Impact of pronase on flow cytometric crossmatch outcome.
Pronase treatment of lymphocytes is used to reduce nonspecific binding of immunoglobulins in flow cytometric crossmatch (FCXM) tests and at higher concentrations to remove CD20 from the cell surface. We examined the effect of pronase treatment on human leukocyte antigen (HLA) expression and on FCXM results. Lymphocytes were tested untreated and after treatment with either 2 mg/mL (10 cell donors) or 1 mg/mL (6 cell donors) of pronase. The 2 mg/mL concentration reduced HLA expression in 28 of 30 (93%) cases. The reduction was statistically significant for HLA class I antigens on T cells (33 ± 10%, p = 0.0006), class I on B cells (23 ± 13%, p = 0.012), and class II on B cells (45 ± 37%, p = 0.005). FCXMs were performed using pronase-treated and untreated cells. The 2 mg/mL concentration of pronase reduced reactivity in 5 of 16 (31%) tests of T cells and 15 of 16 (94%) tests of B cells. Of the remaining 11 T-cell tests, the reactivity was unchanged (≤ 10% difference) in 5 and increased by 18-73% in 6. Treatment with 1 mg/mL of pronase significantly increased reactivity in 20 of 23 tests of T cells (87%, p = 6.0 × 10(-5)). These data indicate that pronase treatment may result in erroneous FCXM results.
Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
Sarah J
SJ
Hetrick
Karl P
KP
Schillinger
Andrea A
AA
Zachary
Annette M
AM
Jackson
D001402:B-Lymphocytes / Q000187:drug effects*; D005434:Flow Cytometry / Q000592:standards*; D005786:Gene Expression Regulation / Q000187:drug effects; D006680:HLA Antigens / Q000276:immunology / Q000378:metabolism; D006650:Histocompatibility Testing / Q000592:standards*; D006801:Humans; D007166:Immunosuppressive Agents / Q000494:pharmacology; D011402:Pronase / Q000494:pharmacology*; D013601:T-Lymphocytes / Q000187:drug effects*
D006680:HLA Antigens; D007166:Immunosuppressive Agents; D011402:Pronase