Gandhi, Manish

AbstractBackgroundSolid‐phase platelet crossmatch (PXM) testing is used to help manage patients with platelet transfusion‐refractoriness. Recently, we published the first report of false‐negative PXM results from prozone effect that was mitigated using sample dilution. This study aimed to describe the prevalence of PXM prozone effect and the levels of class I HLA antibodies (HLA‐Abs) associated with positive PXM results and with false‐negative PXM results from prozone effect.Study Design and MethodsA cross‐sectional study of patients undergoing PXM testing from July 2019 through December 2020 was performed. All PXM tests were run simultaneously using undiluted and 1:4 diluted patient plasma. Prozone effect was defined as a negative PXM result using undiluted patient plasma but a positive PXM result using 1:4 diluted patient plasma.ResultsAmong 59 patients, 830 individual ABO‐compatible PXM results yielded an overall positivity rate of 25.8% (214/830) and a false‐negative rate from prozone effect of 4.7% (10/214). Among the 28 patients with class I HLA‐Ab testing and no other anti‐platelet antibodies, maximum HLA‐Ab mean fluorescence intensity (MFI) was significantly associated with a positive PXM result (p < .0001; AUC approx. 0.9) and categorized into negative (<3700), indeterminate (3700–10300), and positive (>10300) maximum HLA‐Ab MFI zones. Maximum HLA‐Ab MFI, however, was not associated with prozone effect (p = .17; AUC approx. 0.6).DiscussionWhile there is a strong predictive association between class I HLA‐Ab levels and positive PXM results, PXM prozone effect is a common occurrence not associated with class I HLA‐Ab levels, so additional testing with diluted samples should be considered.

BackgroundProzone is a known phenomenon affecting immunoassays causing falsely low or negative results when excess target is present in the test system. For assays used to evaluate immune‐mediated platelet (PLT) transfusion refractoriness, prozone‐like phenomenon has been described in solid‐phase human leukocyte antigen (HLA) antibody testing and can be mitigated by diluting samples or pretreating samples with ethylenediaminetetraacetic acid (EDTA) or dithiothreitol. Prozone phenomenon has not yet been described in solid‐phase red blood cell (RBC) adherence PLT crossmatch assays.Case ReportA 40‐year‐old female with myeloid sarcoma and PLT transfusion refractoriness underwent repeated solid‐phase PLT crossmatches; however, crossmatch‐compatible PLTs units did not yield adequate PLT count responses. Class I HLA antibody testing with neat, diluted, and EDTA‐pretreated serum demonstrated significant prozone‐like effect and the presence of numerous high strength HLA antibodies. Based on this HLA antibody profile, HLA antigen–negative PLTs gave an adequate PLT count response. It was noted that the HLA types of her crossmatch‐compatible PLTs were incompatible with her HLA antibody profile (eg, HLA‐A2). With ABO‐identical, HLA‐A2–positive PLT units, a solid‐phase PLT crossmatch was repeated using undiluted and diluted EDTA plasma. Undiluted EDTA plasma demonstrated negative or weakly positive PLT crossmatches while the diluted EDTA plasma demonstrated strongly positive PLT crossmatches.ConclusionThe prozone phenomenon can cause false‐negative results in solid‐phase RBC adherence PLT crossmatch assays, which can be mitigated with sample dilution. In immune‐mediated PLT transfusion–refractory patients with high‐strength HLA antibodies, sample dilution should be considered to correctly identify compatible PLT inventory.

AbstractBackgroundPlatelet (PLT) transfusion refractoriness increases bleeding complications, hospital stays, and PLT inventory usage. Immune‐mediated refractoriness can be evaluated for using a physical PLT crossmatch with ABO‐compatible inventory and, if positive, managed with HLA‐compatible PLT inventory and donors. Manual completion of these complex tasks can be time‐consuming and potentially error‐prone. This study was conducted to determine if a Web‐based software application could improve process efficiency and accuracy.Study Design and MethodsWorkflow analysis was performed to identify process, data, and analytic requirements for a software application for three PLT transfusion‐refractoriness associated tasks: (a) physical PLT crossmatch inventory selection, (b) HLA‐compatible inventory selection, and (c) HLA‐compatible donor selection. After software application development, a comparison study was performed over 10 consecutive days, with each task performed manually and with the software application (Platelet Virtual Crossmatch [PLT VXM]) for a different unique immune‐mediated PLT transfusion–refractory recipient. Task completion time, number of incompatible units/donors presented, and number of documentation errors were compared.ResultsPLT VXM is a Web‐based software application developed using R and the Shiny Web application framework. PLT VXM significantly reduced median task completion times by 4.5 (49%), 11.2 (79%), and 59.1 minutes (94%), respectively. PLT VXM did not present any incompatible PLT units or donors for user consideration. PLT VXM also had a lower number of documentation errors than the manual process, and none of these documentation errors were software generated.ConclusionComputer‐aided evaluation and management of immune‐mediated PLT transfusion–refractory recipients can significantly improve workflow and reduce manual errors in this complex process.

