In Vitro Diagnostic Medical Device. Store at 2-8 C. Store at C. CAUTION: Consult accompanying documents

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1 E AxSYM HbA1c 3L93-20 ABOL051/R2 Read Highlighted Changes Revised March, 2008 AxSYM HbA1c Customer Service United States: ABBOTT International: Call your Abbott Representative This package insert must be read carefully prior to product use. Package insert instructions must be followed accordingly. Reliability of assay results cannot be guaranteed if there are any deviations from the instructions in this package insert. Key to symbols used List Number Expiration Date In Vitro Diagnostic Medical Device Lot Number Store at 2-8 C Standard Calibrator (A-F) Store at C Control Low, High (L, H) CAUTION: Consult accompanying documents Reagent Pack Consult Instructions for Use Sample Cups Upper limit of Storage Temperature -20 C Matrix Cells Manufacturer Reaction Vessels See REAGENTS section for a full explanation of symbols used in reagent component naming.

2 NAME HbA1c INTENDED USE AxSYM HbA1c is an immunoassay for the quantitative determination of percent hemoglobin A1c (HbA1c) in whole blood samples on the AxSYM System. Percent HbA1c measurements are used in the clinical management of diabetes to assess the long-term efficacy of diabetic control. SUMMARY AND EXPLANATION OF THE TEST HbA1c is formed by the reaction of glucose with the N-terminal amino group of the hemoglobin beta chain. The Diabetes Control and Complications Trial (DCCT) Research Group previously reported a relationship between percent HbA1c and mean blood glucose levels during the preceding 2 3 months. 1 The DCCT study also demonstrated that long-term control of diabetes can prevent complications such as cardiovascular disease, retinopathy, nephropathy, and neuropathy. Measurement of percent HbA1c is the method of choice for monitoring therapy of diabetic patients. 2,3 BIOLOGICAL PRINCIPLES OF THE PROCEDURE In the AxSYM HbA1c assay, whole blood sample is lysed, releasing hemoglobin and HbA1c analyte. Lysed sample is added to the glass fiber matrix that has been coated with Blocking Buffer in a previous step. Hemoglobin and HbA1c analyte are captured on the glass fiber matrix by the binding reaction that occurs between the analyte and the Blocking Buffer. HbA1c is quantified by measuring the amount of HbA1c analyte captured on the matrix cell, using a conjugate of Anti-HbA1c and Alkaline Phosphatase as the signal-generating molecule, and the substrate, 4-Methylumbelliferyl Phosphate (MUP). The AxSYM HbA1c reagents and sample are pipetted in the following sequence: SAMPLING CENTER The whole blood samples can be processed from AxSYM sample cups or from primary blood collection tubes (fluoride oxalate and fluoride EDTA). Potassium EDTA blood collection tubes that have undergone a single freeze-thaw cycle may also be processed from AxSYM sample cups or from the primary blood collection tube. Fresh (non-frozen) potassium EDTA primary whole blood collection tubes may be used if testing is performed in STAT mode and run in groups of eight tubes or less. For further instructions on the use of potassium EDTA whole blood samples, refer to the SAMPLE COLLECTION AND PREPARATION FOR ANALYSIS section. The sample and all AxSYM HbA1c reagents required for one test are pipetted by the Sampling Probe into various wells of a Reaction Vessel (RV). The RV is immediately transferred into the Processing Center. Further pipetting is done in the Processing Center by the Processing Probe. PROCESSING CENTER The