Hemoglobin A1c .....................................................................................................................................

2
HgbA1c_VIITurbo_2.0_7-9-12.doc ..................................................................................................................................2

1 of 21

Hemoglobin A1C on Bio-Rad Variant II Turbo 2.0

(Original In-Use Date: July 9, 2012)
I. PRINCIPLE
The BIO-RAD VARIANT II TURBO HbA1c Kit 2.0 utilizes principles of ion-exchange
high-performance liquid chromatography (HPLC). The samples are automatically diluted on
the VARIANT II TURBO Sampling Station (VSS) and injected into the analytical cartridge.
The VARIANT II TURBO Chromatographic Station (VCS) dual pumps deliver a
programmed buffer gradient of increasing ionic strength to the cartridge, where the
hemoglobins are separated based on their ionic interactions with the cartridge material. The
separated hemoglobins then pass through the flow cell of the filter photometer, where
changes in the absorbance at 415 nm are measured. An additional filter at 690 nm corrects the
background absorbance.
The VARIANT II TURBO Clinical Data Management (CDM) software performs
reduction of raw data collected from each analysis. Two-level calibration is used for
adjustment of the calculated HbA1c values. A sample report including retention times of
detected peaks and a chromatogram are generated by CDM for each sample. The A1c peak is
shaded. This area is calculated using an exponentially modified Gaussian (EMG) algorithm
that excludes the labile A1c and carbamylated peak area from the A1c peak area. The
VARIANT II TURBO HbA1c Kit 2.0 is for use only with the Bio-Rad VARIANT II
TURBO Hemoglobin Testing System.

II. POLICY/SCOPE
This is intended for the China Basin Chemistry section of the Clinical Laboratories and
intended for testing by licensed Clinical Laboratory Scientists and Clinical Laboratory
staff.
III. SPECIMEN REQUIRMENTS
a. Blood is collected in a lavender top tube (EDTA) and refrigerated at 2-8C.
Whole blood is stable 7 days at 2-8 C or 24 hours at room temperature (15 30 C).
Lipemia up to a level of 6000 mg/dL of triglycerides does not interfere.
Icterus up to a level of 20 mg/dL does not interfere.
Hemolysis of the sample is not relevant, as whole blood is hemolyzed in the course
of the analysis.
b. Acceptable container sizes are 5 mL, 7 mL and 10 mL
c. Samples with volume < 2.0 mL (or height less than 25 mm), or clotted samples, require
pre-dilution before being placed on the VARIANT II TURBO.
d. Allow sample tubes to reach room temperature (1530 C) before performing the assay.

2 of 21

IV. EQUIPMENT, REAGENTS, AND SUPPLIES

a. The VARIANT II TURBO HbA1c Kit 2.0 (2500 tests), Catalog 270-2455. The
following supplies are included in the test kit:
i. Elution Buffer A Catalog 270-2456. Five bottles containing 2500 mL of a
sodium perchlorate buffer. Contains <0.05% sodium azide as preservative. Store
at 15-30C.
ii. Elution Buffer B Catalog 270-2457. One bottle containing 2000 mL of a
sodium perchlorate buffer. Contains <0.05% sodium azide as a preservative.
Store at 15-30C.
iii. Wash/Diluent Set Catalog 270-2730. Four bottles containing 2500 mL of
deionized water with <0.05% sodium azide as a preservative. Store at 15-30C.
iv. Cartridge Set Catalog 270-2462. One cation exchange (2500 tests) Analytical
Cartridge (4.6 mm ID x 27.5 mm) and 5 prefilters (500 tests each)
v. Calibrator/Diluent Set Catalog 270-2458. One set consisting of two vials of
Calibrator Level 1, two vials of Calibrator Level 2, and one bottle of Calibrator
diluent. The calibrator vials contain lyophilized human red blood cell
hemolysate with gentamicin, tobramycin, and EDTA as preservatives. Store at 28C.
Preparation:
Reconstitute HbA1c Calibrator Levels 1 and 2 with 7 mL of cold Calibrator
Diluent using a volumetric pipette or other accurately calibrated device. Allow
the calibrators to stand for 5-10 minutes at 15-30C. Swirl gently to dissolve.
Label the bottles with preparation and expiration dates. Reconstituted
calibrators are stable for 24 hours at 2-8 o C. Do not use the calibrators past the
24-hour expiration date.
vi. Whole Blood Primer (270-0350) Two vials of lyophilized human red blood cell
hemolysate with gentamicin, tobramycin, and EDTA as preservatives. Store at
2-8C.
Preparation:
Reconstitute Whole Blood Primer by adding 1 mL of CLRW to the vial. Allow
to stand for 10 15 minutes at 15-30C. Swirl gently to dissolve and ensure
complete mixing. Reconstituted Whole Blood Primer is stable for 24 hours
when stored at 2-8 o C.
vii. Sample Vials (270-2149) One pack of 100 sample vials with piercable caps, 1.5
mL each.
viii. CD-ROM (270-2461) Contains Variant II Turbo HbA1c Kit 2.0 test
parameters.

