USING THE ACCESS AMH ASSAY IN YOUR LABORATORY

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1 INFORMATION BULLETIN USING THE ACCESS AMH ASSAY IN YOUR LABORATORY /////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////// ACCESS AMH ASSAY FOR USE WITH THE ACCESS 2 IMMUNOASSAY SYSTEM * * The Access AMH assay will be available for use with the UniCel DxI 600 and 800 systems and the UniCel DxC 600i system in the fourth quarter of Beckman Coulter is pleased to announce the release of our new Access AMH assay for use with the Access 2 Immunoassay System. Access AMH is the latest development from our rich fifteen year history and experience with AMH testing, which includes the AMH Gen II ELISA assay and the Immunotech EIA AMH/MIS assay. The Access AMH assay offers your laboratory: Consistent and standardized results with AMH Gen II through the use of identical antibodies and calibration. Improved support of fertility assessment through increased sensitivity and precision at the low end of the analytical measuring range. Improved accuracy of patient results from calibrators prepared with recombinant human AMH. Efficient and cost effective results with less technician handling time and increased ease of use compared to manual assays. Speed and flexibility through fully automated instrumentation. Increased stability from lyophilized calibrators. LAB FORWARD 2014 Beckman Coulter, Inc. Page 1 of 10

2 ASSAY CHARACTERISTICS ASSAY CHARACTERISTIC AMH ASSAY Reagent Name Access AMH Reagent Part Number B13127 Calibrator Name Access AMH Calibrators Calibrator Part Number Calibrator Concentrations (Approximate) QC Name QC Part Number QC Concentrations (Approximate) B , 0.16, 0.6, 4, 10, and 24 ng/ml (0, 1.1, 4.3, 29, 71, and 171 pmol/l) Access AMH QC B , 5, 15 ng/ml (7.1, 36, 107 pmol/l) Assay Format One-step sandwich Sample Type Serum, plasma (lithium heparin) Sample Volume 20μL Time to First Result (Approximate) Limit of Blank (LoB) Limit of Detection (LoD) Reportable Measuring Range (Including Dilution) Imprecision Hook Effect 40 minutes 0.01 ng/ml (0.07 pmol/l) 0.02 ng/ml (0.14 pmol/l) 0.02 to ~ 24 ng/ml (0.14 to ~ 171 pmol/l) 10.0% for values 0.16 ng/ml No hook effect up to 260 ng/ml LABORATORY OPERATING ESSENTIALS ORDERING INFORMATION Access AMH Reagent Packs (2 reagent packs, 50 tests/pack) Access AMH Calibrators (S0 - S5; 1 vial/level, 2 ml/vial) Access AMH Controls (3 levels; 2 vials/level, 2 ml/vial) CATALOG NUMBER B13127 B13128 B13129 ACCESS AMH SOFTWARE CODES FOR THE ACCESS 2 SYSTEMS Test Name / LIS Code / DxC Mnemonic AMH Test ID 293 Calibrator ID DL2000 and Remisol Advance Data Manager AMHCal A Beckman Coulter, Inc. Page 2 of 10

3 SENSITIVITY Representative data for Limit of Blank, Limit of Detection, and Limit of Quantitation are provided for illustration only. The performance obtained in individual laboratories may vary. LIMIT OF BLANK (LoB) The Access AMH assay was designed to have a Limit of Blank (LoB) of 0.01 ng/ml ( 0.07 pmol/l). In one study, LoB was tested using a protocol based on CLSI EP17-A2. 1 A total of 240 replicates of a zero analyte sample, the Access AMH S0 Calibrator, were measured in 12 runs using multiple reagent packs and calibrator lots on multiple Access 2 Systems. This study determined the LoB for Access AMH to be ng/ml (0.017 pmol/l). LIMIT OF DETECTION (LoD) The Access AMH assay was designed to have a Limit of Detection (LoD) of 0.02 ng/ml ( 0.14 pmol/l). In one study, LoD was tested using a protocol based on CLSI EP17-A2. 1 Nine replicates each from seven low-level samples were measured using multiple reagent pack lots and one calibrator lot in ten runs on multiple Access 2 Systems. This study determined the LoD for Access AMH to be ng/ml (0.035 pmol/l). LIMIT OF QUANTITATION (LoQ) The Access AMH assay was designed to have a Limit of Quantitation (LoQ) of 0.08 ng/ml ( 0.57 pmol/l). In one study, LoQ was tested using a protocol based on CLSI EP17-A2. 1 Nine replicates of seven low-level samples were measured using multiple reagent pack lots and one calibrator lot in ten runs on multiple Access 2 Systems. This study determined the LoQ for Access AMH to be ng/ml (0.071 pmol/l). LINEARITY Representative data for linearity are provided for illustration only. The performance obtained in individual laboratories may vary. Based on CLSI EP6-A, 2 one high sample (> 24 ng/ml) and one low sample (< 0.02 ng/ml) were mixed to make seven evenly distributed sample concentrations. Four replicates of the seven mixed samples, eight replicates of the low sample, and four replicates of the high sample were run on a single Access 2 Immunoassay System. The Access AMH assay was designed to be linear, with a maximum deviation from linearity of 5.0% for samples > 0.16 ng/ml, and 0.04 ng/ml for samples 0.16 ng/ml. One study, analyzed using a polynomial regression method demonstrated a maximum deviation from linearity of 2.45% for samples > 0.16 ng/ml, and < 0.00 ng/ml for samples 0.16 ng/ml Beckman Coulter, Inc. Page 3 of 10