whole blood sample is combined with the Lysis Buffer and incubated for 450 seconds. The matrix cell is coated with Blocking Buffer. The lysed sample is diluted with Sample Diluent and transferred to the matrix cell. Hemoglobin and the HbA1c analyte are captured on the glass fiber matrix through interactions with the blocking buffer overcoat. The matrix cell is washed to remove unbound materials. The Anti-HbA1c:Alkaline Phosphatase Conjugate is dispensed onto the matrix cell and binds to the analyte, forming an antigen-antibody complex. The matrix cell is washed to remove unbound materials. The substrate, 4-Methylumbelliferyl Phosphate, is added to the matrix cell and the fluorescent product is measured by the MEIA optical assembly. The concentration of HbA1c in the sample is determined using a previously generated calibration curve. For further information, refer to the AxSYM System Operations Manual, Section 3, Principles of Operation. REAGENTS REAGENT PACK, 100 Tests AxSYM HbA1c Reagent Pack (3L93-20)* 1 Bottle (15.9 ml) Anti-HbA1c Antibody:Alkaline Phosphatase Conjugate in TRIS buffer with protein (bovine) stabilizers. Minimum concentration: 1 µg/ml. Preservative: sodium azide. (Reagent Bottle 1) 1 Bottle (10.9 ml) Sample Diluent in TRIS buffer. Preservative: sodium azide. (Reagent Bottle 2) 1 Bottle (20.5 ml) Blocking Buffer in TRIS buffer. Preservative: sodium azide. (Reagent Bottle 3) 1 Bottle (38.4 ml) Lysis Buffer containing detergent. Preservative: sodium azide. (Reagent Bottle 4) * 3L93-66 includes an AxSYM HbA1c Reagent Pack (100 tests), RVs (100 each) and Matrix Cells (100 each). 3L93-20 includes these items for international shipments. CALIBRATORS AxSYM HbA1c Standard Calibrators (3L93-01) 6 Bottles (2.0 ml each) of AxSYM HbA1c Standard Calibrators. Calibrators A-F contain processed human blood to yield the concentrations (% HbA1c) in the following table. Infection risk. Standard Calibrator HbA1c Concentration (% HbA1c) Preservative: sodium azide. The AxSYM HbA1c Standard Calibrators are matched to internal reference standards. The internal reference standards are traceable to the International Federation of Clinical Chemistry (IFCC) reference standards. The IFCC reference standards are provided with % HbA1c values that are equated to the National Glycohemoglobin Standardization Program (NGSP) % HbA1c values, thus aligning AxSYM % HbA1c values with NGSP/DCCT % HbA1c values. The percent HbA1c for IFCC values can be determined using the following master equation. 4 IFCC Value (% HbA1c) = NGSP Value (% HbA1c) % CONTROLS AxSYM HbA1c Controls (3L93-10) 2 Bottles (5.0 ml each) of AxSYM HbA1c Controls. Controls L and H contain processed human blood to yield the concentrations (% HbA1c) in the following table. Infection risk. Control Preservative: sodium azide. HbA1c Concentration (% HbA1c) HbA1c Concentration Range (% HbA1c) OTHER REAGENTS AxSYM Probe Cleaning Solution (9A35-05) 2 Bottles (220 ml each) AxSYM Probe Cleaning Solution containing 2% Tetraethylammonium Hydroxide (TEAH). Solution 1 (MUP) (8A47-04) 4 Bottles (230 ml each) Solution 1 (MUP) containing 4-Methylumbelliferyl Phosphate, 1.2 mm, in AMP buffer. Preservative: sodium azide. Solution 3 (Matrix Cell Wash) (8A81-04) 4 Bottles (1000 ml each) Solution 3 (Matrix Cell Wash) containing 0.3 M Sodium Chloride in TRIS buffer. Preservatives: sodium azide and antimicrobial agents. Solution 4 (Line Diluent) (8A46) 1 Bottle (10 L) Solution 4 (Line Diluent) containing 0.1 M Phosphate buffer. Preservatives: sodium azide and antimicrobial agents.

