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BACKGROUND: Use of nucleic acid testing (NAT) in donor infectious disease screening improves transfusion safety. Advances in NAT technology include improvements in assay sensitivity and system automation, and real-time viral target discrimination in multiplex assays. This article describes the sensitivity and specificity of cobas MPX, a multiplex assay for detection of human immunodeficiency virus (HIV)-1 Group M, HIV-2 and HIV-1 Group O RNA, HCV RNA, and HBV DNA, for use on the cobas 6800/8800 Systems. STUDY DESIGN AND METHODS:The specificity of cobas MPX was evaluated in samples from donors of blood and source plasma in the United States. Analytic sensitivity was determined with reference standards. Infectious window periods (WPs) before NAT detectability were calculated for current donor screening assays. RESULTS:The specificity of cobas MPX was 99.946% (99.883%-99.980%) in 11,203 blood donor samples tested individually (IDT), 100% (99.994%-100%) in 63,012 donor samples tested in pools of 6, and 99.994% (99.988%-99.998%) in 108,306 source plasma donations tested in pools of 96. Seven HCV NAT-yield donations and one seronegative occult HBV infection were detected. Ninety-five percent and 50% detection limits in plasma (IU/mL) were 25.7 and 3.8 for HIV-1M, 7.0 and 1.3 for HCV, and 1.4 and 0.3 for HBV. The HBV WP was 1 to 4 days shorter than other donor screening assays by IDT. CONCLUSION: cobas MPX demonstrated high specificity in blood and source plasma donations tested individually and in pools. High sensitivity, in particular for HBV, shortens the WP and may enhance detection of occult HBV. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes. ABBREVIATIONS: CASThe studies described in this article were sponsored by Roche Molecular Systems, Inc.Received for publication May 25, 2017; revision received November 13, 2017; and accepted November 17, 2017. doi:10.1111/trf.14457 (HIV, HBV, or HCV) during the primary multiplex screen, eliminating the need for a second round of discriminatory testing.Assays used for testing blood donations require a high level of sensitivity to assure detection of infectious blood units but at the same time must also have high specificity to avoid wastage of safe donations and unnecessary donor deferral. The cobas MPX test for use on the cobas 6800/8800 Systems (Roche Molecular Systems, Inc.[RMS]) is a qualitative in vitro nucleic acid screening test (NAT) for the direct detection of HIV Type 1 (HIV-1) Group M RNA, HIV-1 Group O RNA, HIV Type 2 (HIV-2) RNA, HCV RNA, and HBV DNA in human plasma and serum. Results are simultaneously detected and discriminated for HIV, HCV, and HBV. This article describes the results of preclinical and clinical performance studies evaluating the sensitivity and specificity of this new donor screening test. MATERIALS AND METHODS Assays and syste...

Background: Limit of detection (LOD) is an important performance characteristic of clinical laboratory tests.Verification, as recommended by the CLSI EP17-A2 guideline, is done by testing a sample with a claimed LOD concentration. Claimed LOD is verified if the 95% CI for the population proportion, calculated from observed proportion of positive results, contains the expected detection rate of 95% (CLSI EP17-A2; Clin Chem 2004;50:732-40). Claimed LOD, verification sample concentration, and observed rate of positive results are subjects to systematic and random errors that can cause false failure or false acceptance of the LOD verification. The aim of this study was to assess the probability to pass or fail verification of claimed LOD with various numbers of tests as function of the ratio of test sample concentration and actual LOD for PCR-based molecular diagnostics tests and provide recommendations for study design. Methods:A method of calculating the probability of passing the claimed LOD verification following CLSI EP17-A2 guideline recommendations, based on the Poisson-binomial probability model, have been developed for PCRbased assays. Results: Calculations and graphs have shown that the probability of passing LOD verification depends on the number of tests and has local minima and maxima between 0.975 and 0.995 for the number of tests from 20 to 1000 on samples having actual LOD concentration. The probability of detecting the difference between claimed LOD and actual LOD increases with the number of tests performed. Graphs and tables with examples are included. Conclusions: Method, tables, and graphs helping in planning LOD verification study in molecular diagnostics are provided along with the recommendations on what to do in case of failure to verify the LOD claim. IMPACT STATEMENTProposed methods will help with planning manufacturer-claimed LOD verification with high probabilities of passing verification when claimed LOD does not exceed the actual LOD significantly and failing verification otherwise. Verification of LOD will assure that the analytical sensitivity of molecular diagnostics assays is adequate for (a) minimizing risk for recipients of blood donations, (b) reliable screening of patients for cervical cancer, (c) reliable companion diagnostics, (d) reliable monitoring of treatment of patients with viral and bacterial infections, etc. The proposed method advances the method recommended in the CLSI EP17-A2 guideline consisting of testing the hypothesis of equality between the actual and claimed LOD in verification studies.

Background: Errors of calibrator-assigned values lead to errors in the testing of patient samples. The ability to estimate the uncertainties of calibrator-assigned values and other variables minimizes errors in testing processes. International Organization of Standardization guidelines provide simple equations for the estimation of calibrator uncertainty with simple value-assignment processes, but other methods are needed to estimate uncertainty in complex processes. Methods: We estimated the assigned-value uncertainty with a Monte Carlo computer simulation of a complex value-assignment process, based on a formalized description of the process, with measurement parameters estimated experimentally. This method was applied to study uncertainty of a multilevel calibrator value assignment for a prealbumin immunoassay. Results:The simulation results showed that the component of the uncertainty added by the process of value transfer from the reference material CRM470 to the calibrator is smaller than that of the reference material itself (<0.8% vs 3.7%). Varying the process parameters in the simulation model allowed for optimizing the process, while keeping the added uncertainty small. The patient result uncertainty caused by the calibrator uncertainty was also found to be small. Conclusions: This method of estimating uncertainty is a powerful tool that allows for estimation of calibrator uncertainty for optimization of various value assignment processes, with a reduced number of measurements and reagent costs, while satisfying the requirements to uncertainty. The new method expands and

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