The effect of serum pretreatment regimens for the detection of HLA class I antibodies in platelet‐refractory patients

Abstract

BACKGROUND Single 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 METHODS Five 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. RESULTS Interlaboratory 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. CONCLUSION Our 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.

Platelet transfusion refractoriness: how do I diagnose and manage?

“…Unfortunately, there is wide interlaboratory variability, with a range of 500 to 6,000 MFI used as a cutoff value. 28 In an effort to identify clinically significant antibodies, an adaptation of the solid phase assay was developed that targets complement-fixing antibodies. Although some platelets are removed from the circulation by macrophages stimulated by antigen-antibody interactions, a subset is bound by the CIq protein, which activates the classic complement cascade, ending with direct platelet lysis.…”

False‐negative solid‐phase platelet crossmatch results due to prozone phenomenon

“…To further investigate these repeated false-negative solid-phase PLT crossmatch results, 6 ABO-identical HLA-incompatible (HLA-A2-positive) PLT units were tested against the patientʼs undiluted EDTA plasma, EDTA plasma diluted 1-in-8 with RPMI buffer, and EDTA plasma diluted 1-in-16 with RPMI buffer (Figure 2). While only 2 PLT units (Units 1 and 4) showed weak positive reactivity against the undiluted EDTA plasma, there was a robust increase in PLT reactivity against all 6 PLT units from the undiluted EDTA plasma to the 1-in-8 and 1-in- 16 diluted plasma solid-phase PLT crossmatch results aligned with the EDTA-pretreated serum Class I HLA antibody results for the Class I HLA antigens expressed on these 6 PLT units (Table 1).…”

“…12,13,17 This prozone-like effect has been mitigated by diluting the serum sample or treating the serum sample with ethylenediaminetetraacetic acid (EDTA) or dithiothreitol (DTT). 13,14,16,18 A similar prozone phenomenon causing falsenegative results in solid-phase red blood cell (RBC) adherence PLT crossmatches has not previously been reported in the literature. In this case report, we describe an instance of the prozone phenomenon causing falsely negative PLT crossmatch results with the use of undiluted patient EDTA plasma and correspondingly inadequate PLT count increases but positive PLT crossmatch results with the use of diluted patient EDTA plasma.…”

“…11 A similar prozone-like phenomenon has also been described for solid-phase assays used to detect HLA antibodies. [12][13][14][15][16] In these instances, serum substances like complement C1, in the presence of high-titer IgG HLA antibodies, interfere with the detection of antibody binding causing falsely low or negative results. 12,13,17 This prozone-like effect has been mitigated by diluting the serum sample or treating the serum sample with ethylenediaminetetraacetic acid (EDTA) or dithiothreitol (DTT).…”

Development and performance characteristics of Platelet Virtual Crossmatch (PLT VXM), a software application for the evaluation and management of platelet transfusion–refractory patients

“…1,2,[5][6][7][8] Laboratory testing for immune-mediated PLT transfusion refractoriness can include physical PLT crossmatch assays, recipient Class I HLA antibody testing, and donor and recipient Class I HLA antigen typing. [9][10][11][12][13][14][15] An algorithm integrating the use of these assays for the identification and management of immune-mediated PLT transfusion-refractory patients has been previously published ( Figure 1). 16 The execution of such an algorithm in routine clinical practice requires the integration of multiple data sources with large and complex data sets, which is a time-consuming and potentially error-prone process for transfusion medicine personnel.…”