Korean J Transplant.  2023 Nov;37(Suppl 1):S27. 10.4285/ATW2023.F-5944.

Higher concentration of pronase can effectively reduce false positive results of B cell flow-cytometric crossmatch in patients with rituximab treatment and no donor-specific human leukocyte antigen antibodies

• Oh I
• Min SI
• Jeong CW
• Kim YS
• Lee H
• Song EY

• KMID: 2551601
• DOI: http://doi.org/10.4285/ATW2023.F-5944

Abstract

Background
Pronase treatment reduces nonspecific binding in B cell flow cytometric crossmatch (B-FCXM). Higher concentration of pronase might reduce false positivity from rituximab, but also can decrease the sensitivity. We evaluated the effect of variable pronase concentration on B-FCXM with sera from patients with various conditions.
Methods
We analyzed 63 sera, including 30 from patients with rituximab treatment before 7–63 days ago (17 with donor-specific antibody [DSA] [MFI 1,260–10,325 for A, B, DR, and 5,226–19,033 for DQ] and 13 with non-DSA). Controls comprised 29 sera from patients with DSA but nonrituximab-treated. Additionally, we spiked 4 sera from DSA-negative and nonrituximab-treated patients with rituximab (Mabthera, Roche; 100 ug/mL). We isolated peripheral blood mononuclear cells from 38 kidney trans-plantation donors (Seoul National University Hospital, June 2022 to January 2023) and treated them with six different pronase concentrations (0, 0.5, 1.0, 2.0, 3.0, and 4.0 mg/mL). Donor cells underwent crossmatch with three patient groups: rituximab and DSA (RD, n=21), rituximab without DSA (RN, N=13), and DSA with no rituximab (ND, n=33). NDs DSA matched those of RDs specificity and MFI. The days between rituximab and blood sampling were matched between RD and RN.
Results
We tested 40 DSA (six human leukocyte antigen [HLA]-A, 15 HLA-B, 12 HLA-DR, 7 HLA-DQ). The false positive rates in RN with 2.0 mg/mL (12/38, 31.6%), 3.0 mg/mL (2/38, 5.3%), and 4.0 mg/mL (0/38, 0.0%) pronase were significantly lower than 1.0 mg/mL (30/38, 78.9%) in B-FCXM (P<0.001). Sensitivity in ND with 3.0 mg/mL (23/38, 60.5%) and 4.0 mg/mL (22/38, 57.9%) pronase did not differ significantly from 1.0 mg/mL (25/38, 65.8%; P>0.05).
Conclusions
Higher pronase concentration can effectively reduce false positive results in B-FCXM for patients with rituximab treatment and DSA, without a significant decrease in DSA detection sensitivity.