Abstract
HLA compatibility is assessed by crossmatching (XM) and donor specific antibody (DSA) testing. Flow cytometric XM (FXM) results vary among centers due to variation in reagents and sample preparation. We evaluated FXM variability and effects of test standardization in 6 US centers participating in the HALT study.
6 labs performed FXM using 3 exchanges (Exch) including sera with weak DSA and either frozen (1) or fresh (2) cells. All samples were sent from 1 lab, which screened sera by single antigen beads and typed cells for HLA-A,B,C,DRB1/3/4/5,DQB1. HLA typing and DSA results were not revealed to the other labs until study completion. FXM data assessed included cell viability, pronase and anti-hIgG concentrations, instrument and channel settings, and fluorescence (FL) values. Four different cytometer types were used by the labs. Labs used their own methods for Exch 1 and 2. In Exch 3, sample preparation and reagents were standardized: pronase (2mg/ml); anti-hIgG dilution (1:500).
Each Exch had 10-24 T/B FXM comparisons. Exch 2 (S1 vs C1) was a negative control. Table 1 shows range of variation in FL values. Table 2 shows expected vs observed interpretations.
The discrepant results produced by varying FXM methods raise concerns about the assessment of HLA compatibility results from multicenter clinical trials. Standardization of reagents and sample preparation improved FXM result concordance among centers demonstrating that standardization can reduce the variation of results often seen in a multicenter clinical trial. Partially funded by the NIH, NHLBI grant R34 HL10541.