3 of 21

V. WARNINGS AND PRECAUTIONS

Caution: This product requires the handling of human specimens. It is recommended that all
human sourced materials are considered potentially infectious and be handled in accordance
with the OSHA Standard on Bloodborne Pathogens. Appropriate biosafety practices should
be used for materials that contain or are suspected of containing infectious agents.
VI. CALIBRATION/CALIBRATION VERIFICATION
a. A calibration is performed for every installation of a new cartridge (2500 injections).
CDM will prompt the user when 2500 injections have been reached. Calibration must be
performed after priming a new analytical cartridge and/or installing a new lot of buffer.
Thereafter, calibration should be repeated every 90 days or as needed.
b. Refer to Section V. EQUIPMENT, REAGENTS, AND SUPPLIES for information on
preparation for Calibrator/Diluent Set and Whole Blood Primer needed to perform a
calibration.
c. Refer to Appendix A Reagent Lot and Cartridge Change for instructions on priming
and calibrating a new lot of reagent and/or cartridge.
VII.

QUALITY CONTROL
a. BioRad LYPHOCHEK Diabetes Control Levels 1 and 2 (Cat. No. 740), stored at 2-8C,
is stable until the manufacturers expiration date. Reconstitute each vial with 0.5 mL of
CLRW. Reconstituted controls are stable for 7 days at 2-8o C.
b. Each run should be bracketed by a set of controls, one at the beginning and end of each
run.

VIII.

PROCEDURE
a. Refer to Appendix B BioRad Variant II Turbo Maintenance for instructions on daily,
monthly and as needed instrument maintenance.
b. VARIANT II TURBO requires a warm-up, from the inactive state, once per day prior to
beginning a procedure. Cartridge temperature must be within specifications before
starting the days run.
c. Allow patient samples to reach room temperature before loading onto the VARIANT II
TURBO. No sample preparation is required. Mixing the tubes prior to loading is not
necessary.
d. Two levels of quality control material should be included at the beginning and end of the
each patient run to check the performance of the assay.
i. Load a sample rack with microtube adapters, barcode labeled for Blank, C-DB1
(Level 1 Control) and C-DB2 (Level 2 Control).
ii. Fill a sample vial with a 1:300 dilution of control (either DB1, DB2 or whole
blood) and place into the Blank adapter.
iii. Make a 1:300 dilution, into sample vials, of both control levels using 5uL of
control to 1.5 mL of Wash/Diluent. Place controls into corresponding barcode
labeled adapters.

4 of 21

iv. Small volume patients (less than 2.0 mL or 25 mm sample height) are also
diluted 1:300 in sample vials, with Wash/Diluent, and place into non-barcoded
microvial adapters labeled with patient ID before being placed into the sample
rack.
Sample #
1
2
3
4 to N*
N+1
N+2

Sample
BLANK (QC sample)
C-DB1 (QC Level 1)
C-DB2 (QC Level 2)
Patient Samples
C-DB1 (QC Level 1)
C-DB2 (QC Level 2)

N+3

STOP Tube/Empty Rack

*Last Patient Sample

v. Place up to 10 racks onto the belts of the VSS (5 on each side). Loading more
than 10 racks at a time will create rack jams that will abort the run. Verify that
rack handlers and belt are stationary before loading the racks. The VARIANT II
TURBO sampling station is continuous feed. CDM will build up to 500 samples
in a worklist. Place a barcoded STOP adapter, or an empty sample rack, at the
end of the run. If a STOP adapter is not used and the samples are not removed
when completed, the sampling station will continually rerun the samples until the
user stops the run or the worklist builds 500 samples.
vi. Start the Run:
1. Under the Run Icon,
2. Select Start
CDM will build the worklist as the barcodes are read. A sample with no barcode label will
build as an unknown under Sample ID/Lot number.
**NOTE: Unlike the Variant II, patient sample ID numbers CANNOT be edited after
the sample has finished analysis and the print has turned to grey. **
The VARIANT II TURBO will read the rack barcode first and then start sampling. Prediluted samples are directly aspirated, 500uL, with no additional dilution by the VSS. The
sample needle simultaneously pierces, vents, and aspirates the sample from each primary tube
after its barcode is read. A 1:200 dilution is made in the dilution chamber. The syringe in the
VCS aspirates 500uL of the dilution for transport to the VCS.
e. If the instrument has stopped, you must include a blank sample at the beginning of your
next run before your two levels of controls.
f.