3 WARNINGS AND PRECAUTIONS For In Vitro Diagnostic Use. SAFETY PRECAUTIONS CAUTION: This product contains human sourced and/or potentially infectious components. For a specific listing, refer to the REAGENTS section of this package insert. No known test method can offer complete assurance that products derived from human sources or inactivated microorganisms will not transmit infection. Therefore, all human sourced materials should be considered potentially infectious. It is recommended that these reagents and human samples be handled in accordance with the OSHA Standard on Bloodborne Pathogens. 5 Biosafety Level 2 6 or other appropriate biosafety practices 7,8 should be used for materials that contain or are suspected of containing infectious agents. The human whole blood used in Calibrators A-F and in the Controls L and H is nonreactive for HBsAg, HIV-1 Ag or HIV-1 RNA, anti-hiv-1/hiv-2 and anti-hcv or HCV RNA. The Conjugate contains 2-methyl-4-isothiazolin-3-one and is classified per applicable European Community (EC) Directives as: Irritant (Xi). The following are the appropriate Risk (R) and Safety (S) phrases. R43 May cause sensitization by skin contact. S24 Avoid contact with skin. S35 This material and its container must be disposed of in a safe way. S37 S46 Wear suitable gloves. If swallowed, seek medical advice immediately and show this container or label. Calibrators A-F and Controls L and H contain sodium azide and are classified per applicable European Community (EC) Directives as: Harmful (Xn). The following are the appropriate Risk (R) and Safety (S) phrases. R22 Harmful if swallowed. R32 Contact with acids liberates very toxic gas. S35 This material and its container must be disposed of in a safe way. S36 S46 Wear suitable protective clothing. If swallowed, seek medical advice immediately and show this container or label. For product not classified as dangerous per European Directive 1999/45/EC as amended - Safety data sheets are available for professional users on request. Refer to AxSYM System Operations Manual, Section 8, for the safe disposal of these materials. HANDLING PRECAUTIONS Do not use reagent packs beyond the expiration date, or a maximum of 336 cumulative hours on-board the AxSYM System. Do not mix reagents from different reagent packs. AxSYM HbA1c reagents are susceptible to bubbles/foaming and require inspection and removal of bubbles before loading. If bubbles are present, refer to the AxSYM System Operations Manual, Section 9, for instructions on removing bubbles from reagent packs. Do not use Solution 1 (MUP) beyond the expiration date or a maximum of 14 days on-board the AxSYM System. When loading new Solution 1 (MUP), it is important to immediately tighten the instrument cap for MUP to minimize exposure to air. Prolonged exposure of MUP to air may compromise performance. Do not mix matrix cells from different lots. Recalibrate the AxSYM HbA1c assay when matrix cells with a new lot number are used. Refer to the AxSYM System Operations Manual, Sections 7 and 8, for a more detailed discussion of the safety and handling precautions during system operation. STORAGE INSTRUCTIONS REAGENT PACK The AxSYM HbA1c Reagent Pack must be stored at 2-8 C (do not freeze). The Reagent Pack may be used immediately after removal from the refrigerator. When stored and handled as directed, reagents are stable until the expiration date. The AxSYM HbA1c Reagent Pack may be on-board the AxSYM System for a maximum of 336 cumulative hours (for example, 42 eight hour shifts). Recalibration may be required to obtain maximum on-board reagent stability. More frequent use of controls may be required to monitor reagent performance within the same lot. After 336 hours, the reagent pack must be discarded. Refer to the AxSYM System Operations Manual, Sections 2 and 5, and Appendix C, for further information on tracking on-board time. STANDARD CALIBRATORS AND CONTROLS Prior to use, the AxSYM HbA1c Standard Calibrator Pack and AxSYM HbA1c Control Pack must be stored at -20 C or colder. Remove from box and allow calibrators and controls to thaw at room temperature (15-30 C) for 60 to 90 minutes until completely thawed. Mix thoroughly by LOW speed vortexing or by gently inverting prior to use. After each use, immediately return the thawed AxSYM HbA1c Standard Calibrators and Controls to refrigerated storage (2-8 C). Do not refreeze. AxSYM HbA1c Standard Calibrators and Controls can be stored at 2-8 C for up to 30 days or until labeled expiration date (whichever comes first). After 30 days, they must be discarded. OTHER REAGENTS Solution 1 (MUP) must be stored at 2-8 C (do not freeze). It may be used immediately after removal from the refrigerator. It may be on-board the AxSYM System for a maximum of 14 days. After 14 days it must be discarded. The AxSYM Probe Cleaning Solution, Solution 3 (Matrix Cell Wash), and Solution 4 (Line Diluent) must be stored at C. INSTRUMENT PROCEDURE Assay File Installation The AxSYM HbA1c Assay File must be installed on the AxSYM System from the software disk, 3L94-01 or higher, prior to performing the AxSYM HbA1c assay. Refer to the AxSYM System Operations Manual, Section 2, for proper installation procedures. NOTE: AxSYM HbA1c must only be used with AxSYM System Software version 5.0 or higher. AxSYM HbA1c Assay Parameters Assay parameters can be displayed and edited according to the procedure in the AxSYM System Operations Manual, Section 2. Selected assay parameters used for the AxSYM HbA1c assay are listed below. Assay Parameters 1 Long Assay Name (English): Hemoglobin_A1c 6 Abbrev Assay Name (English): HbA1c 11 Assay Number: Selected Result Decimal Places > 1 Refer to the AxSYM System Operations Manual for a detailed description of Instrument Procedures.

