ABX Pentra. HbA1c WB. Reagent for quantitative in-vitro determination of Hemoglobin A1c (HbA1c). A11A01702

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1 (Whole blood) Réf.: A11A01702 Volume R1: 1 x 23 ml Volume R2: 1 x 23 ml Volume R3: 1 x 110 ml Volume R4: 2 x 21 ml Volume R5: 1 x 25 ml ABX Pentra Whole blood Reagent for quantitative in-vitro determination of Hemoglobin A1c (HbA1c). 2009/10/06 A93A01022M EN A11A x 23 ml Clinical interest (1-7) HbA1c results from the non-enzymatic fixation of the N-terminal fragment of the β chain of hemoglobin A0. HbA1c levels, which are proportional to blood glucose levels, reflect the average daily glucose concentration during the two months preceding the blood sample. Recent studies have shown that regular HbA1c measurement can lead to changes in the treatment of diabetes and to improved metabolic control as shown by a reduction in HbA1c values. The present in-vitro diagnostic method is thus used for the quantitative determination of hemoglobin A1c (HbA1c), a marker of diabetes, in human blood. The measurements obtained with this procedure can be used for the long-term control of diabetics. Method HbA1c is expressed as a percentage of the total hemoglobin content. In order to determine THb concentrations by spectrophotometry, the different forms of hemoglobin must be converted into a single form having a uniform absorbance spectrum. The HbA1c and total hemoglobin (THb) values in μmol obtained with the test are used to calculate the HbA1c/THb ratio and must not be used individually for the diagnosis. Principle (8): HbA1c and total hemoglobin concentrations are measured, and the ratio is given as a percentage of HbA1c. In order to determine HbA1c as a percentage, five reagents are used: Antibody Reagent (R1) diluted with Diluent I (R5), Agglutinator Reagent (R2), Hemolysis Reagent (R3) and Total Hemoglobin Reagent (R4). The whole-blood sample is mixed with the Hemolysis Reagent(R3) by the analyser: The red blood cells are lysed and the hemoglobin chain is hydrolysed by the action of a protease present in the reagent. The Total Hemoglobin Reagent (R4) is used to determine total hemoglobin. The method is based on the conversion of all forms of hemoglobin into alkaline haematin in an alkaline solution of non-ionic detergent as described in the original procedure by Wolf et al. The reaction is triggered off by the addition of a blood sample pre-treated with the Total Hemoglobin Reagent (R4), resulting in a green colouration of the solution. The conversion of the different types of hemoglobin into alkaline haematin with a defined absorbance spectrum allows the calculation of the total hemoglobin concentration. Hemoglobin is measured using an end-point method at 550 nm. 1 x 23 ml 1 x 110 ml 2 x 21 ml 1 x 25 ml HORIBA ABX SAS BP Montpellier - cedex 4 - France The latex agglutination inhibition test is used to measure specific HbA1c. An agglutinin (synthetic polymer containing multiple copies of the immunoreactive portion of HbA1c) causes the agglutination of the latex particles covered with monoclonal mouse antibodies specific for HbA1c. In the absence of HbA1c in the sample, the agglutinin in the Agglutinator Reagent (R2) and the microparticles covered with Antibody Reagent (R1) agglutinate. The agglutination leads to an increase in the absorbance of the suspension. The presence of HbA1c in the sample reduces the rate of agglutination, for HbA1c enters into competition with the Agglutinator Reagent (R2) at the microparticles' antibody docking sites. The greater the amount of HbA1c in the sample, the lower the agglutination rate. The reaction is measured by absorbance at 550 nm and the agglutination rate is used to calculate the concentration from a calibration curve. The percentage of HbA1c is then calculated using HbA1c and total hemoglobin values in µmol. Form-0846 Rev. 3 Hemoglobin levels are directly proportional to an increase in the OD observed.