Print a worklist for each run performed.

g. Print individual chromatograms for samples that were:

i. Pre-diluted
ii. No barcode read
iii. Flagged by CDM

5 of 21

IX. RESULTING/REPORTABLE RANGE

a. The run and chromatograms should be reviewed for acceptability.
ITEM
Total Area
Range

CRITERIA

ACTION

1.0 million to 3.5 million

OK to report

Outside range

HbA1c should not be reported.

Manually dilute sample and reanalyzed.

Values should be within range.

OK to report

Controls not acceptable

Repeat controls and any patient samples.

3.5 19.0 %

OK to report

Any sample with >15% HbA1c should

be suspected of having a hemoglobin
variant.

Check HbA1c and glucose history. Then

consult with a specialist or supervisor.

Results less than 3.5%

Report as <3.5%

Results greater than 19.0%

Report as >19.0% after consulting with a

specialist or supervisor

Hb A1a/A1b, LA1c, A1c, P3, P4 and Ao

OK to report

Correctly identified

OK to report

Missing A1c or Ao peaks

Repeat analysis

Consistently in range (refer to current

Cartridge Insert for retention time
windows)

OK to report

Inconsistent

Repeat analysis

Sharp and symmetrical

OK to report

If not sharp and symmetrical

Repeat analysis

Properly constructed
(i.e. stable, not drifting)

OK to report

Not properly constructed

Repeat analysis

25% does not interfere with assay

OK to report

If HbF peak is 25%,

HbA1c should not be reported.

Send ETC "MHAB". Send out for
alternate testing method.

Quality Control

HbA1c
Reportable
Range

All normal
peaks are
present
A1c and Ao
peaks

A1c and Ao
retention times

A1c peak shape

Baseline

HbF (Fetal
Hemoglobin)

6 of 21

ITEM

CRITERIA

ACTION

LA1c 6% does not interfere with assay

CHb 4% does not interfere with assay

OK to report

NOTE: CHb elutes in the LA1c window

Labile A1c
(LA1c) and
Carbamylated
Hemoglobin
(CHb)

LA1c >6% interfere with assay

CHb >4% interfere with assay
There is a peak >4% in the LA1c
window

Heterozygous
hemoglobins E,
D, S, and C

P3 or P4 Peak

Variant and/or C
windows

Unknown
peak following
Ao peak

HbA1c should not be reported.

Allow a small aliquot of sample to sit
overnight at room temperature and
reanalyze with this aliquot.
If repeat result is 4%, OK to report.
If repeat result is >4%, send ETC
"USUB". Send out for alternate testing
method.

HbA1c result is reportable.

Append ETC MUHB to HbA1c result.

(MUHB = Mutant hemoglobin present)

P3 peak 5% for hemoglobin variant

samples
(i.e. HbS-, HbC-, HbD-, and HbE- trait)

OK to report

P3 peak >5% for hemoglobin variant

samples
(i.e. HbS-, HbC-, HbD-, and HbE- trait)

HbA1c should not be reported.

Send ETC "MHAB". Send out for
alternate testing method.

P3 peak 10% for non-variant samples

OK to report

P4 peak 10%

OK to report

If either peak exceeds the cutoff, consult

with a specialist or supervisor before
reporting HbA1c result.

HbA1c should not be reported.

Send ETC "MHAB". Send out for
alternate testing method.

Combined area of 60% should be

suspected of having a homozygous
variant or variant--thalassemia
phenotype

HbA1c should not be reported.

Send ETC "MHAB". Send out for
alternate testing method.

HbA1c result with an unknown peak is

not reportable. Possible carryover.

Manually dilute sample and reanalyze; if

result is still questionable (i.e. unknown
peak), consult with a specialist or
supervisor.

7 of 21

b. All samples that were not flagged by CDM can be released using the OEM function in
Sunquest after the tech has reviewed the chromatograms.
c. For results on samples that were prediluted, did not have a barcode read or flagged by
CDM, the OEH or MEH function should be used in Sunquest to report these values.
d. The reportable range for VARIANT II TURBO Hemoglobin A1c is 3.5 to 19.0%.
e. Results greater than 19.0% are reported as >19.0%.
f.

Results less than 3.5% are reported as < 3.5%.

g. For samples with unreportable results due to HPLC interference

i. Send one of the following English Text Codes:
1. MHAB -Mutant hemoglobin present interfering with this HbA1c assay.
Sample will be sent for testing by an alternate method. See "MISC Lab
Test" (result to follow).
2. USUB - Unidentified interfering substance present, unable to obtain
HbA1c result in this assay. Sample will be sent for testing by an alternate
method. See MISC Lab Test (result to follow).
ii. Credit the HBA1 test and order MOLT with HbA1c to Quest in the modifier
and SENDO on the same accession number. On the following result screen, reenter HbA1c to Quest on the MOLT prompt. Label an aliquot and give it to
Send Outs. Minimum volume is 0.5mL.
iii. Save an aliquot (approx. 0.5mL) for Chemistry in case further hemoglobinopathy
testing is needed.