4 SAMPLE COLLECTION AND PREPARATION FOR ANALYSIS Follow manufacturer s processing instructions for sample collection tubes. Human whole blood (sodium fluoride/potassium oxalate (fluoride oxalate), sodium fluoride/sodium EDTA (fluoride EDTA)) can be used in the AxSYM HbA1c assay. Provided the following criteria are met, whole blood samples collected in potassium EDTA can be used. Potassium EDTA specimens that have been frozen for a minimum of 2 hours at -20 C and then thawed and mixed completely. Fresh (non-frozen) whole blood samples collected in potassium EDTA can be used if processed from the primary blood collection tube and if run in STAT mode only, which will minimize the potential for red blood settling. Refer to the instructions below for running fresh (non-frozen) potassium EDTA whole blood specimens in STAT mode. Before running an HbA1c test as STAT, check the ORDER STATUS screen. There can be no STAT tests or calibrators pending in the ORDER STATUS screen before a STAT HbA1c test is ordered. Desired HbA1c tests in STAT must not total more than 8 tests. Do not request any assay calibration until STAT HbA1c test requests have completed sampling on the AxSYM system. All potassium EDTA samples (non-frozen and freeze-thaw cycled) should be mixed thoroughly immediately prior to analysis on the AxSYM system. To minimize the effects of red blood cell settling and evaporation, all samples (patient samples, calibrators and controls) should be tested within 3 hours of being thoroughly mixed and placed on board the AxSYM System. Note: AxSYM System Software Version 3.60 and higher offers an Auto Retest/Auto Dilution feature. Due to the mixing requirements discussed above, this feature must not be used with this assay. The AxSYM System does not provide the capability to verify sample type. It is the responsibility of the operator to verify that the correct sample type(s) is (are) used in the AxSYM HbA1c assay. For optimal results, samples should be free of clots or other particulate matter. Do not use heat-inactivated samples. Analyze fresh samples if possible. If testing will be delayed, whole blood samples may be stored at 2-8 C or frozen (-20 C or colder) for up to 14 days prior to being tested. Fresh and stored samples should be mixed thoroughly immediately prior to analysis on the AxSYM System. Multiple freeze/thaw cycles should be avoided. Samples may undergo up to 3 freeze/thaw cycles. Samples must be mixed thoroughly after thawing to ensure consistency in the results. 9 Refer to the AxSYM System Operations Manual, Section 5, for a detailed discussion of on-board sample storage constraints. Inspect samples for bubbles. Remove bubbles prior to analysis. When shipped, samples must be packaged and labeled in compliance with applicable state, federal, and international regulations covering the transport of clinical samples and infectious substances. Samples may be shipped on wet or dry ice. Do not exceed storage temperature and time limitations as cited above. SAMPLE VOLUME The sample volume required to perform a single AxSYM HbA1c test on the AxSYM System varies according to the type of sample container used. For sample cups, a ROUTINE and STAT test requires 150 µl. For every additional HbA1c test performed (ROUTINE or STAT) from the same sample container, an additional 100 µl of sample is required. The sample cup minimum volumes for both STAT and ROUTINE tests are calculated by the AxSYM System. They are displayed on the Order screen at the time the test(s) is (are) ordered and printed in the Orderlist Report. When using Host Order Query, the Order screen information and Orderlist Reports are not available. Refer to the AxSYM System Operations Manual, Section 5, for a description of the Host Order Query Option. For sample volume requirements in Primary or Aliquot Tubes and control volume requirements for multiple AxSYM HbA1c reagent lots, refer to the AxSYM System Operations Manual, Section 5. To obtain the recommended volume requirements for the AxSYM HbA1c Standard Calibrator and Controls, hold the bottles