2 Reagents ABX Pentra is a multi-reagent kit. Kit contents: Reagent 1: Antibody Reagent 1 x 23 ml Reagent 2: Agglutinator Reagent 1 x 23 ml Reagent 3: Hemolysis Reagent 1 x 110 ml Reagent 4: Total Hemoglobin Reagent 2 x 21 ml Reagent 5: Diluent I 1 x 25 ml Antibody Reagent (R1) contains: particles coupled with HbA1c antibodies (mouse); Bovine Serum Albumin; Buffer; Surfactants; Preservatives. Agglutinator Reagent (R2) contains: Covalent polymer-bonding hapten; Bovine Serum Albumin; Buffer; Preservative; Surfactant. Hemolysis Reagent (R3) contains: Porcine pepsin; Buffer; Preservative. This reagent is also sold separately under the reference A11A Total Hemoglobin Reagent (R4) contains: Sodium Hydroxide; Surfactant. Diluent I (R5) Contains: Preservative ; NaCl. ABX Pentra should be used according to this reagent notice. The manufacturer cannot guarantee its performance if used otherwise. Handling a 1. Mix 1 volume of Antibody Reagent (R1) with 1 volume of Diluent I (R5) into a MBU 100 ml vial, Ref. A11A Carefully mix Agglutinator Reagent (R2) by inverting, and pour into a reagent bottle. 3. Carefully mix Hemolysis Reagent (R3) by inverting, and pour into a reagent bottle. 4. Carefully mix Total Hemoglobin Reagent (R4) by inverting, and pour into a reagent bottle. 5. Allow the reagents to stabilise in the refrigerated ABX Pentra 400 reagent compartment for an hour before use. See diagram below (sector A, B and C are taken as an example): Hemolysis Reagent Reagent 3 Total Hemoglobin Reagent Reagent 4 Agglutinator Reagent Reagent 2 Antibody Reagent Reagent 1 (diluted) Only use the required quantity, and after use immediately place the reagents in a cold-temperature environment in a closed vial. Whole blood samples, calibrator and control do not require any pretreatment. The use of a positive displacement pipette is recommended for collecting whole blood. Note: It is possible to test simultaneously up to 4 specimens. If more than 4 specimens have to be tested, wait for the instrument to turn to the Analysing status before placing the next specimens after homogenization. Assays have to be requested only using the ratio, never request HbA1c and THb assays separately. The ratio formula must be created by using the NGSP ratio (see ABX Pentra 400 User Manual ). Calibrator For calibration, use: ABX Pentra Cal, Ref. A11A01703 (not included) 1 x 8 ml + 5 x 2 ml The total hemoglobin is calibrated with the ABX Pentra Cal (CAL 1) using a target total hemoglobin value. Calibrators and reagents are not lot-dependent and are submitted to an internal quality control at HORIBA Medical. Calibration of the HbA1c method is carried out by using the ABX Pentra Cal, which contains six HbA1c calibrator levels. These calibration procedures are compatible with the NGSP/DCCT certification. Position of the calibrators on the calibrator sample rack: 1. CAL 1 (200 µl) for THb 2. CAL 1 (200 µl) for HbA1c 3. CAL 2 (150 µl) 4. CAL 3 (150 µl) 5. CAL 4 (150 µl) 6. CAL 5 (150 µl) 7. CAL 6 (150 µl) Note: the CAL 1 in position 1 is taken as an example, the CAL 1 can also be placed in position 8, 9 or 10. Control For internal quality control, use: ABX Pentra HbA1C WB Control, Ref. A11A01704 (not included) Normal control: 2 x 0.25 ml (lyophilisate) Pathological control: 2 x 0.25 ml (lyophilisate) a.modification from index L to M: handling modification.