X. EXPECTED VALUES
The non-diabetic reference range for VARIANT II TURBO Hemoglobin A1c is 4.3 to 5.6%.
All hemoglobin A1c results will automatically be appended with the following table:
_______________________________________
HbA1c cutoffs for diagnosing diabetes:
4.3% - 5.6% = normal
5.7% - 6.4% = increased risk for diabetes
>6.4% = diabetes
___________________________________________
HbA1c goals in treatment of diabetes:
Ages 0-6 years: 7.6% - 8.4%
Ages 6-12 years: <8%
Ages 13-19 years: <7.5%
Adults: <7%

8 of 21

XI. LIMITATIONS OF PROCEDURE

a. Samples from patients with hemolytic anemias will exhibit decreased glycosylated
hemoglobin values due to the shortened life span of the red cells. Samples from patients
with polycythemia or post-splenectomy may exhibit increased glycosylated hemoglobin
values due to a somewhat longer life span of the red cells.
XII.

PERFORMANCE
a. SPECIFICITY/INTERFERENCES
b. PRECISION
The precision of the VARIANT II TURBO HbA1c Kit - 2.0 was evaluated in a study
based on the Clinical and Laboratory Standards Institute (CLSI) EP5-A2 guideline
Evaluation of Precision Performance of Quantitative Measurement Methods. The study
design was modified to include two different VARIANT II TURBO instruments at each
of three different laboratories, for a total of six instruments. The same set of normal and
diabetic samples were run in duplicate, in each of 2 runs per day, for 10 days, on each
instrument, using a single kit lot. A single calibration was performed on each instrument,
during the first run of the study. The results of the precision study are summarized in the
table below:

Normal Patient

Diabetic Patient

Mean (% A1c)

5.6

11.4

Within-Run (% CV)

0.78

0.39

Between-Day (%CV)

0.66

0.69

Between-Run (% CV)

0.53

0.45

Within-Device (% CV)

1.15

0.91

c. ACCURACY
The VARIANT II TURBO HbA1c Kit 2.0 was compared to the previous VARIANT II
TURBO Hemoglobin A1c Program. 40 whole blood patient samples were run using the
VARIANT II TURBO HbA1c Kit 2.0 and the previous VARIANT II TURBO
Hemoglobin A1c program. The range of values on the VARIANT II TURBO HbA1c Kit
2.0 was 4.8 12.5% HbA1c. The correlation results are as follows:
n = 40
slope = 1.0957
intercept = -0.7375
R2 = 0.9952

9 of 21

To demonstrate accuracy within the linear range of 3.5 19.0% HbA1c, the VARIANT
II TURBO HbA1c Kit 2.0 was compared to the VARIANT II Hemoglobin A1c
Program. 40 EDTA whole blood patient samples and 12 samples prepared by mixing
EDTA whole blood patient samples with Lyphochek Hemoglobin A1c Linearity Set
samples in various ratios were run using the VARIANT II TURBO HbA1c Kit 2.0 and
the VARIANT II Hemoglobin A1c Program. The range of values on the VARIANT II
TURBO HbA1c Kit 2.0 was 2.6 19.0% HbA1c. The correlation results are as
follows:
n = 52
slope = 0.9621
intercept = 0.4443
R2 = 0.994
d. LINEARITY
To demonstrate the linearity of the HBA1c measurement throughout the reportable range, a
normal and a diabetic HbA1c whole blood patient sample were used to prepare dilutions, and
the diluted samples were analyzed with the VARIANT II TURBO HbA1c Kit 2.0. The
linearity was assessed following the CLSI EP6-A guideline Evaluation of the Linearity of
Quantitative Measurement Procedures: A Statistical Approach. The results of the study
demonstrate HbA1c linearity from 3.5 19.0% within a maximum measured difference of
0.24% in this interval.
Sample Pool
1
2
3
4
5
6
7
8
9
XIII.

Predicted 1st
Order
2.82
5.26
7.28
9.66
11.20
13.70
15.59
17.61
19.56

Predicted 3rd
Order
2.58
5.34
7.45
9.79
11.26
13.64
15.47
17.52
19.63

Difference
0.24
-0.08
-0.17
-0.13
-0.06
0.06
0.12
0.09
-0.07

TECHNICAL NOTES
a. Samples from patients with hemolytic anemias will exhibit decreased glycosylated
hemoglobin values due to the shortened life span of the red cells. Samples from patients
with polycythemia or post-splenectomy may exhibit increased glycosylated hemoglobin
values due to a somewhat longer life span of the red cells.
b. Each analytical cartridge is good for about 2500 injections.
c. Column temperature is approx. 35 2 C.
d. -thalassemia trait, as indicated by increased HbA2 concentrations up to 10%, does not
interfere with the assay.
e. No significant interference from variant hemoglobin was observed at the following
concentrations: HbS 67%; HbC 72%; HbD 55%; HbE 41%.

