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

Abstract

Background Solid‐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 Methods A 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. Results Among 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). Discussion While 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.

Three patients highlighting potential pitfalls in platelet refractory testing

“…17 However, on a follow up study from the same group, the authors examined a cohort of 59 PR patients undergoing PXM and concluded that maximum mean fluorescence intensity (MFI) and MFI sum (an arguable surrogate for antibody titer) on solid-phase HLA testing were not predictors of the prozone effect. 18 Nonetheless, out of 830 ABO-compatible PXMs, they found a false-negative rate of ~5% attributable to prozone. Thus, prozone effect in PXM appears to be a rather common occurrence with an underlying mechanism that remains to be fully elucidated.…”

“…Horton et al presented a case of false‐negative solid phase PXM and postulated that the mechanism is more likely to be classic prozone 17 . However, on a follow up study from the same group, the authors examined a cohort of 59 PR patients undergoing PXM and concluded that maximum mean fluorescence intensity (MFI) and MFI sum (an arguable surrogate for antibody titer) on solid‐phase HLA testing were not predictors of the prozone effect 18 . Nonetheless, out of 830 ABO‐compatible PXMs, they found a false‐negative rate of ~5% attributable to prozone.…”

“…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.…”

“…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.…”