3 Each control should be assayed daily and/or after each calibration. The frequency of controls and the confidence intervals should correspond to laboratory guidelines and country-specific directives. The results must be within the range of the defined confidence limits. Each laboratory should establish a procedure to follow if the results exceed these confidence limits. Materials required but not provided Automated clinical chemistry analyzer: ABX Pentra 400 Calibrator: ABX Pentra Cal, Ref. A11A01703 Control: ABX Pentra Control, Ref. A11A01704 ABX Pentra Hemolysis Reagent, Ref. A11A01730 MBU 100 ml vial, Ref. A11A01759 Standard laboratory equipment Specimen a (9) Whole blood in EDTA. Stability (10): 3 days at C 8 days at 2-8 C 60 days at -20 C Frozen samples must be thawed at room temperature and then carefully mixed before use. Carefully mix the blood samples immediately before carrying out the test so as to guarantee accurate THb results. Reference range (4,11,13,14,15,16) Each laboratory should establish its own reference ranges. The values given here are used as guidelines only. Each laboratory should verify that its standard values are compatible with the NGSP reference values. This technique has been NGSP-certified with 4 to 6% HbA1c as a standard value in non-diabetics, with the therapeutic objective of obtaining values < 7%, as recommended by the ADA (American Diabetes Association). All patient results must be interpreted in the light of the overall clinical examinations of the patient. Limits of the method (11): Most patients have haemoglobin levels of between 4.4 mmol/l and 14.4 mmol/l. However, specialist publications have shown that the haemoglobin levels of patients with severe anaemia may be less than 4.4 mmol/l, and that the haemoglobin levels of patients with polycythaemia may exceed 14.4 mmol/l. Patients known to have these symptoms must be tested using another method if their haemoglobin values are outside of the measurement range. a. Modification from index L to M: modification of specimen stability. Any condition affecting the life cycle of red blood cells (such as haemolytic anaemia or other haemolytic diseases, pregnancy or a significant blood loss...) leads to a reduction in the exposure of these cells to glucose and consequently to a drop in the % of glycated haemoglobin levels (GHb). Ghb % results are not reliable in patients suffering from chronic blood loss and resultant variable erythrocyte life cycles. Samples containing haemoglobin C and S may lead to an increase of the expected HbA1c value. Additionally, samples containing high levels of haemoglobin F (> 15%) may yield HbA1c results lower than those expected. There is no interference with carbamylated and acetylated hemoglobin. This test is not reliable for the diagnosis of diabetes mellitus (14,16). This test is not useful in judging day-to-day glucose control, and should not be used to replace daily home testing of blood glucose (14). Storage and Stability Protect from extreme heat, light and frost. Reagent shelf life (sealed and after opening): The reagents are stable until the expiry date indicated on the label if stored between 2 and 8 C in their original packs and protected from contamination. Stability of reconstituted latex solution: refer to the paragraph "Performance on ABX Pentra 400". Assay Procedure Test instructions for automated systems other than ABX Pentra 400 are available on request. Waste Management Please refer to local legal requirements. General Precautions 1. Reagent, for professional in-vitro diagnostic use only. 2. Antibody Reagent (R1) : Human-derived components have been used in the preparation of this product. Each donor unit has been tested individually and shown by FDA-approved methods to be negative for Hepatitis B surface antigen (HBsAg) and for antibodies to Human Immunodeficiency Virus (HIV-1 and HIV-2) and to Hepatitis C Virus (HCV). Caution. However, as no testing method can rule out the potential risk of infection with absolute certainty, the material should be treated just as carefully as patient specimen, as potentially infectious and handled with appropriate caution. In case of exposure, follow the guidelines of the competent health authorities. Irritant. S24: Avoid contact with skin. S37: Wear suitable gloves. R43: Skin contact may lead to a sensitisation. 3. Agglutinator Reagent (R2): Irritant. S24: Avoid contact with skin. S37: Wear suitable gloves. R43: Skin contact may lead to a sensitisation.