10 of 21

XIV.

ALTERNATE METHODS
Hemoglobin A1c with eAG (Immunoturbidmetry) performed by Quest Diagnostics.

REFERENCES
a. VARIANT II TURBO HbA1c Kit 2.0 Instruction Manual: Bio-Rad Laboratories,
effective November 2012.
b. American Diabetes Association Home Page. http://www.diabetes.org (accessed April
2012).
c. Fowler, M.J. Microvascular and Macrovascular Complications of Diabetes. Clin.
Diabetes 2008, 26 (2), 77-82.
d. Whiting, D.R.; Guariguata, L.: Weil, C.; Shaw, J. IDF Diabetes Atlas: Global Estimates
of the Prevalence of Diabetes for 2011 and 2030. Diabetes Res. Clin. Pract. 2011, 94,
311-321.
e. Forsham, P. H. Diabetes Mellitus: A Rational Plan for Management. Postgrad. Med.
1982, 71, 139-154.
f.

Hollander, P. The Case for Tight Control in Diabetes. Postgrad. Med. 1984, 75, 80-87.

g. Baynes, J. W.; Bunn, H. F.; Goldstein, D.; Harris, M.; Martin, D. B.; Peterson, C.;
Winterhalter, K. NationalDiabetes Data Group: Report of the Expert Committee on
Glucosylated Hemoglobin. Diabetes Care 1984, 7, 602-606.
h. Nathan, D. M.; Singer, D. E.; Hurxthal, K.; Goodson, J. D. The Clinical Information
Value of the Glycosylated Hemoglobin Assay. N. Engl. J. Med. 1984, 310, 341-346.
i.

Mayer, T. K.; Freedman, Z. R. Protein Glycosylation in Diabetes Mellitus: A Review of

Laboratory Measurements and of Their Clinical Utility. Clin. Chim. Acta 1983, 127, 147184.

j.

Rohlfing, C. L.; Little, R. R.; Wiedmeyer, H. M.; England, J. D.; Madsen, R.; Harris, M.
I.; Flegal, K. M.; Eberhardt, M. S.; Goldstein, D. E. Use of GHb (HbA1c) in Screening
for Undiagnosed Diabetes in the U.S. Population. Diabetes Care 2000, 23, 187-191.

k. International Federation of Clinical Chemistry and Laboratory Medicine Home Page.

http//www.ifcchba1c.net (accessed April 2012).
l.

Hoelzel, W.; Weykamp, C.; Jeppsson, J. O.; Miedema, K.; Barr, J. R.; Goodall, I.;
Hoshino, T.; John, W. G.; Kobold, U.; Little, R.; Mosca, A.; Mauri, P.; Paroni, R.;
Susanto, F.; Takei, I.; Thienpont, L.; Umemoto, M.; Wiedmeyer, H. M.; IFCC Working
Group on HbA1c Standardization. IFCC Reference System for Measurement of
Hemoglobin A1c in Human Blood and the National Standardization Schemes in the
United States, Japan, and Sweden: A Method-Comparison Study. Clin. Chem. 2004, 50
(1), 166-174.

m. American Diabetes Association, European Association for the Study of Diabetes,

International Federation of Clinical Chemistry and Laboratory Medicine, and
International Diabetes Federation. Consensus Statement on the Worldwide

11 of 21

Standardization of the Hemoglobin A1c Measurement. Diabetes Care 2007, 30 (9),

2399-2400.
n. Sacks, D.B.; Bruns, D.E.; Goldstein, D.E., Maclaren, N.K.; McDonald, J.M.; Parrott, M.
Guidelines and Recommendations for Laboratory Analysis in the Diagnosis and
Management of Diabetes Mellitus. Clin.Chem. 2002, 48 (3), 436-472.
o. Biss, E.; Huaman-Guillen, P.; Hrth, P.; Busse-Grawitz, A.; Lizama, M.; Krmer-Guth,
A.; Haehnel, W.; Wieland, H. Heterogeneity of Hemoglobin A1d: Assessment and Partial
Characterization of Two New Minor Hemoglobins, A1d3a and A1d3b, Increased in
Uremic and Diabetic Patients, Respectively. J. Chromatogr. B, Biomed. Appl. 1996, 687,
349-356.
p. Panzer, S.; Kronik, G.; Lechner, K.; Bettelheim, P.; Neumann, E.; Dudczak, R.
Glycosylated Hemoglobins (GHb): An Index of Red Cell Survival. Blood 1982, 59,
1348-1350.
q. Color Atlas of Hemoglobin Disorders; Hoyer, J.D., Kroft, S.H., Eds.; College of
American Pathologists (CAP): Northfield, IL, 2003; pp 43, 47,51, and 59.
r.

