Standard Operating Procedure
Subject Hemoglobin A1c – Cobas c501/c502
Index Number Lab-4004
Section Laboratory
Subsection Chemistry
Category Departmental
Contact Amy VanLin
Last Revised 7/16/2019
References
Required document for Laboratory Accreditation by the College of American Pathologists (CAP), Centers
for Medicare and Medicaid Services (CMS) and/or COLA.
Applicable To
Employees of the Gundersen Health System and Gundersen Tri-County Hospital laboritories.
Detail
INTENDED USE:
In vitro test for the quantitative determination of % hemoglobin A1C (standardized according to IFCC
transferable to DCCT/NGSP) in whole blood on Roche/Hitachi cobas c systems.
PRINCIPLE:
This method uses Tetradecyltrimethylammonium bromide (TTAB) as the detergent in the hemolyzing
reagent to eliminate interference from leukocytes (TTAB does not lyse leukocytes). Sample pre-
treatment to remove labile HbA1c is not necessary. All hemoglobin variants which are glycated at the β-
chain N-terminus and which have antibody-recognizable regions identical to that of HbA1c are
measured by this assay. Consequently, the metabolic state of patients having uremia or the most
frequent hemoglobinopathies (HbAS, HbAC, HbAE) can be determined using this assay.
Hemoglobin A1C – The HbA1c determination is based on the turbidimetric inhibition immunoassay
(TINIA) for hemolyzed whole blood.
1. Sample and addition of R1 (buffer/antibody reagent): Glycohemoglobin (HbA1c) in the sample
reacts with anti-HbA1c antibody to form soluble antigen-antibody complexes. Since the specific
HbA1c antibody site is present only once on the HbA1c molecule, formation of insoluble
complexes does not take place.
2. Addition of R 3 (buffer/polyhapten reagent) and start of reaction: The polyhaptens react with
excess anti-HbA1c antibodies to form an insoluble antibody-polyhapten complex which can be
determined turbidimetrically.
Hemoglobin – Liberated hemoglobin in the hemolyzed sample is converted to a derivative having a
characteristic absorption spectrum which is measured bichromatically during the pre-incubation
phase (sample + R1) of the above immunological reaction. A separate Hb reagent is consequently
not necessary.
The final result is expressed as % HbA1c and is calculated from the HbA1c/Hb ratio as follows:
Protocol 2 (% HbA1c acc. To DCCT/NGSP): HbA1c (%) = (HbA1c/Hb) x 91.5 + 2.15
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Standard Operating Procedure
CLINICAL SIGNIFICANCE:
Hemoglobin (Hb) consists of four protein subunits, each containing a heme moiety, and is the red-
pigmented protein located in the erythrocytes. Its main function is to transport oxygen and carbon
dioxide in blood. Each Hb molecule is able to bind four oxygen molecules. Hb consists of a variety of
subfractions and derivatives. Among this heterogeneous group of hemoglobins HbA1c is one of the
glycated hemoglobins, a subfraction formed by the attachment of various sugars to the Hb molecule.
HbA1c is formed in two steps by the nonenzymatic reaction of glucose with the N-terminal amino group
of the β-chain of normal adult Hb (HbA). The first step is reversible and yields labile HbA1c. This is
rearranged to form stable HbA1c in a second reaction step.
In the erythrocytes, the relative amount of HbA converted to stable HbA1c increases with the average
concentration of glucose in the blood. The conversion to stable HbA1c is limited by the erythrocyte’s life
span of approximately 100 to 120 days. As a result, HbA1c reflects the average blood glucose level
during the preceding 2 to 3 months. HbA1c is thus suitable to monitor long-term blood glucose control
in individuals with diabetes mellitus. Glucose levels closer to the time of the assay have a greater
influence on the HbA1c level.
The risk of diabetic complications, such as diabetic nephropathy and retinopathy, increases with poor
metabolic control. In accordance with its function as an indicator of the mean blood glucose level,
HbA1c predicts the development of diabetic complications in diabetes patients.
For routine clinical use, testing every 3 to 4 months is generally sufficient. In certain clinical situations,
such as gestational diabetes, or after a major change in therapy, it may be useful to measure HbA1c in 2
to 4 week intervals.
SPECIMEN:
EDTA whole blood. Mix specimen thoroughly before use. The minimum volume required for analysis
directly from collection tubes is 1 mL of whole blood. In case of obvious blood clots in whole blood
sample, either remove the clot(s) manually or reject the sample. Whole blood samples as small as 125
μL may be run in sample cups. Universal precautions apply.
Stability:
3 days at 15-25°C, 7 days at 2-8°C, 6 months at – 15˚ to -25˚C. Freeze only once.
REAGENTS/MATERIALS:
Tina-quant Hemoglobin A1c Gen.3, 150 tests – the reagent cassette is labeled as A1C-3. R1 is in position
A and R3 is in position C. Position B contains H2O for technical reasons.
