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Hemoglobin A1c .....................................................................................................................................2HgbA1c_VIITurbo_2.0_7-9-12.doc ..................................................................................................................................2

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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 (15–30 °C) before performing the assay.

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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 15-30°C.

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 15-30°C.

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 15-30°C.

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 2-8°C.

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 15-30°C. 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 2-8°C.

Preparation:Reconstitute Whole Blood Primer by adding 1 mL of CLRW to the vial. Allow to stand for 10 – 15 minutes at 15-30°C. Swirl gently to dissolve and ensure complete mixing. Reconstituted Whole Blood Primer is stable for 24 hourswhen 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.

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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 manufacturer’s 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 day’s 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.

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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 # Sample

1 BLANK (QC sample)2 C-DB1 (QC Level 1)3 C-DB2 (QC Level 2)4 to N* Patient SamplesN+1 C-DB1 (QC Level 1)N+2 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. Pre-diluted 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

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IX. RESULTING/REPORTABLE RANGE

a. The run and chromatograms should be reviewed for acceptability.

ITEM CRITERIA ACTION

Total Area Range

1.0 million to 3.5 million OK to report

Outside rangeHbA1c should not be reported.Manually dilute sample and reanalyzed.

Quality ControlValues should be within range. OK to report

Controls not acceptable Repeat controls and any patient samples.

HbA1c Reportable

Range

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

All normal peaks are present

Hb A1a/A1b, LA1c, A1c, P3, P4 and Ao OK to report

A1c and Ao peaks

Correctly identified OK to report

Missing A1c or Ao peaks Repeat analysis

A1c and Ao retention times

Consistently in range (refer to current Cartridge Insert for retention time windows)

OK to report

Inconsistent Repeat analysis

A1c peak shapeSharp and symmetrical OK to report

If not sharp and symmetrical Repeat analysis

Baseline

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

OK to report

Not properly constructed Repeat analysis

HbF (Fetal Hemoglobin)

≤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.

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ITEM CRITERIA ACTION

Labile A1c (LA1c) and

Carbamylated Hemoglobin

(CHb)

LA1c ≤6% does not interfere with assayCHb ≤4% does not interfere with assay

NOTE: CHb elutes in the LA1c window

OK to report

LA1c >6% interfere with assayCHb >4% interfere with assay

There is a peak >4% in the LA1c window

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.

Heterozygoushemoglobins E,

D, S, and CHbA1c result is reportable.

Append ETC “MUHB” to HbA1c result.(MUHB = Mutant hemoglobin present)

P3 or P4 Peak

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.

Variant and/or C windows

Combined area of ≥60% should be suspected of having a homozygous variant or variant-β-thalassemia phenotype

HbA1c should not be reported.Send ETC "MHAB". Send out for alternate testing method.

“Unknown” peak following

Ao peak

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.

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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, re-enter “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% = normal5.7% - 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%

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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

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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 PoolPredicted 1st

OrderPredicted 3rd

OrderDifference

1 2.82 2.58 0.242 5.26 5.34 -0.083 7.28 7.45 -0.174 9.66 9.79 -0.135 11.20 11.26 -0.066 13.70 13.64 0.067 15.59 15.47 0.128 17.61 17.52 0.099 19.56 19.63 -0.07

XIII. 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. β-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%.

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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, 147-184.

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

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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. Bissé, E.; Huaman-Guillen, P.; Hörth, P.; Busse-Grawitz, A.; Lizama, M.; Krämer-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.

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BioRad Variant II TURBO Hemoglobin A1C 2.0Appendix 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.

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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 reagentsfor 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; hand-tighten 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!

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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.

Prefilter

Cartridge

Prefilter Adapter

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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.

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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.

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BioRad Variant II TURBO Hemoglobin A1C 2.0

Appendix BBioRad 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.

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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?”

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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.

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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