Bain, B.J. Haemoglobinopathy Diagnosis; Blackwell Science, Ltd.: Malden, MA, 2001;
pp 154 and 164.

s. American Diabetes Association. Standards of Medical Care in Diabetes - 2012. Diabetes

Care 2012, 35 (Suppl. 1), S11 S63.
t.

Fairbanks, V.F. Thalassemias and Related Disorders. Hemoglobinopathies and

Thalassemias; Brian C. Decker; New York, 1980; pp 18-27.

12 of 21

BioRad Variant II TURBO Hemoglobin A1C 2.0

Appendix A
Reagent Lot and Cartridge Change
A CD is packaged with every Variant II TURBO Hemoglobin A1C Kit 2.0. The CD contains
information related to lot numbers and test parameters. The CD is loaded once for each kit in order to
update the reagent lot numbers and test.
One Analytical cartridge and 5 prefilters are packaged with every Variant II TURBO 2500-test
Hemoglobin A1c kit 2.0. Each Hemoglobin A1C Analytical cartridge is good for 2500 injections of
patient samples, and each prefilter is good for 500 injections of patient samples (calibrator and
controls are excluded from the count). The new Analytical cartridge needs to be primed twice before
calibration. Once calibrated, the calibration is good for 90 days.
1) Prepare the following reagents:
a) Reconstitute 2 bottles of Whole Blood Primer by adding 1 mL of CLRW to each bottle. Swirl
gently to dissolve. Allow the primer to stand for 10 minutes; swirl again to ensure complete
mixing. The reconstituted primer is stable for 24 hours when stored at 2-8C.
b) Reconstitute Calibrators Level 1 and 2 by adding 7 mL of Cold Calibrator Diluent to each
bottle. Allow the calibrators to stand for 10 to 15 minutes; swirl gently to dissolve. The
reconstituted calibrators are stable for 24 hours at 2-8C. The calibrators are ready for use
without further preparation. Do not use calibrators past the 24 hour expiration date.
c) Lyphochek Diabetes Control Levels 1 and 2 must be diluted 1:300 prior to analysis. Pipette
1.5 mL of Wash/Sample Diluent Solution into a labeled microvial, followed by 5 L of the
reconstituted control. Cap each control vial and mix thoroughly.
2) Install the CD parameters:
Under the Setup Icon,
a) Select the Test screen
b) Insert CD into the drive
c) Select UPDATE KIT
d) Select Drive e and File name V2 A1C.cab
e) Press OK
f) When the information on the CD is loaded, the screen will prompt:
Update Kit has finished successfully
g) Press OK
h) Under Table Settings, verify the lot numbers of the cartridges and reagents.

13 of 21

3) When a new buffer lot is installed, a short system flush has to be performed. When new
buffers of the same lot are installed, a system flush is not necessary. PROCEED TO STEP
#6.
4) To perform a System Flush:
Under the Setup Icon,
a) Select the Test screen
b) Select REAGENTS
c) Select START SYSTEM FLUSH
d) The screen will prompt:
Choose one of the System Flush Types: Use the Short flush when changing reagents
for the same test. Use the extended flush when changing test.
e) Select SHORT
f) The screen will prompt:
The first step of the System Flush action is over.
Put reagent lines in the new buffer and continue
g) Follow the prompts and replace the new buffers
h) Select OK
i)

The purge valve will open automatically and the reagent lines will be flushed with the new
buffers at the rate of 6 mL/min for 20 minutes (an onscreen timer will count down remaining
time). The buffers do not go through the cartridge.

j)

When the flush is complete, the screen will prompt:

The System Flush action has finished

k) Select DONE
l)

The instrument will be in manual mode

m) Under the Maintain Icon,

n) Select Return to READY State
5) Installing a New Analytical Cartridge:
NOTE: Do not use tools during the installation of the analytical cartridge and prefilter; handtighten only.
a) Open the door to the VCS. Open the cartridge thermal block cover.
b) Lift the cartridge holder out of the clip.
c) Grasp the bottom (inlet cap) of the holder and turn it counterclockwise to remove it from the
PEEK Housing. Remove the used prefilter. DO NOT DISCARD THE STAINLESS
STEEL PREFILTER ADAPTER!

14 of 21

d) Grasp the PEEK Housing and turn it counterclockwise to remove it from the top (outlet cap)
of the holder. Remove the used analytical cartridge.
e) Remove a new prefilter from the package. The prefilter can be installed in either direction.
f) Push the prefilter firmly onto the end WITH THE O-RING of the Stainless Steel Prefilter
Adapter . Insert the adapter into the bottom (inlet) cap, prefilter facing upward.
g) Place the PEEK Housing into the inlet cap with the arrow pointing in the direction of flow
(bottom to top). Turn it clockwise until it is secured.
h) Remove the end caps from a new analytical cartridge. Position the cartridge with the arrow
pointing in the direction of flow (bottom to top). Push the cartridge firmly into the PEEK
Housing. Connect the PEEK Housing to the outlet cap by turning the outlet cap clockwise
until it is secured.
i)

Go to the Maintain/Instruments screen. Select Do Startup Actions from the Execute

Commands list. Click Start.

j)

Check for leaks and gradually tighten loose fittings as needed.

k) Place the assembled cartridge holder into the clip inside the cartridge thermal block.
l)

Close the cartridge thermal block cover, turning the lever clockwise to lock it.

m) After the startup actions are completed, return the instrument to Ready state.

