M1078. Case Study: Comprehensive Incurred Sample Reanalysis Investigation for Clinical Sample Analysis Nadezhda Kulagina, John Kamerud and Joseph Bower Immunochemistry Services, Covance Laboratories Inc., Chantilly, Virginia Introduction ISR Investigation Results Sample Relationship Table 2. ISR Investigation for Analyte in Human Serum: Comparison of Original and Repeat Results The performance of a validated method employing ligand-binding assay is monitored during samples analysis using appropriate quality control (QC) samples that are typically prepared by spiking the analyte in the matrix of the study samples. Although performance of the spiked QC samples provides a reasonably good measure of method reproducibility, there may still be uncertainty about the reproducibility of results generated for study samples (incurred samples) in which the analyte may have been subjected to a more complex biological system. The February 2008 AAPS Workshop provided recommendations related to the conduct and expectations of the incurred sample reanalysis (ISR) in order to demonstrate reproducibility of bioanalytical or immunochemistry methods for quantification of both small and large molecule drug entities. Figure 3. Evaluation of failed ISR samples per subject and cohort Purpose ■ To discuss incurred sample reanalysis (ISR) investigation for failed ISR samples ■ To evaluate impact on clinical sample analysis Method ■ ■ Sandwich bioassay with electrochemiluminescence (ECL) detection was used in the study (Figure 1) An in-study assessment of the analytical method was performed through reanalysis of a sub-set of samples. The criteria for reanalysis included but was not limited to: • At least 2/3 of reanalyzed samples should be within ± 30 of the original value • Samples (106) selected for reanalysis samples had original concentration values between the Low and High QC values Figure 2. Comparison of original and ISR values. ■ ISR samples demonstrated a relative over recovery as compared to the initial result (11 of 106 ISR samples were lower than the initial and 95 ISR samples were greater than the original result). ■ ISR Plates ISR samples were analyzed on plates ISR-1, ISR-2, ISR-3, and ISR4. All ISR plates contained samples which demonstrated >30% difference from the initial result indicating that the ISR plate was not a primary factor in out-of-range performance. Samples from Caucasian (C) and Asian (A) subjects (3 cohorts) included in ISR. Each subject was represented by at least 6 samples. Asian and Caucasian subjects had a total of 41% and 21 % of ISR samples that exceeded 30% difference, respectively. Out-of-range samples appeared to be grouped by subject (i.e.º a subject has several samples >30% difference or a subject has no samples out-of-range). The relationship of subject or cohort specific to ISR cannot be excluded. This performance difference could potentially be related to selectivity which could be impacted by a change in coating reagent lot. Variability between the original and investigation repeat results with respect to original result, determined by two analysts using both lot A and B coating materials, was within 30% acceptance for the majority of results. Table 3. ISR Investigation for Analyte in Human Serum: Comparison of ISR and Repeat Results Twenty samples from the original ISR analysis were assayed again by analysts I and II that contributed to original and ISR data, respectively. Each analyst’s plate contained the same 20 samples of which 10 samples demonstrated >30% difference from original in the ISR evaluation and 10 samples were within 30% difference from original. Table 1. ISR Investigation for Analyte in Human Serum: Analyst Variability Various Parameters There were no apparent association of using different equipment, lots of Calibrators and QCs, lots of detection reagent and sample freeze-thaw cycle (analyzed within 2–3 cycles) in ISR out-of-range performance. Analyst All ISR plates were completed by analyst II while all initial results were by analysts I and III. Due to the general over recovery of ISR relative to initial results, the impact of an analyst cannot be excluded. Figure 1. Schematics of sandwich assay for detection of analyte. ISR Investigation Plan ■ > 1/3 of the results (38 of 106) were outside 30% difference of original value ■ Initiation of comprehensive ISR investigation. The following variables were evaluated: ISR runs, calibrator and QC performance, sample freeze/thaw, coating and detection reagents, equipment, analyst performance variability, and sample relationship Variability between the ISR and investigation repeat results with respect to ISR result, determined by two analysts using both lot A and B coating materials, showed higher variability especially for previously failed samples suggesting a possible error during ISR run. Coating Material Lot A of coating material was used for all initial results and lot B was used for all ISR plates. Standard reagent qualification was successfully applied prior to changing coating reagent lots however the impact of coating reagent lot cannot be excluded. This reagent difference has the potential to impact selectivity. Conclusions Investigation of sample repeat showed that although all results were within 30% difference of analyst I value, higher result variability (8.7% to 26.2%) was observed between analysts I and II on plates coated with lot A (used to generate original results) coating material. In addition, higher result variability (10.2 to 33.1%) was observed between analyst’s II data generated on plates coated with A and B materials. ■ Despite a thorough investigation for failed ISR no assignable cause was identified. The repeat analysis suggested no apparent association of ISR failure with the use of different lots of coating materials or other assay reagents. Repeat analysis of failed samples demonstrated agreement with the original results, as well as no analyst bias. ■ While the composition of the study population did show some evidence for potential bias, this could not be concluded from the data. Based on the results it was concluded that original results are valid and the method could be used to support a clinical study. Later the method reproducibility was confirmed by ISR results in two other independent clinical studies. M1078 Case Study: Comprehensive Incurred Sample Reanalysis Investigation for Clinical Sample Analysis Nadezhda Kulagina, John Kamerud and Joseph Bower Immunochemistry Services, Covance Laboratories Inc., Chantilly, Virginia Presented at the 2011 AAPS National Biotechnology Conference San Francisco, California 16–18 May 2011 Covance is an independent, publicly held company with headquarters in Princeton, New Jersey, USA. Covance is the marketing name for Covance Inc. and its subsidiaries around the world. The Americas +1.888.COVANCE ( +1.888.268.2623) +1.609.419.2240 Europe/Africa +800.2682.2682 +44.1423.500888 Asia Pacific +800.6568.3000 +65.6.5677333 Web Site: www.covance.com © COPYRIGHT 2011, COVANCE INC.
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