vertically and dispense 6 to 8 drops of each Standard Calibrator or each Control into the respective sample cup. AxSYM HbA1c PROCEDURE Materials Provided 3L93-66 (US) 3L93-20 (International) AxSYM HbA1c Reagent Pack, containing: AxSYM HbA1c Materials Required But Not Provided AxSYM System 3L93-01 AxSYM HbA1c Standard Calibrators 3L93-10 AxSYM HbA1c Controls 8A A A46 9A35-05 AxSYM 8A76-01 Pipettes or pipette tips (optional) to deliver the volumes specified on the Orderlist screen. CAUTION: AxSYM HbA1c Standard Calibrators, AxSYM HbA1c Controls and whole blood samples should be mixed by gentle inversion prior to use. When manually dispensing samples into sample cups, verify that dispensing equipment does not introduce cross-contamination and that it delivers the specified sample volume. Use a separate pipette tip for each sample. Use accurately calibrated equipment. For optimal performance, it is important to follow the routine maintenance procedures defined in the AxSYM System Operations Manual, Section 9. If your laboratory requires more frequent maintenance, follow those procedures. Assay Procedure Sections 5 and 6 of the AxSYM System Operations Manual can be easily removed for use at the instrument. They contain detailed steps for performing assay calibration and sample testing procedures. Prior to ordering tests, confirm that the System inventory of Matrix Cells, bulk solutions, and waste levels are acceptable. The Orderlist Report contains sample placement information and sample cup requirements for all ordered tests. It is recommended that this report be referenced when loading samples into sample segments. When using Host Order Query, the Orderlist Report is not available. Refer to the AxSYM System Operations Manual, Section 5, for a description of the Host Order Query Option. CAUTION: When operating the AxSYM System, always observe the following: The System status must be WARMING, PAUSED, READY or STOPPED before adding or removing sample segments, reagent packs or RVs. Do not open the Interior Waste Door or the AxSYM Processing Center Cover while any test is in process. If opened, all processing will stop. All tests will be terminated and must be repeated. When testing is completed, it is recommended that the AxSYM HbA1c Reagent Pack is removed from the Sampling Center to maximize the on-board reagent pack use. Store at 2-8 C. SAMPLE DILUTION PROCEDURES Patient samples with HbA1c values exceeding 14.5% are flagged with the code > 14.5%. To quantitate the concentration of these samples, perform the Manual Dilution Protocol.

5 Manual Dilution Protocol Patient samples with HbA1c concentrations reported as greater than 14.5% may be diluted using a manual dilution of 1:2. Add 100 µl of the patient sample to 100 µl of a low HbA1c sample (< 6% HbA1c) and mix thoroughly before testing. Calculate the value of the patient sample using the following equation: Sample Value (%) = (Observed Concentration x 2) QUALITY CONTROL PROCEDURES - Low Sample Concentration CALIBRATION The AxSYM HbA1c assay must be calibrated using the AxSYM HbA1c Standard Calibration (6-point) procedure. Standard Calibration To perform an AxSYM HbA1c Standard Calibration, test the Standard Calibrators A, B, C, D, E, and F in duplicate. A single sample of both levels of AxSYM HbA1c controls must be tested as a means of evaluating the assay calibration. Once the AxSYM HbA1c calibration is accepted and stored, all subsequent samples may be tested without further calibration unless: A reagent pack with a new lot number is used. Controls are out of range. Matrix cells with a new lot number are used. The MEIA Optics Verification Update has been performed. Refer to the AxSYM System Operations Manual, Section 6, for: Setting up an assay calibration. When recalibration may be necessary. Calibration Verification. The AxSYM System verifies that the results of an assay calibration meet the specifications assigned to selected validity parameters. An error message occurs when the calibration fails to meet a specification. Refer to the AxSYM System Operations Manual, Section 