4 EXPECTED VALUES Each laboratory should establish its own reference intervals and limits to ensure the proper representation of specific populations. In one study, AMH concentrations were measured in serum samples collected from apparently healthy adult females, adult males, and newborns using the Access AMH assay on the Access 2 Immunoassay System. ADULT REFERENCE GROUP AGE RANGE (YEARS) N MEDIAN ng/ml (pmol/l) 95% RI ng/ml (pmol/l) Females Females Females Females Females Females Males > (26.49) 2.27 (16.21) 1.88 (13.43) 1.62 (11.60) 0.29 (2.05) 0.01 (0.06) 4.87 (34.77) ( ) ( ) ( ) ( ) ( ) ( ) ( ) PEDIATRIC REFERENCE GROUP AGE RANGE (DAYS) N MEDIAN ng/ml (pmol/l) 95% RI ng/ml (pmol/l) Males Females (335.17) 0.16 (1.17) ( ) ( ) 2014 Beckman Coulter, Inc. Page 4 of 10

5 IMPRECISION Representative data for imprecision are provided for illustration only. The performance obtained in individual laboratories may vary. The Access AMH assay exhibits total imprecision 10.0% at concentrations 0.16 ng/ml, and total standard deviation (SD) ng/ml at concentrations < 0.16 ng/ml. One study, using human serum samples involved a total of 40 assays, with two replicates per assay, over 20 days. The following data were calculated based on CLSI EP5-A2 3 guidelines. SAMPLE GRAND MEAN (n = 80) SD WITHIN RUN BETWEEN RUN TOTAL IMPRECISION % CV SD % CV SD % CV Sample N/A N/A N/A Sample Sample Sample DILUTION RECOVERY Representative data for dilution recovery are provided for illustration only. The performance obtained in individual laboratories may vary. SAMPLE TYPE SAMPLE DILUTION LEVEL OBSERVED DOSE EXPECTED DOSE RECOVERY (%) MEAN RECOVERY (%) Plasma 1 Plasma 2 Neat / % 1/ % 1/ % 1/ % Neat / % 1/ % 1/ % 1/ % 94.0% 92.1% 2014 Beckman Coulter, Inc. Page 5 of 10

6 SAMPLE TYPE SAMPLE DILUTION LEVEL OBSERVED DOSE EXPECTED DOSE RECOVERY (%) MEAN RECOVERY (%) Plasma 3 Serum 4 Serum 5 Serum 6 Neat / % 1/ % 1/ % 1/ % Neat / % 1/ % 1/ % 1/ % Neat / % 1/ % 1/ % 1/ % Neat / % 1/ % 1/ % 1/ % 88.5% 99.0% 92.8% 94.3% 2014 Beckman Coulter, Inc. Page 6 of 10

