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Abstract: Solid phase antibody assays are increasingly used to provide quantitative measures of donor-specific HLA antibodies for assessment of pretransplant risk, although cell-based crossmatches continue to serve as gold standards for determination of donor HLA antibody strength. This study determined the ability of HLA antibody solid phase assays to predict the strength of cell-based flow cytometric (FC) and complement-dependent cytotoxicity (CDC) crossmatches. Eighty-two recipient/donors pairs were analyzed using receiver operating characteristic (ROC) curve analyses to determine the accuracy of donor-specific median fluorescence intensity values (Σ MFI) from single antigen bead assays for predicting strong FC and CDC crossmatches. Diagnostic sensitivity and specificity of optimal Σ MFI values were highest for predicting strong T cell FCs. Σ MFI values showed good sensitivity for predicting positive direct and AHG-augmented CDC crossmatches (91% and 94%, respectively), but with lower specificity (67% each). Specificity and sensitivity for predicting positive B cell CDC crossmatches were 73% and 84%. Σ MFI values derived from single antigen bead assays can predict strong flow and positive CDC crossmatches, but with tradeoffs between sensitivity and specificity. The results support the use of solid phase assays for quantitative virtual crossmatching and as a replacement for cell-based crossmatching. [Copyright &y& Elsevier]

Aim: The FDA has issued new draft guidance on molecular testing around Research Use Only (RUO) and non-Quality System Regulation (QSR) products in clinical labs. Promega has introduced a new QSR-compliant nucleic acid extractor and kit specifically to address this issue. At BloodCenter of Wisconsin we have evaluated this new extractor, the Maxwell® CSC, in terms of its performance in HLA typing assays as well as its fit in the molecular HLA workflow. Methods: Patient samples (buccal swabs) were extracted using the Maxwell® CSC, and then evaluated for total yield and purity of DNA. They were compared to the existing extraction method and evaluated by sequence-based typing, rSSO and STR assays for overall success and results concordance. The data handling capabilities of the instrument were also evaluated, such as how the run reports affected technician time, downstream operations and integration with electronic records. Results: DNA concentrations from double-buccal swab isolations ranged from 13ng/μl to 247ng/μl, with a mean concentration of 76.31ng/μl. DNA purities ranged from OD260/280 ratios of 1.69 to 2.00, with a mean of 1.89. These results are comparable to those obtained on the Maxwell® MDx instrument in our laboratory. All samples tested in downstream HLA STR assay, KIR rSSO, HLA rSSO and sequence-based typing assays were successful and concordant with previously obtained results. Conclusions: The Maxwell® CSC is a QSR-compliant system which will allow molecular laboratories to adhere to the draft FDA guidance for molecular testing. It provides a solution for nucleic acid extraction which performs equivalently to Promega’s current extraction systems with regards to DNA yield, quality and utility in downstream assays. The Maxwell® CSC also helps reduce data entry tasks with exportable sample-specific results. This is useful for tracking samples throughout the workflow, reducing the need to re-enter sample identifiers, saving technicians time and reducing errors. [Copyright &y& Elsevier]