin complex concentrate should specify the total number of units of factor IX in each vial. To assess the appropriateness of the dose, the plasma prothrombin level should be measured approximately 30 minutes after the infusion. Since the presence of an LA is expected to shorten the half-life of prothrombin, it is advisable to check factor levels prior to deciding on subsequent doses. Patient #4: As with the other 3 patients, the results of the coagulation screening tests (PTT and PTT-LA), the PTT mixing study, and/or the Staclot LA delta confirmatory test were abnormal [T1]. However, this patients dRVVT test was normal, and the patient had a significant bleeding history, a classic picture of acquired hemophilia. This combination of findings is consistent with the presence of a specific inhibitor to factor VIII, a condition known as acquired hemophilia.5 This suspicion was confirmed by this patients extremely low (<1%) factor VIII activity [T1]. Acquired hemophilia is a rare condition caused by the development of a neutralizing autoantibody against factor VIII, which leads to factor VIII deficiency. Most patients with acquired hemophilia are over age 50 and about half have an associated disorder such as rheumatoid arthritis, systemic lupus erythematosus, malignancy, or a drug reaction. Peripartum women may also develop IgG anti-factor VIII. Patients with this disease often have a dramatic clinical presentation with spontaneous bleeding and/or delayed hemostasis. Other common sites of bleeding include the gastrointestinal and the urinary tracts. Such patients are usually managed with immunosuppressive agents such as corticosteroids. Serious hemorrhage requires additional treatment. If the level of inhibitor is low, the factor VIII level may be raised with factor concentrates purified from plasma or derived by recombinant technology. In patients with high inhibitor levels (>5 BU), hemostasis may be attained with alternative compounds which bypass the need for factor VIII, such as activated prothrombin complex concentrates or recombinant factor VIIa. If the antibody to factor VIII does not cross-react with porcine factor VIII, this product may also be of benefit to control bleeding. The role of the laboratory in this disease is to monitor factor VIII and factor VIII inhibitor levels over time. A summary of the most likely diagnosis and its rationale for each of the four patients presented in this case study is included in T3.

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Summary of Most Likely Diagnosis and Rationale for the Diagnosis in Patients #1 through #4
Patient # 1 Most Likely Diagnosis LA with thrombocytopenia* Rationale

T3

2 3 4

Antiphospholipid (APL) syndrome Acquired prothrombin deficiency due to presence of an LA Acquired hemophilia

Fever, malaise, and cervical lymphadenopathy suggest infectious mononucleosis. Viral infections are commonly associated with decreased platelet count, while aspirin is an inhibitor of platelet function and, therefore, a promoter of bleeding such as epistaxis High Staclot LA delta value Normal PT and prothrombin activity values Phospholipid antibodies (e.g., LA) are found in 18% of ischemic stroke patients less than 44 years old dRVVT screening and confirmatory test results consistent with the presence of a phospholipid inhibitor Markedly increased Staclot LA delta value INR > 1.4 Markedly low prothrombin activity level Occurs typically in individuals over the age of 50 50% of patients with acquired hemophilia have an associated disorder (eg, RA, SLE, a malignancy, or a drug reaction) Associated with spontaneous bleeding and/or delayed hemostasis Markedly low factor VIII activity Markedly increased factor VIII inhibitor level

*Due to the unavailability of the data from the patients complete blood count (CBC), thrombocytopenia could not be confirmed by a platelet count. RA, rheumatoid arthritis; SLE, systemic lupus erythematosis.

Keywords: lupus anticoagulant, coagulopathies, acquired hemophilia, antiphospholipid syndrome, acquired prothrombin deficiency
1. Greaves M. Antiphospholipid antibodies and thrombosis. Lancet. 1999;353:1348-1353. 2. Brandt JT, Triplett DA, Alving B, et al. Criteria for the diagnosis of lupus anticoagulant: An update. Thromb Haemostas. 1995;74:1185-1190.

3. Male C, Lechner K, Eichinger S, et al. Clinical significance of lupus anticoagulants in children. J Pediatr. 1999;134:199-205. 4. Levine SR, Deegan MJ, Futrell N, et al. Cerebrovascular and neurologic disease associated with antiphospholipid antibodies: 48 cases. Neurology. 1990;40:1181-1189. 5. Boggio LN, Green D. Acquired hemophilia. Rev Clin Exp Hematol. 2001;5:389404.

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