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FO 4
OPTIMIZATION OF PRONASE TREATMENT FOR X-MATCH TESTING
Gansuvd Balgansuren, MD, PhD 1 , Prof. Namid Munkhtuvshin MD,PhD 2
1 Clinical Transplantation Immunology Laboratory, Duke University Health System, Duke University,
2 Central Scientific Research Laboratory, Nat’l Institute of Medicine, Mongolia
Objectives: Due to increased receipt of crossmatch test requests from patients treated with rituximab
desensitization therapy we aimed to optimize pronase concentration for routine testing that could
significantly reduce non-specific background binding CD20 while keeping CD19.
Materials and Methods: Peripheral blood mononuclear cells (PBMNC) were incubated with different
concentrations (0.5; 1.0; 1.5 and 20 mg/ml) of pronase for 30 minutes at 37°C. Two kinds of normal
human sera (GEN & GTI) and positive pooled serum (PPS) were used as negative and positive controls.
10 µg/ml rituximab spiked NHS and rituximab treated patient’s sera were used as test samples.
Anti-human CD19 and CD20 antibodies were used for phenotypic analysis.
Results: 15 min incubation of donor cells with various concentrations of pronase resulted no change on
CD20 expression, however extended incubation of donor cells with pronase at the dose of 1.5 mg/ml
and 2.0 mg/ml for 30 min completely removed CD20 from B cell surface. The latter dose also markedly
reduced CD19 expression (55%±26%, n=8) as well as its intensity. Therefore, 1.5mg/ml of pronase has
been chosen for further X-Match testing. MCS of rituximab spiked NHS (GEN & GTI) with
un-pronased donor cells in B cell X-Match was 512±50 & 452±53 and which has reduced up to 68±28
& 47±18 with 1.5 mg/ml pronase treated donor cells, respectively (n=20). Rituximab treated patients
sera with un-pronased donor cells in B cell X-Match showed MCS of 445, 447 and 468 which has also
reduced up to 20, 48 and 6 with 1.5mg/ml pronase treated donor cells. The pronase treatment
significantly reduced non-specific background binding in both T and B cell X-Match and significantly
increased sensitivity only in T cell X-Match.
Conclusion: 1.5 mg/ml pronase treatment of donor cells for 30 min at 37°C could completely remove
CD20 from B cell surface while keeping sufficient number of CD19 positive cells to safely X-Match
rituximab treated patients alone with other patients.
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