4 4. Antibody Reagent (R1), Agglutinator Reagent (R2), Hemolysis Reagent (R3) and Total Hemoglobin Reagent (R4): Avoid contact with the eyes, skin and clothing. Wash hands carefully with water after handling the product. Do not ingest. 5. Diluent I (R5): Irritant. R36/38: May cause eye and skin irritations. R43: Skin contact may lead to a sensitisation. 6. The reagent vials should be discarded after use. 7. Please refer to the MSDS associated with the reagent. Performance on ABX Pentra 400 a The performance data listed below has been obtained on the ABX Pentra 400 analyser. Number of tests: 381 Tests Shelf life of reconstituted latex solution: The shelf life of the reconstituted latex solution is 60 days if stored at 2-8 C after reconstitution. Only use the required quantity, and immediately place the reagents in a cold-temperature environment after use. Sample volume (after treatment): Hb: 25 µl/test HbA1c: 3 µl/test Detection limit: The detection limit of total hemoglobin is determined according to CLSI (NCCLS), EP17-A protocol (17) and equals 1.60 µmol/l (0.42 g/dl). Minimum interpretation limit: The minimum interpretation limit (MIL) of HbA1c is evaluated using multiple determination of low concentration specimen and equals 0.17 µmol/l (0.05 g/dl). Accuracy and Precision: Repeatability (within-run precision) 3 specimens of low, medium and high concentration and 2 controls are tested 20 times according to the recommendations found in the Valtec protocol (18). Mean value %HbA1c CV % Control specimen Control specimen Specimen Specimen Specimen Reproducibility (run-to-run precision) 3 specimens of low, medium and high levels and 2 controls have been tested in duplicate for 20 days (2 series per day) according to the recommendations found in the CLSI (NCCLS), EP5-A2 protocol (19). Mean value %HbA1c CV % Control specimen Control specimen Specimen Specimen Specimen Linearity and Measuring Range: The reagent linearity is determined according to the recommendations found in the CLSI (NCCLS), EP6-A protocol (20). Low linearity: for Total hemoglobin: 1.60 µmol/l (0.42 g/dl) for HbA1c: 0.17 µmol/l (0.05 g/dl) High linearity: for Total hemoglobin: µmol/l (22.41 g/dl) for HbA1c: 10.8 µmol/l (2.85 g/dl) The range of the total hemoglobin method in which the %HbA1c is accurate is contained between 17.5 and 85.0 µmol/l (4.61 and g/dl). The assay confirmed a measuring range from 4.46 to % HbA1c. Samples must not be diluted in case of HbA1c concentration higher than the highest calibration point of µmol/l (2.85 g/dl). In the case where the concentration in HbA1c is higher than the highest calibration point, samples must not be diluted. However, it is possible to calculate the lowest %HbA1c value of the sample with the concentration in THb obtained and gives the result as: Higher than...xxx. Example: if the THb concentration is 44.0 µmol/l (11.6 g/dl), then the lowest %HbA1c value for the method is: - in µmol/l: (10.80)/44.00 x 100 = %HbA1c (apply on this results the NGSP correction factor = 15.9%). The result of %HbA1c for this sample is : Higher than 15.9%. - in g/dl (2.85)/11.6 x 100 = 24.6 %HbA1c (apply on this results the NGSP correction factor = 15.9%). The result of %HbA1c for this sample is : Higher than 15.9%. Correlation: 118 whole blood patient samples have been correlated with a commercial reagent taken as reference according to the recommendations found in the CLSI (NCCLS), EP9-A protocol (21). Values ranged from 4.53 to % of HbA1c. The equation for the allometric line obtained using Passing-Bablock regression procedure (22) is: Y = 1.03 X (%HbA1c) with a correlation coefficient r 2 = a.modification from index L to M: modification of the performance data.