10, for an explanation of the corrective actions for the error code. Refer to the AxSYM System Operations Manual, Appendix E, for an explanation of the calibration validity parameters that may be used by the AxSYM System. QUALITY CONTROL The minimum control requirement for an AxSYM HbA1c assay is a single sample of both controls tested every 24 hours, each day of use for each reagent lot. Controls may be placed in any position in the Sample Carousel. If the quality control procedures in your laboratory require more frequent use of controls to verify test results, follow those procedures. Ensure that assay controls are within the concentration ranges specified. Refer to the REAGENTS, CONTROLS section of this package insert for AxSYM HbA1c Control ranges. INDICATIONS OF INSTABILITY OR DETERIORATION OF REAGENTS When an AxSYM HbA1c control value is out of the specified range, it may indicate deterioration of the reagents or errors in technique. Associated test results may be invalid and require retesting. Recalibration may be indicated. Refer to the AxSYM System Operations Manual, Section 10, for further troubleshooting information. The AxSYM System has the capability to generate a Levey-Jennings plot of each assay s quality control performance. Refer to the AxSYM System Operations Manual, Section 5 for further information. At the discretion of the laboratory, selected quality control rules may be applied to the quality control data. RESULTS UNITS The results are printed with concentrations expressed in %, where % is equivalent to % HbA1c. CALCULATION AxSYM HbA1c uses a linear regression data reduction method to generate a standard calibration curve. Refer to the AxSYM Systems Operations Manual, Appendix F, for further information. FLAGS Some results may contain information in the Flags field. For a description of the flags that may appear in this field, refer to the AxSYM System Operations Manual Sections 1 and 2. LIMITATIONS OF THE PROCEDURE This assay is not intended for: The diagnosis of diabetes mellitus. 1,2 Monitoring daily glucose control and should not be used to replace daily home testing of urine and blood glucose levels. Analyzing samples from patients with conditions causing shortened red blood cell survival, such as hemolytic diseases, pregnancy, significant acute or chronic blood loss Analyzing samples from patients with total hemoglobin levels < 7 or > 21 g/dl (< 70 or > 210 g/l). Hemoglobinopathies may interfere with glycated hemoglobin (GHb) analysis. 15 It has been shown that samples containing Hemoglobin D or Hemoglobin E variants can interfere when using the AxSYM HbA1c assay. Refer to the SAMPLE COLLECTION AND PREPARATION FOR ANALYSIS section in this package insert for additional information. EXPECTED VALUES Depending on the assay used, HbA1c is approximately 4 to 6% in nondiabetics, 6 to 8% in controlled diabetics and can be as much as 20% in uncontrolled diabetics. 3,16,17 Diabetic patients with HbA1c levels below 7% meet the therapeutic goal of the American Diabetes Association. 18 In a representative study, fluoride oxalate whole blood samples from 100 apparently healthy donors were tested using the AxSYM HbA1c assay. The median AxSYM value was 5.1% HbA1c. Using non-parametric analysis, the central 95% of the population was 4.6 to 6.0% HbA1c. It is recommended that each laboratory establish its own reference range, which may be unique to the population it serves depending upon geographical, patient, dietary or environmental factors. SPECIFIC PERFORMANCE CHARACTERISTICS PRECISION The AxSYM HbA1c assay is designed to have precision of 5% total CV for samples with 8% HbA1c values. Precision was determined as described in the National Committee for Clinical Laboratory Standards (NCCLS) document EP5-A2. 19 The low control, high control, and two samples were assayed in replicates of two at two separate times of the day for 20 days (n = 80 for each panel member). Testing was performed on two AxSYM Systems, using two reagent lots. Data using one reagent lot are summarized in the following tables:* LOW CONTROL NON-DIABETIC SAMPLE DIABETIC SAMPLE HIGH CONTROL