R1 - Antibody Reagent – MES buffer: 0.025 mol/L; TRIS buffer: 0.015 mol/L, pH 6.2; HbA1c
antibody (ovine serum): ≥0.5 mg/mL; detergent; stabilizers; preservatives
R3 - Polyhapten Reagent – MES buffer: 0.025 mol/L; TRIS buffer: 0.015 mol/L, pH 6.2; HbA1c
polyhapten: ≥8 ug/mL; detergent; stabilizers; preservatives
Hemolyzing Reagent Gen. 2, 51 mL – the reagent cassette is labeled as A1CD2.
Aqueous buffered matrix, pH 7.25; tetradecyltrimethylammonium bromide: 36 g/L; sodium
dihydrogenphosphate monohydrate: 16 mmol/L; sodium monohydrogenphosphate dehydrate: 64
mmol/L; stabilizers; preservatives
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Standard Operating Procedure
Precautions and warnings:
For in vitro diagnostic use. Exercise the normal precautions required for handling all laboratory reagents.
Disposal of all waste material should be in accordance with local guidelines. The SDS are available on
Gladiator.
Storage and Stability:
Tina-quant Hemoglobin A1c Gen.3: Unopened at 2-8°C – up to the stated expiration date. On-board in
use and refrigerated on the c501/c502 – 4 weeks.
Hemolyzing Reagent Gen. 2: Unopened at 2-8°C – up to the stated expiration date. On-board in use and
refrigerated on the c501/c502 – 4 weeks.
EQUIPMENT/INSTRUMENTATION:
Roche c501/c502 analyzer – Refer to the Operator's Manual for operating instructions, maintenance and
troubleshooting.
Calibration:
This method has been standardized against the approved IFCC reference method for the measurement
of HbA1c in human blood and can be transferred to results traceable to DCCT/NGSP by calculation.
Calibration mode (Hb): Linear, 2-point calibration. Calibration mode (HbA1c): Spline, full calibration.
Calibrator:
Always calibrate both assays (Hb and HbA1c) in parallel.
C.f.a.s. HbA1c.
Preparation: Add 2.0 mL of DI water and let stand for 30 minutes. Mix carefully, avoiding foam
formation.
Stability: 2 days at 2-8°C.
Calibration Frequency:
Calibration must be performed once per reagent lot using fresh reagent (i.e. not more than twenty-four
hours since the reagent pack was registered on the analyzer). Renewed calibration is recommended as
follows:
1. If necessary after instrument service or repair
2. If dictated by quality control results
3. Every 29 days
QUALITY CONTROL:
BioRad Diabetes controls, levels 1 and 2
Ready to use.
Product stability:
Stored unopened at -10 to -70˚C, until the stated expiration date.
Stored unopened at 2-8˚C, 6 months but should not be used past the expiration date (note on the
bottle, the date refrigeration temperatures storage begins).
Opened and stored tightly capped at 2-8˚C, 14 days.
Once thawed, do not refreeze the control; discard the remaining material.
This product is shipped under frozen conditions.
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Standard Operating Procedure
Refer to Lab-4405 Quality Control Criteria for Chemistry for interpretation of QC. Each level of Quality
Control should be performed at a minimum:
1. once every twenty-four hours
2. if a new pack of reagent is put in use
3. if a calibration is performed
Implementation
Specimens should be at room temperature before analysis. Mix capped specimens by inversion. Remove
caps before placing specimens in racks with blue stickers.
If there is less than 1 mL of specimen, transfer approximately 200 μL of the mixed specimen to a labeled
Cobas cup and place the cup in position 5 of the #3 white QC rack. Click on QC, STATUS, and sort by the
Control column. Highlight both tests for the A1C-PT control. Click SELECT, SAVE. After the analysis of the
patient is complete, print the report.
Cobas 6000
1. Select the result on the Data Review screen.
2. Press the Global Print button.
3. Select Data Monitor/Report on the left side, choose Report under Print Format, then press Print.
Cobas 8000
1. Select the result on the Data Review screen.
2. Press the Global Print button.
3. Press the Preview button.
4. Go to History
5. Select Result list on left side of screen (check patient name and order)
6. Press Print Out
Place a copy of the patient’s label on the report. Scan the barcode to bring up the correct patient and
testing in EPIC. Manually enter the result into the LIS. Save the labeled printout in the “Manually
Entered Results” folder for the appropriate analyzer.
PROCEDURE NOTES:
Results are reported to the nearest tenth in %.
AMR (Analytical Measurement Range): 4.3-18.8%
Values below 4.3 are reported as <4.3%.
Values below 18.8 are reported as >18.8%.
Reasons for Linearity Flags:
Linearity flags for Hb and/or HbA1c are typically caused by one of the following:
1. <Test (Hb and/or HbA1c) due to abnormally low hemoglobin (Hb) levels in the whole blood
sample. This could occur in very anemic patients or due to pre-analytical errors (e.g., when the
blood is drawn from a central venous catheter, i.e., unintended dilution of whole blood sample
by the infusion).
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Standard Operating Procedure
2. >Test (Hb and/or HbA1c) due to falsely high amounts of erythrocytes in the final measuring
cuvette. This can happen when the whole blood sample is sedimented. In the case of flagged
HbA1c, the HbA1c value is often abnormally high as well.