5 Interferences: Triglycerides: No significant influence is observed up to 7 mmol/l (612.5 mg/dl) (as Intralipid, representative of lipemia). Total Bilirubin: No significant influence is observed up to 561 µmol/l (32.8 mg/dl) Direct Bilirubin: No significant influence is observed up to 587 µmol/l (34.3 mg/dl) Ascorbic Acid: No significant influence is observed up to 3.40 mmol/l (59.8 mg/dl) Labile No interference with the labile hemoglobin. hemoglobin: Other limitations are given by Young as a list of drugs and preanalytical variables known to affect this methodology (23, 24). Calibration Stability: The reagent is calibrated on Day 0. The calibration stability is checked by testing 2 control specimens. The calibration stability is 30 days. Note: A recalibration is recommended when reagent lots change, when the latex suspension is changed and when quality control results fall outside the range established. NGSP Ratio a : Y = (581400*(HbA1c/Hb) )/10000 Application release for HbA1c assay (A1c-WB) b : 6.xx Application release for total hemoglobin assay (THb-WB) c : 7.xx Warning It is the user's responsibility to verify that this document is applicable to the reagent used. Reference 1. Mayer TK and Freedman ZR: Protein glycosylation in diabetes mellitus: A review of laboratory measurements and of their clinical utility. Clin. Chim. Acta 127; (1983). 2. Baynes JW.Bunn HF, Goldstein DE, et al : National Diabetes Group; Report of the expert committee on glucosylated hemoglobin. Diabetes Care 7; (1984). 3. Koenig RJ, Peterson CM, Kilo C. et al: Hemoglobin A1c as an indicator of the degree of glucose intolerance in diabetes. Diabetes 25; (1976). 4. Nathan DM, Singer DE, Hurxthal K, and Goodson JD: The clinical information value of the glycosylated hemoglobin assay. NE J. Med. 310; (1984). a.modification from index L to M: new NGSP ratio. b.modification from index L to M: new application release. c.modification from index L to M: new application release. 5. McCarren M: DCCT; and it works: Intensive therapy reduces the risk of diabetic eye, kidney, and nerve disease. Diabetes forecast (September 1993). 6. Larsen ML, Horder M, and Mogensen EF: Effect of long-term monitoring of glycosylated hemoglobin levels in insulindependent diabetes mellitus. NE. J. Med. 323; (1990). 7. Nathan DM: Hemoglobin A 1c. Infatuation or the real thing? NE. J. Med. 323; (1990). 8. Wolf HU, Lang W, and Zander R; Alkaline haematin D-575, a new tool for the determination of haemoglobin as an alternative to the cyanhaemoglobin method. Clin. Chim. Acta. 136; (1984). 9. Guder W.G., Zawta B., The Quality of Diagnostics Samples. Samples: From the Patient to the Laboratory. 1 st ed. Guder W.G., Narayanan S., Zawta B., (WHILEY-VCH, Darmstadt, Germany), (2001), HORIBA Medical Internal studies. 11. Knowles BJ, Haigh WB, and Michaud GC: A monoclonal antibodybased immunoassay for hemoglobin A 1c. Diabetes 35; Supplement: 94A (1986). 12. Sacks D.B., Carbohydrates, Tietz Textbook of Clinical Chemistry and Molecular Diagnostics. 4 th Ed., Burtis C.A., Ashwood E.R., Bruns D.E., (Elsevier Saunders eds., St Louis, USA), (2006), Goldstein DE, Little RR, Wiedmayer HM, et al: Glycated hemoglobin: Methodologies and clinical applications Clin. Chem. 32; (1986). 14. Burtis CA and Ashwood ER, eds: Tietz Textbook of Clinical Chemistry, 2 nd edition, WB Saunders Company, Philadelphia, PA. p.2021 (1994). 15. Diabetes Care 2002; 25 (suppl. 1); Ellis G. Diamandis EP, Giesbrecht EE, et al. An automated «high pressure» liquid chromatographic assay for hemoglobin A 1c. Clin. Chem. 30: (1984). 17. Protocols for determination of limits of detection and limits of quantitation, Approved Guideline, CLSI (NCCLS) document EP17-A, Vol. 24, No. 34, Vassault A., Grafmeyer D. Naudin C. et al., Protocole de validation de techniques (document B), Ann. Biol. Clin., 1986, 44, Evaluation of Precision Performance of Clinical Chemistry Devices, Approved Guideline, CLSI (NCCLS) document EP5-A2, Vol. 24, No. 25, august Evaluation of the Linearity of Quantitative Analytical Methods, Approved Guideline, CLSI (NCCLS) document EP6-A, Vol. 23, No. 16, april Method Comparison and Bias Estimation Using Patient Samples, Approved Guideline, 2 nd ed., CLSI (NCCLS) document EP9-A2, Vol. 22, No. 19, Passing H, Bablock W. A new biometrical procedure for testing the equality of measurements from two different analytical methods. J. Clin. Chem. Clin. Biochem. (1983) 21: Young DS. Effects of Drugs on Clinical Laboratory Tests. 4th Edition, Washington, DC, AACC Press (1997) 3: Young DS. Effects of Preanalytical Variables on Clinical Laboratory Tests. 2nd Edition, Washington, DC, AACC Press (1997) 3:

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