6 LINEARITY/RECOVERY The AxSYM HbA1c assay was designed to have a mean recovery of 100 ± 10% when analyzing samples diluted with a low % HbA1c sample. According to NCCLS document EP6-A 20, five separate pools of high HbA1c samples ( %) were diluted with five separate pools of low HbA1c samples ( %). Additionally, the AxSYM HbA1c Standard Calibrator F (14.5%) was diluted with the AxSYM HbA1c Standard Calibrator A (4.0%), which is represented by sample 4 below. The observed overall mean recovery was 98.9% (observed mean recovery range 93.3% to 103.4%). Data from this study are summarized in the following table:* % Recovery per Dilution Factor (Low:High) ** Sample 4:1 3:2 1:1 2:3 1:4 Mean % Recovery Overall Mean Recovery 98.9 ** % Recovery = Observed Value (% HbA1c) Expected Value (% HbA1c) x 100 SPECIFICITY Effect of Total Hemoglobin Levels on Percent HbA1c The effect of varying amounts of total hemoglobin on the percent HbA1c was assessed using International Federation of Clinical Chemistry (IFCC) reference controls. Samples with varied hemoglobin levels were evaluated. Results from this study showed that the difference was < 0.75% HbA1c for samples with 7 to 21 g/dl hemoglobin in comparison to a sample with a normal hemoglobin level of 14 g/dl.* Hemoglobin Derivatives The AxSYM HbA1c assay has no interference due to labile HbA1c (which has a Schiff base attachment of glucose to HbA). Whole blood samples with percent HbA1c values in the normal and diabetic range were treated with high concentrations of glucose (1400 mg/dl, 3 hours at 37 C) to generate labile HbA1c. Results from this study showed the difference between treated and untreated samples was < 0.75% HbA1c when assayed in the AxSYM HbA1c assay.* Hemoglobin Variants The AxSYM HbA1c assay is designed to have a mean % HbA1c difference from the reference method of < 0.75% HbA1c. Samples with the following hemoglobin variants have been tested on the AxSYM HbA1c assay. Data from this study are summarized in the following table.* Hemoglobin variant n Mean % HbA1c difference from reference method HbC % HbD %** HbE %** HbF 5-0.4% HbJ 8 0.3% HbS % ** Refer to the LIMITATIONS OF THE PROCEDURE section of this package insert. CARRYOVER In a representative study, carryover from a sample with a high HbA1c value (estimated to be 22.7% HbA1c) that was assayed adjacent to a sample of AxSYM HbA1c Low Control (5.0% HbA1c) was < 0.75% HbA1c.* INTERFERENCE The AxSYM HbA1c assay is designed to have a mean potential interference from acetylsalicylate, ascorbic acid, bilirubin, gamma globulin, rheumatoid factor, sodium cyanate, triglycerides and urea of < 0.75% HbA1c at the levels indicated in the following table* as confirmed by a study based on guidance from Clinical and Laboratory Standards Institute (CLSI) document EP7-A2. 21 Interfering Substance Interfering Substance Concentration Acetylsalicylate 50 mg/dl Ascorbic acid 50 mg/dl Bilirubin 20 mg/dl Gamma Globulin 5 g/dl Rheumatoid Factor 200 IU/mL Sodium Cyanate 50 mg/dl Triglycerides 1600 mg/dl Urea 667 mg/dl METHOD COMPARISON Correlation studies based on guidance from NCCLS document EP9-A2 22 were performed on 300 samples to compare the AxSYM HbA1c assay to a commercially available HbA1c assay utilizing HPLC method. Fluoride oxalate and potassium EDTA anti-coagulated whole blood samples were used in the study to assay percent HbA1c. Results from the Passing-Bablok 23 linear comparison are summarized in the following graphs and table:* AxSYM (% HbA1c) AxSYM HbA1c vs HPLC HbA1c HPLC (% HbA1c) AxSYM HbA1c vs HPLC HbA1c 95% Confidence Interval Y Intercept Slope Correlation Coefficient Sample Range (AxSYM): % HbA1c. Sample Range (HPLC): % HbA1c. A bias analysis of AxSYM HbA1c vs. the comparison method (HbA1c HPLC) was performed on the same 300 samples. The following representative data is provided to aid in understanding the difference between these two assays. The average % HbA1c difference bias exhibited by AxSYM HbA1c vs. the comparison HbA1c HPLC method in this study was -0.26% HbA1c. The 95% confidence interval of that average bias is -0.32% to -0.20% HbA1c. Results of the study are summarized below.*