Background Daratumumab (DARA), a human IgG1 K monoclonal antibody targeting CD38, is used to treat refractory multiple myeloma patients. CD38 is expressed on many cell types (RBCs, granulocytes, lymphocytes, etc.), and thus, DARA can interfere with serological tests. Information regarding how DARA affects anti‐granulocyte antibody (AGA) testing and optimal neutralization of DARA will help laboratories perform accurate testing. Methods Screening of AGA was performed by the granulocyte agglutination test (GAT) and the flow cytometric granulocyte immunofluorescence test (Flow‐GIFT). Samples were tested from patients on DARA ( n = 7), non‐transfused blood donors (healthy controls, n = 7) and AGA reactive samples (positive controls, n = 5). Two neutralization experiments, CD38 removal with DTT and DARA epitope blockage with mouse anti‐CD38, were evaluated. Results Positive reactivity of human IgG binding was observed in 5/7 DARA cases when tested by Flow‐GIFT; however, all 7 cases had negative GAT agglutination results. Further studies by Flow‐GIFT revealed DARA concentrations >0·63 μg/ml bound to granulocytes. DARA binding was negated by DTT though a reduced Flow‐GIFT sensitivity was observed in positive control samples due to increased background detection of human IgG. Mouse anti‐CD38 neutralized the detection of human IgG observed in DARA‐treated patient serum without effecting controls. Conclusion We established that DARA can interfere with AGA testing, leading to false positive Flow‐GIFT results without causing GAT agglutination. DTT treatment increased background binding of secondary antibodies causing a decrease in Flow‐GIFT sensitivity. In comparison, blockage of the DARA binding epitope using mouse anti‐CD38 antibody was effective in neutralizing DARA interference while maintaining Flow‐GIFT sensitivity.

BACKGROUNDSingle antigen bead (SAB) assays are used to identify human leukocyte antigen (HLA) antibodies in patients with platelet refractoriness due to HLA Class I alloimmunization. Some laboratories use serum pretreatment regimens to eliminate interference from immunoglobulin M antibodies and complement. These modifications may contribute to interlaboratory variability, which is a recognized problem with the SAB assay.STUDY DESIGN AND METHODSFive patientsʼ sera were overnight shipped to 12 laboratories in the United States and internationally. Recipients used their labʼs SAB procedure to identify HLA Class I antibodies. The resultant mean fluorescence intensity (MFI) data were compared by instrumentation, bead lot, and pretreatment regimens. Laboratory‐specific cutoffs for positive antibodies were applied to the results.RESULTSInterlaboratory variability for MFI values appears to be associated with different pretreatment regimens. The coefficient of variation (CV) of MFI from samples pretreated with ethylenediaminetetraacetic acid, dithiothreitol, or heat inactivation (EDHI) were similar, ranging from 14% to 56% (mean, 22%). For samples with no pretreatment, the CVs were significantly higher than EDHI‐treated samples, ranging from 25% to 74% (mean, 39%; 95% confidence interval, 12.10‐21.90; p < 0.0001). An intralaboratory comparison of pretreatment regimens confirmed these findings. Some positive antibody specificities present in EDHI‐treated samples were negative in corresponding samples with no pretreatment when laboratory‐specific cutoffs for positive antibodies were applied.CONCLUSIONOur results show that greater interlaboratory precision can be achieved when samples are pretreated with EDHI as opposed to no pretreatment, likely because these pretreatments eliminate interference from inhibitors. Inhibitors may mask antibodies, leading to missed (or uncalled) specificities when no pretreatment is used.