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

Abstract

Prozone 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 Report: A 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. Conclusion: The 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. Platelet (PLT) transfusion refractoriness, defined as repeated inadequate PLT count increases after PLT transfusion, can be immune mediated or nonimmune mediated. 1-3 Immune-mediated PLT destruction occurs predominantly due to antibodies against Class I human leukocyte antigens (HLA). 1,3-5 For immune-mediated cases, multiple in vitro testing approaches exist, such as solid-phase PLT crossmatches, Class I HLA antibody testing, and Class I HLA typing, to help characterize immune-mediated cases and provide appropriate PLT

Prozone rates in the solid‐phase platelet crossmatch assay and correlation with class I HLA antibody levels

“…Third, there were several negative PXM reactions with maximum MFIs well above the bead saturation limit of the class I HLA‐Ab assay (Figure 4). In our previously published case report, one of the false‐negative prozone examples demonstrated only weak positive reactivity using 1:8 diluted patient EDTA plasma but 4+ positive reactivity using 1:16 diluted patient EDTA plasma 20 . Thus, 1:4 diluted patient EDTA may be insufficient to detect all such instances and, therefore, several additional false‐negative PXM reactions may have been missed.…”

“…Our previous case report describing prozone effect in the PXM assay used patient EDTA plasma diluted 1:8 and 1:16 with RPMI medium 20 . Based on subsequent recommendations from the assay manufacturer, undiluted EDTA patient plasma and EDTA patient plasma diluted 1:4 with 6% albumin were tested simultaneously to detect any false‐negative PXM results from prozone effect.…”

“…Recently, we published a case report on a newly discovered limitation of the solid‐phase red blood cell (RBC) adherence platelet crossmatch (PXM) assay – prozone effect due to high levels of patient anti‐platelet antibodies 20 . In that case report, a patient with high levels of class I HLA‐Abs had multiple false‐negative PXM results using undiluted patient plasma leading to unsatisfactory 1‐h corrected count increments (CCIs).…”

Three patients highlighting potential pitfalls in platelet refractory testing

“…The differential clinical response between crossmatched-and HLA-matched platelets was curious, as both should lead to similar transfusion increments. 16 Since the prozone effect has been described in PXM, 17,18 we asked the reference laboratory to repeat PXM using diluted and undiluted serum against HLAincompatible donors from the suspected false-negative PXM. Undiluted serum demonstrated compatibility; however, a 1:8 dilution resulted in positive crossmatches.…”

“…Since the prozone effect has been described in PXM, 17,18 we asked the reference laboratory to repeat PXM using diluted and undiluted serum against HLA‐incompatible donors from the suspected false‐negative PXM. Undiluted serum demonstrated compatibility; however, a 1:8 dilution resulted in positive crossmatches.…”