Frequent false-positive reactions in pronase-treated T-cell flow cytometric cross-match tests.
OBJECTIVE
Pretransplantation cross-match (XM) is essential in organ transplantation. The flow cytometric XM (FCXM) is the most sensitive cell-based XM technique. Pronase treatment is used to improve the sensitivity and specificity of the B-cell FCXM. Thus, pronase-treated (PT) T cells are tested in a single tube T-cell/B-cell technique. Observing discrepancies between PT and pronase-nontreated (PN) T- FCXM results, we investigated their incidence, clinical significance, and possible causes.


METHODS
We tested 226 serum samples from 167 kidney transplantation candidates or posttransplantation follow-up patients using PT and PN T-FCXM in parallel using 3-color and 2-color immunofluorescence staining, respectively. We reviewed panel-reactive antibody (PRA) and donor-specific antibody (DSA) status as well as HLA data and clinical outcomes.


RESULTS
The T-FCXM positive rate was significantly higher among PT versus PN tests (24.3% vs 11.1%; P < .001). Less than half of the PT-positive cases were positive in the PN test (45.5%; 25/55). Discrepancies were observed in 30 cases (13.3%), all of which gave PT(+)/PN(-) results. Our findings suggested that PT(+)/PN(-) results might arise from non-HLA antibodies. Class I DSA-positive rate (6.3% vs 2.2%; P = .45) and antibody-mediated rejection rate (0% vs 16.3%; P = .32) were not different between PT(+)/PN(-) and PT(-)/PN(-) groups. Moreover, 2 cases of PT(+)/PN(-) were observed among HLA-A, B, DR-identical donor-recipient pairs.


CONCLUSIONS
Pronase treatment is prone to give false-positive reactions in T-FCXM test probably due to the participation of non-HLA antibodies including autoantibodies. Patients might be inappropriately excluded from receiving organs. In laboratories using PT single tube T/B FCXM, caution is needed to avoid false-positive reporting of results.
Department of Laboratory Medicine, Seoul National University College of Medicine, Seoul, Korea. parkmhee@snu.ac.kr
H
H
Park
Y M
YM
Lim
B Y
BY
Han
J
J
Hyun
E Y
EY
Song
M H
MH
Park
D000328:Adult; D016009:Chi-Square Distribution; D005189:False Positive Reactions; D005260:Female; D005434:Flow Cytometry; D006084:Graft Rejection / Q000276:immunology; D006680:HLA Antigens / Q000276:immunology*; D006648:Histocompatibility; D006650:Histocompatibility Testing / Q000379:methods*; D006801:Humans; D007518:Isoantibodies / Q000097:blood*; D016030:Kidney Transplantation / Q000276:immunology*; D008297:Male; D008875:Middle Aged; D011237:Predictive Value of Tests; D011402:Pronase; D056910:Republic of Korea; D013601:T-Lymphocytes / Q000276:immunology*; D016896:Treatment Outcome
D006680:HLA Antigens; D007518:Isoantibodies; D011402:Pronase