Cartridge

Prefilter
Prefilter Adapter

15 of 21

To update the prefilter injection counter in CDM:

i)

From the SETUP/Test screen, select Cartridges.

ii) Change the In Use column entry from Yes to No for the current prefilter; a new line will
automatically be generated for the new prefilter.
iii) In the new prefilter entry, enter its lot number (if applicable) in the Lot # column, and type
500 in the Inj. Limit column.
iv) In the In Use column, select Yes.
6) Priming and calibration:
a) A new Analytical cartridge is primed twice before the calibration run.
b) Fill 2 microvials with 1 ml Whole Blood Primer in each and 3 vials with 1 ml of CLRW in
each.
c) Place the vials in barcoded microvial adapters in a sample rack as indicated below (using
BLANK adapters for the CLRW will not count against the number of injections on the
cartridge, nor will results print out):
1. PRIMER
2. PRIMER
3. BLANK (CLRW)
4. BLANK (CLRW)
5. BLANK (CLRW)
6. STOP tube or empty rack
d) Place Blank, Calibrator 1 and 2, and QC in barcoded microvial adapters in a second rack as
indicated below, and place this rack behind the primer rack on the VSS.
NOTE: Allow the instrument to return to READY after the prime is complete before
performing calibration, to eliminate any carryover from the primer:
1. BLANK (QC material or patient sample)
2. CALIBRATOR LEVEL 1
3. CALIBRATOR LEVEL 2
4. CONTROL LEVEL 1 (DB1)
5. CONTROL LEVEL 2 (DB2)
6. STOP tube or empty rack
e) Start the run. When the calibration is complete, check the Slope and Intercept of the
Calibrators on the printout. Slope and intercept are calibrator lot-specific and are updated via
the CD-ROM. If the calibration failed and delta factors are enabled in test setup, the slope
and/or intercept will flag as out of range and the analyzer will stop; if the calibration passed,
analysis will continue. Check current QC ranges for acceptability of results before running
patient samples.

16 of 21

f) Print and file the calibration summary report:

i)

Under the Data Icon, select View Run.

ii) Select the run with the calibration

iii) Select View Sample, select a calibrator
iv) Click Print, select Calibration Summary Report, select OK
v) Date and initial and file the calibration summary report in appropriate binder.

17 of 21

BioRad Variant II TURBO Hemoglobin A1C 2.0

Appendix B
BioRad Variant II TURBO Maintenance
DAILY MAINTENANCE
1) Perform Piston Seal Flush.
a) Fill a 10 mL syringe with CLRW. Insert the syringe into the piston/seal wash port.
b) Slowly depress the syringe plunger until all the water has been dispensed. Leave the water
in the lines.
c) Remove the empty syringe.
2) Check Buffer and Wash levels.
3) Check Waste Container Level and empty as needed.
4) Check Printer Paper Supply.
5) Record Injection Count for Analytical cartridge.
a) Under Setup Icon, select the Test screen
b) Select CARTRIDGES
6) Return the instrument to the READY state.
a) Under Maintain Icon, select RETURN TO ACTIVE
b) The Screen will prompt:
Do you want to perform the automatic warm-up operations?
c) Select NO. Instrument should now be in READY state.
d) Record Pump Pressures:
For Pump A: Under Maintain Icon/PUMP, change %B to 0 (zero) and flow rate to 2.0
mL/min. Select START FLOW and let pump run ~ 2 min. Check for leaks. Select
STOP FLOW if no leaks are detected. Record pump pressure on maintenance log.
For Pump B: Repeat as above but change %B to 100. (Note: Changes to settings here do
not affect settings used for actual analysis.) Difference between Pump A and Pump B
readings should not vary by more than 5%.
e) Under Execute Commands, select DO STARTUP ACTIONS
f) Select START. This process takes approximately 4 minutes, and only needs to be
performed once a shift. If starting a second run on the same day and instrument has gone
into the INACTIVE state, only steps 6a) through d) above are required.
g) When completed, instrument returns to READY state.