7 Bias (% HbA1c) AxSYM HbA1c vs HPLC HbA1c Bias % HbA1c BIBLIOGRAPHY 1. The Diabetes Control and Complications Trial Research Group. The effect of intensive treatment of diabetes on the development and progression of long-term complications in insulin-dependent diabetes mellitus. N Eng J Med 1993;329(14): Lester E. The clinical value of glycated haemoglobin and glycated plasma proteins. Ann Clin Biochem 1989;26: Goldstein DE, Little RR, Weidmeyer H-M, et al. Glycated hemoglobin: methodologies and clinical applications. Clin Chem 1986;32(10): B Hoelzel W, Weykamp C, Jeppsson J-O, et al. IFCC reference system for measurement of hemoglobin A1c in human blood and national standardization schemes in the United States, Japan, and Sweden: A method-comparison study. Clin Chem 2004; 50(1): US Department of Labor, Occupational Safety and Health Administration, 29 CFR Part , Occupational Exposure to Bloodborne Pathogens. 6. US Department of Health and Human Services. Biosafety in Microbiological and Biomedical Laboratories, Fifth Edition. Washington, DC: US Government Printing Office, January World Health Organization. Laboratory Biosafety Manual. Geneva: World Health Organization, Clinical and Laboratory Standards Institute. Protection of Laboratory Workers from Occupationally Acquired Infections; Approved Guideline Third Edition. CLSI Document M29-A3. Wayne, PA: Clinical and Laboratory Standards Institute, Constantine NT, Abouseif NE, Fox E. To mix or not to mix: the effects of not mixing sera on HIV serologic results. Lab Medicine 1990;21(11): Goldstein DE, Wiedmeyer H-M, England JD, et al. Recent advances in glycosylated hemoglobin measurements. Crit Rev Clin Lab Sci 1984;21(3): Peacock I. Glycosylated haemoglobin: measurement and clinical use. J Clin Pathol 1984;37(8): Horton BF, Huisman TH. Studies on the heterogeneity of haemoglobin, VII. Minor haemoglobin components in haematological diseases. Br J Haematol 1965;II: Lind T, Cheyne GA. Effect of normal pregnancy upon the glycosylated haemoglobins. Br J Obstet Gynaecol 1979;86: Shenouda FS, Cockram CS, Baron MD, et al. Importance of shortterm changes in glycosylated haemoglobin. Br Med J 1982;284(6322): Sacks DB. Hemoglobin variants and hemoglobin A1c analysis: problem solved?. Clin Chem 2003;49(8): Nathan DM, Singer DE, Hurxthal K, et al. The clinical information value of the glycosylated hemoglobin assay. N Engl J Med 1984;310(6): Sacks DB. Carbohydrates. In: Burtis CA, Ashwood ER, editors. Tietz Textbook of Clinical Chemistry. Philadelphia, PA; WB Saunders; 1994: Goldstein DE, Little RR, Lorenz RA, et al. American Diabetes Association. Tests of Glycemia in Diabetes. Diabetes Care 2003;26(1):S National Committee for Clinical Laboratory Standards. Evaluation of precision performance of quantitative measurement methods; Approved Guideline-Second Edition. NCCLS document EP5-A2. Wayne, PA: NCCLS, National Committee for Clinical Laboratory Standards. Evaluation of the linearity of quantitative measurement procedures: a statistical approach; Approved Guideline. NCCLS document EP6-A. Wayne, PA: NCCLS, Clinical and Laboratory Standards Institute. Interference testing in Clinical Chemistry; Approved Guideline-Second Edition. CLSI document EP7-A2. Wayne, PA: Clinical and Laboratory Standards Institute, National Committee for Clinical Laboratory Standards. Method comparison and bias estimation using patient samples; Approved Guideline-Second Edition. NCCLS document EP9-A2. Wayne, PA: NCCLS, Passing H, Bablok W. A new biometrical procedure for testing the equality of measurements from two different analytical methods. J Clin Chem Clin Biochem 1983;21(11): Axis-Shield Diagnostics Ltd, The Technology Park, Dundee, DD2 1XA, UK Distributed by: Abbott Laboratories Inc., Abbott Park, IL 60064, USA and ABBOTT, Wiesbaden, Germany March, , 2008 Axis-Shield For additional product information, please contact your local customer service organization.