Ann Lab Med.  2024 Nov;44(6):545-552. 10.3343/alm.2024.0132.

Effects of Various Concentrations of Pronase on Flow Cytometric Crossmatching Patients Treated With Rituximab and Donor HLA-Specific Antibodies

• Kim TS
• Oh I
• Choi YJ
• Nam M
• Lee H
• Song EY

• KMID: 2560801
• DOI: http://doi.org/10.3343/alm.2024.0132

Abstract

Background
Pronase pretreatment can reduce rituximab (RTX) interference by degrading CD20 in B-cell flow cytometry crossmatch (FCXM) testing. However, it may also reduce the assay sensitivity by degrading HLA molecules. We investigated the effects of various pronase concentrations on RTX interference and the analytical sensitivity of B-cell FCXM testing.
Methods
Using 59 patient serum samples and 38 donor lymphocyte samples, we designed 97 recipient–donor pairs and divided them into three groups according to RTX use and the presence of weak-to-moderate donor HLA-specific antibody (DSA) reactions: RTX+/ DSA−, RTX+/DSA+, and RTX−/DSA+. FCXM was performed after pretreating lymphocytes with six different pronase concentrations (0, 0.5, 1, 2, 3, and 4 mg/mL).
Results
With B-FCXM testing, false-positive results due to RTX in the RTX+/DSA– group markedly decreased with increasing pronase concentrations. The median channel shift values in the RTX+/DSA+ and RTX−/DSA+ groups did not significantly decrease when the pronase concentration was increased from 1 mg/mL to 2 or 3 mg/mL. All eight RTX+/ DSA+ cases that were positive at 1 mg/mL pronase but negative at 2 or 3 mg/mL had mean fluorescence intensity (MFI) DSA values of less than 3,000 except for DQ5 (MFI: 5,226). With T-cell FCXM, false-positive results were observed in 2.9% of 315 FCXM tests with pronase pretreatment.
Conclusions
Higher concentrations (2 or 3 mg/mL) of pronase effectively eliminated RTX interference but still carried a risk for false negativity for weak DSA reactions in B-cell FCXM. Higher pronase concentrations can be used as an auxiliary method to detect moderate-to-strong DSA reactions in RTX-treated patients.

Keyword

Flow cytometry; Histocompatibility testing; Kidney transplantation; Pronase; Rituximab

Figure

• 

  Fig. 1 B-cell flow cytometry crossmatch testing results. Sera from three groups, RTX+/DSA– (A), RTX+/DSA+ (B), and RTX−/DSA+ (C), were crossmatched with lymphocytes treated with the indicated pronase concentrations. The gray circles represent the results for each sample, and the black lines represent the median and interquartile range. The dotted line indicates the median channel shift (MCS) cutoff (120) for positive results. The median MCSs of the 2, 3, and 4 mg/mL pronase groups were compared with that of the 1 mg/mL pronase group using the Kruskal–Wallis test, followed by Dunn’s multiple-comparison test. The table below each graph shows the total number of samples tested (Total), the number of positive samples (Pos.), and the percentage of positive results (Pos. %). *P<0.05; ****P<0.0001. Abbreviations: RTX, rituximab; DSA, donor HLA-specific antibody; ns, not significant.

• 

  Fig. 2 Comparison of the mean fluorescence intensities (MFIs) of donor HLA-specific antibodies (DSAs) present in RTX+/DSA+ and RTX−/DSA+ sera. (A) The MFI values of all DSAs in the RTX+/DSA+ and RTX−/DSA+ groups at 1, 2, and 3 mg/mL pronase were plotted according to the B-cell flow cytometry crossmatch (FCXM) results (positive or negative). For cases with two DSAs, the MFI sum of both DSAs was used. (B) The FCXM-negative cases represented in (A) were divided into the RTX+/DSA+ and RTX−/DSA+ groups and subdivided according to 1, 2, and 3 mg/mL pronase treatment (P1, P2, and P3, respectively). The horizontal lines and error bars represent the median and interquartile range of each column, respectively. Abbreviations: RTX, rituximab; XM, crossmatches; ns, not significant.

• 

  Fig. 3 T-cell flow cytometry crossmatch testing results. Sera from the RTX+/DSA– (A), RTX+/DSA+ (B and D), and RTX−/DSA+ (C and E) groups were crossmatched with lymphocytes treated with the indicated pronase concentrations. The crossmatching results for RTX+/DSA+ and RTX−/DSA+ sera were plotted in separate graphs based on the MHC class of the DSAs present in each serum sample because MHC class II molecules are usually not expressed on naive T cells (B and C for MHC class I; D and E for MHC class II). The dotted lines indicate the median channel shift (MCS) cutoff (90) for positive results. The median MCSs of each column were compared with that of the control column (0 mg/mL pronase) using the Kruskal–Wallis test, followed by Dunn’s multiple-comparison test. The table below each graph shows the total number of samples tested (Total), the number of positive samples (Pos.), and the percentage of positive results (Pos. %). *P<0.05; **P<0.01. Abbreviations: RTX, rituximab; DSA, donor HLA-specific antibodies; ns, not significant.

Reference

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