References Required document for Laboratory Accreditation by the College of American Pathologists.

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1 Subject Hemoglobin A1c Cobas c501 Index Number Lab-4004 Section Laboratory Subsection Chemistry Category Departmental Contact Benjamin Michel Last Revised 9/12/2016 References Required document for Laboratory Accreditation by the College of American Pathologists. Applicable To Employees of the Gundersen Health System clinical laboratory. Detail INTENDED USE: In vitro test for the quantitative determination of % hemoglobin A1C (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 pretreatment 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 (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 R2 (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 Lab-4004 Hemoglobin A1c Cobas c501 Page 1 of 6

2 CLINICAL SIGNIFICANCE: Hemoglobin (Hb) consists of four protein subunits, each containing a heme moiety, and is the redpigmented 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. Whole blood samples as small as 125 μl may be run in sample cups. Universal precautions apply. Stability: 3 days at C, 7 days at 2-8 C, 6 months at -20 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 H 2 O for technical reasons. R1 - Antibody Reagent MES buffer: mol/l; TRIS buffer: mol/l, ph 6.2; HbA1c antibody (ovine serum): 0.5 mg/ml; detergent; stabilizers; preservatives R3 - Polyhapten Reagent MES buffer: mol/l; TRIS buffer: 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 Lab-4004 Hemoglobin A1c Cobas c501 Page 2 of 6

3 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. 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 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 4 weeks. EQUIPMENT/INSTRUMENTATION: Roche c501 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: C.f.a.s. HbA1c. Always calibrate both assays (Hb and HbA1c) in parallel. 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 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. Lab-4004 Hemoglobin A1c Cobas c501 Page 3 of 6

4 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. 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. Place a copy of the patient s label on the report. 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): % 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). 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 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. CALCUATIONS: 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 Lab-4004 Hemoglobin A1c Cobas c501 Page 4 of 6

5 INTERPRETATION: Expected Values: %. Hemoglobin A1c values of % 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 judging day-to-day glucose control and should not be used to replace daily home testing of 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 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. 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. 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, 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 Lab-4004 Hemoglobin A1c Cobas c501 Page 5 of 6

6 4. Roche Cobas 6000 Operator s Manual 5. Translating the A1c Assay Into Estimated Average Glucose Values. Diabetes Care 31: (2008) Lab-4004 Hemoglobin A1c Cobas c501 Page 6 of 6