Handling Recommendations for Linearity Flags (Reference #7):
Handling recommendations for <Test flags (measured value is under the technical limit):
1. Repeat the analysis with the same settings.
2. In case of obvious blood clots in the whole blood sample, either remove the clots manually or
reject the sample.
3. If a <Test flag occurs again, let the erythrocytes in the sample settle without mixing, and repeat
the analysis with the same settings.
Handling Recommendations for >Test flags (measured value is over the technical limit):
1. Remix the whole blood sample by inversion and repeat the analysis with the same settings.
2. If a >Test flag occurs again, dilute the whole blood sample 1:2 with 0.9% NaCl. Remix and repeat
the analysis with the same settings. Report the %HbA1c result from this measurement without
using any dilution correction factors.
CALCULATIONS:
% HbA1c
The reported result for the % HbA1c is calculated on the analyzer from the HbA1c/Hb ration as follows:
(% HbA1c acc. To DCCT/NGSP): HbA1c (%) = (HbA1c/Hb) x 91.5 +2.15
Estimated Average Glucose (Reference #6)
Based on the results of the A1c-Derived Daily Glucose (ADAG) study, the ADA recommends the use of a
new term in diabetes management, estimated average glucose, or eAG. This new calculation is intended
to help health care provides report HbA1c results to patients using the same units that patients see
routinely in blood glucose measurements. The following equation is used to calculate the eAG (mg/dL):
eAG (mg/dL) = (28.7 x HbA1c) – 46.7
INTERPRETATION:
Expected Values: 4.0-5.6%. Hemoglobin A1c values of 5.7-6.4% indicate an increased risk for developing
diabetes mellitus. Hemoglobin A1c values ≥6.5% are diagnostic for diabetes mellitus. In diabetic
patients, HbA1c goals should be discussed with a healthcare provider.
LIMITATIONS:
1. The test is designed only for accurate and precise measurement of HbA1c (%). The individual
results for total Hb and HbA1c concentration should not be reported.
2. The test is not intended for the diagnosis of diabetes mellitus or for judging day-to-day glucose
control and should not be used to replace daily home testing of urine or blood glucose.
3. As a matter of principle, care must be taken when interpreting any HbA1c result from patients
with Hb variants. Abnormal hemoglobins might affect the half-life of the red cells or the in vivo
glycation rates. In these cases even analytically correct results do not reflect the same level of
glycemic control that would be expected in patients with normal hemoglobin.
4. Any cause of shortened erythrocyte survival will reduce exposure of erythrocytes to glucose
with a consequent decrease in HbA1c (%) values, even though the time-averaged blood glucose
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Standard Operating Procedure
level may be elevated. Causes of shortened erythrocyte lifetime might be hemolytic anemia or
other hemolytic diseases, homozygous sickle cell traits, pregnancy, recent significant or chronic
blood loss, etc. Caution should be used when interpreting the HbA1c results from patients with
these conditions.
5. Glycated HbF is not detected by the assay as it does not contain the glycated beta-chain that
characterizes HbA1c. However, HbF is measured in the Total Hb assay and as a consequence,
specimens containing high amounts of HbF (>10%) may result in lower than expected HbA1c
values.
6. For diagnostic purposes, %HbA1c values should be used in conjunction with information from
another diagnostic procedures and clinical evaluations.
Icterus: No significant interference up to an I index of 60 (approximate total bilirubin concentration: 60
mg/dL).
Lipemia: No significant interference up to an L index of 500. There is poor correlation between the L
index (corresponds to turbidity) and triglycerides concentration. (Intralipid concentration 600
mg/dL)
See package insert for additional interference and cross-reactivity studies. The results should always be
assessed in conjunction with the patient’s medical history, clinical examination and other findings.
Special Wash Requirements:
The determination of certain analytes interferes with this assay requiring a special wash step. Refer to
the NaOHD/SMS/SmpCln1+2/SCCS method sheet and the operator manual for further instructions.
REVIEW AND CHANGES:
This document and all attached forms should be reviewed optimally on an annual basis, with 2 years as
the maximum review date. Review will be done by the Technical Leader, Supervisor, Manager, Medical
Director or designated person. Changes require retyping document or form and review by the Medical
Director.
REFERENCES:
1. Roche Tina-quant Hemoglobin A1c Gen.3 – Hemolysate and Whole Blood Application package
insert
2. Roche C.f.a.s. HbA1c package insert
3. Roche Hemolyzing Reagent Gen.2 package insert
4. Roche Hemolyzing Reagent Gen. 3 package insert
5. Roche Cobas 6000 Operator’s Manual
6. Translating the A1c Assay Into Estimated Average Glucose Values. Diabetes Care 31: 1473-1478
(2008)
7. Roche Reagent Bulletin: 07-035UA VV-08872 11/10/17 Tina-quant™ Hemoglobin A1c (HbA1c)
Assays – Linearity Flags on the cobas c 311, 501, 502 and 513 analyzers.
8. Roche NaOHD/SMS/SmpCln1+2/SCCS method sheet
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