18 of 21

MONTHLY MAINTENANCE
1) Exterior and interior surface cleaning
a) Use gauze moistened with CLRW to wipe the exterior surface of the system.
b) Wipe up any fluid inside the chromatography station. Tighten any leaking connections.
c) Clean the interior base with gauze moistened with CLRW.
2) Clean the conveyor belt
a) Under the Maintain Icon, select the Instruments screen
b) Under Execute commands, select CLEANING BELTS
c) Select START
d) When the belts start moving, use gauze moistened with CLRW to wipe the belts.
e) Select STOP when both belts are clean.
3) Clean Sample Needle and Barcode Reader
a) Under the Maintain Icon, select the Instruments screen
b) Under Sampler, select REPLACE NEEDLE
c) After the needle is repositioned, open the automatic sampler cover of the Sampling Station
(when the conveyor belt stops moving).
d) The screen will prompt:
Variant Front End door is open
Select OK
e) Clean the needle with a Kimwipe moistened with CLRW, removing debris. DO NOT USE
BLEACH. Be careful not to bend the needle.
f) Using Kimwipe or lens paper dampened with CLRW, gently wipe the barcode reader face.
BE CAREFUL NOT TO SCRATCH THE BARCODE READER FACE.
g) Replace the sampler cover. Power should be restored to the unit. A self-check is initiated.
h) Click on the Instrument #1 Variant II Turbo icon.
i)

Select RETURN TO READY

j)

Select OK

k) When the instrument returns to the READY state,

l)

Select MOVE NEEDLE HOME

m) The screen will prompt:

It is highly recommended to flush the sampler after replacing the needle.
Would you like to do it now?

19 of 21

n) Select YES
o) When process is complete, return the instrument to READY.
4) Clean the Dilution Well
a) Power off the Variant II TURBO Sampling Station (VSS) using the toggle switch on the right
side of the VSS.
b) Remove the needle cover door.
c) Push the needle gantry back, away from the dilution chamber.
d) Clean the dilution chamber using cotton swabs and CLRW. Remove particulate by filling the
tower with CLRW, then using cotton swabs to clean and soak up loose particulate.
e) Replace the needle cover door.
f) Power on VSS, wait for sampler to complete initialization and flush cycle.
g) Select OK to CDM communication error box, sampler resetting box and rack resetting box,
returning the system back to READY state.

AS NEEDED MAINTENANCE
1) Clean and decontaminate the probe and sampling fluid path
This procedure is performed as needed, generally for troubleshooting purposes. Perform the
decontamination procedure when any of the following symptoms occur:
Carryover from a hemoglobin variant sample generates an identified peak in the next
injection.
High total area counts occur across multiple samples.
Buildup is visible inside the injection valve tubing.
Blood is visible in the dilution chamber.
NOTE: The prefilter must be discarded after the decontamination procedure is
completed.
a) Remove the Analytical cartridge and replace it with the plastic PEEK dummy cartridge. If
the Analytical cartridge is not due for replacement, place green end caps on cartridge and
store for reinstallation after procedure completed. If the Analytical cartridge is due for
replacement, it may be discarded.
b) Disconnect cartridge-to-detector tubing from the cartridge holder. Install decontamination
tubing to the outlet of the cartridge holder.
c) Place outlet side of decontamination tubing into a beaker to collect waste from the
decontamination procedure.

20 of 21

d) Under the Setup Icon,

i)

Select the Test screen

ii) Select DECON_T for New test

iii) The screen will prompt:
Verify that the correct cartridge and reagents are selected for this test. Allow
sufficient time for instrument equilibration
iv) Select OK.
e) Place 5 sample microvials of 5% sodium hypochlorite solution (undiluted bleach) into
adapters in the first 5 positions of a sample rack.
f) Place 5 sample microvials filled with CLRW into adapters in the last 5 positions of the same
sample rack.
g) Prepare 5 mL of a 1:10 dilution of whole blood in CLRW by adding 500 uL of whole blood
to 4.5 mL of CLRW. Mix thoroughly.
h) Place 5 sample microvials, containing 1 mL each of the 1:10 hemolysate solution, into
adapters in the first five positions of a second sample rack.
i)

Place the STOP adapter with an empty sample microvial into position six of the second
sample rack, and place both sample racks on the Sampling Station.

j)

Start the worklist.

i) Under the Run Icon,
ii)

Select START

iii) Select OK
k) When the status returns to READY, remove the plastic PEEK dummy cartridge. Clean the
Analytical cartridge and prefilter holders with a cotton swab moistened with DI water. The
prefilter must be discarded and replaced after the decontamination procedure is completed.
If Analytical cartridge injection limit has been reached, install a new Analytical cartridge and
perform required prime and calibration.
l)

Remove decontamination tubing from the cartridge holder. Discard beaker waste. Reconnect
the cartridge-to-detector tubing, securing both tubing end connections.

m) Under the Setup Icon,

i) Select the Test screen
ii) Select V2_TURBOA1C for New test
iii) The screen will prompt:
Verify that the correct cartridge and reagents are selected for this test. Allow
sufficient time for instrument equilibration
iv) Select O

21 of 21