7 ANALYTICAL SPECIFICITY / INTERFERENCES Representative data for analytical specificity and interferences are provided for illustration only. The performance obtained in individual laboratories may vary. Serum samples with AMH concentrations of 1 and 5 ng/ml (7.1 and 36 pmol/l) were spiked with multiple concentrations of the substances listed in the table and run on a single Access 2 Immunoassay System. The values were calculated as described in CLSI EP7-A2. 4 Interference was determined by testing controls (no interfering substance added) and matched test samples (with interfering substance added). There was no significant interference (exceeding a 10% shift in dose) observed when the following substances were tested at the indicated concentrations. SUBSTANCE HIGHEST CONCENTRATION ADDED Acetaminophen Acetylsalicylic Acid Ascorbic Acid Bilirubin (conjugated) Bilirubin (unconjugated) Biotin Gamma Globulin Hemoglobin Heparin (low molecular weight) Ibuprofen Multi-vitamin Intralipids Total Protein (human serum albumin) Uric Acid 20 mg/dl 65 mg/dl 170 μmol/l 43 mg/dl 40 mg/dl 735 nmol/l 60 mg/dl 300 mg/dl 3000 U/L 50 mg/dl 1% (v/v) 37 mmol/l 12 g/dl 1.4 mmol/l Serum samples with AMH concentrations of 1 and 5 ng/ml (7.1 and 36 pmol/l) were spiked with multiple concentrations of the substances listed in the table and run on a single Access 2 Immunoassay System. The values were calculated as described in CLSI EP7-A2. 4 There was no significant cross-reactivity (exceeding 5.0% cross-reactivity) observed when the following substances were tested at the indicated concentrations. SUBSTANCE HIGHEST CONCENTRATION ADDED Inhibin A Activin A hlh hfsh TGF β ng/ml μg/ml 100 miu/ml 115 miu/ml 65 ng/ml 2014 Beckman Coulter, Inc. Page 7 of 10

8 METHODS COMPARISON The representative data for method comparisons are provided for illustration only. The performance that is observed in individual laboratories may vary. ACCESS AMH (REF B13127) vs. AMH GEN II (REF A79765) N RANGE OF OBSERVATIONS INTERCEPT SLOPE (95% CI) CORRELATION COEFFICIENT (r) BIAS to (0.89 to 0.94) % to (0.92 to 0.97) % 25 Access AMH vs AMH Gen II Method Comparison (0.16 to 21.7 ng/ml samples) 10 Access AMH vs AMH Gen II Method Comparison (0.16 to 9.88 ng/ml samples) 20 8 Access AMH Access AMH Passing & Bablok fit = ( x) r = 0.99, n = Passing & Bablok fit = ( x) r = 0.99, n = AMH Gen II AMH Gen II 2014 Beckman Coulter, Inc. Page 8 of 10

9 EVALUATION OF FRESH SERUM SAMPLES AND FROZEN STORAGE In one study using the Access AMH assay, 49 serum samples were tested within eight hours of a blood draw and then frozen at -20 C for varying time points between one and five weeks. A comparison between samples that were tested fresh and samples that had been frozen gave the following statistical data, using Passing Bablok regression: BIAS 95% CI Constant to 0.01 Proportional to Access 2 Serum Frozen Identity Passing & Bablok (I) fit ( x) Access 2 Serum Fresh 2014 Beckman Coulter, Inc. Page 9 of 10

10 AMH MEASUREMENT IN WHOLE BLOOD In one study using the Access AMH assay, five samples were drawn in serum gel tubes and remained unseparated at room temperature for over 24 hours. The following results were obtained: SERUM SAMPLE DAY 0 24 HOURS % DIFFERENCE % % % % % CONTACT INFORMATION For more information about the Access AMH assay, please contact your local Beckman Coulter representative. REFERENCES 1. Approved Guideline - Evaluation of Detection Capability for Clinical Laboratory Measurement Procedures, EP17-A2. June Clinical and Laboratory Standards Institute. 2. Approved Guideline - Evaluation of Linearity of Quantitative Measurement Procedures: A Statistical Approach, EP6-A. April Clinical and Laboratory Standards Institute. 3. Approved Guideline - Evaluation of Precision Performance of Quantitative Measurement Methods, EP5-A2. August Clinical and Laboratory Standards Institute. 4. Approved Guideline - Interference Testing in Clinical Chemistry, EP7-A2. November Clinical and Laboratory Standards Institute. Beckman Coulter, the stylized logo, Access, UniCel, and DxI are trademarks of Beckman Coulter, Inc., and are registered with the USPTO. For Beckman Coulter s worldwide office locations and phone numbers, please visit 8/ Beckman Coulter, Inc. Printed in U.S.A. Page 10 of 10