4-Color Flow Cytometric Crossmatch Using Whole Blood Lysis

초록

Background. In lymphocyte crossmatch using fl ow cytometry (flow fl ow cytometric crossmatch, FCXM), the conventional tricolor FCXM protocol requires a mononuclear cell isolation step. To develop a new, more streamlined protocol, we introduced whole blood lysis (WBL) and CD45 fl uorescence-triggered acquisition using 4-color fl ow cytometry. Methods. A total of 186 donor/recipient pairs for transplantation were classified fi ed into donor- specific fi c human leukocyte antigen (HLA) alloantibody-positive (DSA+, n = 78) and DSA-negative (DSA-,- , n = 108) groups. The latter group was reclassified fi ed into blood group ABO-incompatible (ABOi, n = 56) and ABO-compatible (n = 52) subgroups. The WBL FCXM protocol with CD45 V500-C was optimized using a FACSLyric cytometer (BD Biosciences) with 3 lasers. Measurements for T cells or B cells were calculated as a mean fl uorescence intensity (MFI) ratio (test divided by control). WBL FCXM was compared with conventional FCXM in each group. Results. WBL FCXM showed no difference quantitatively compared with conventional FCXM, except for the B cell FCXM in the DSA-- group (B cell MFI ratio: 1.06 0.44 and 0.92 0.41, respectively [P = .0001]). There was no ABO antibody interference in the ABOi subgroup. Similar results were observed in the qualitative determinations of FCXM as follows: 1) In the DSA+ group, the sensitivity of B cell WBL FCXM (96.2%) showed no difference compared with that of conventional FCXM (91.0%, P = .2188) and 2) In the DSA-- group, the specificity fi city of T cell WBL FCXM (96.3%) showed no difference compared with that of conventional FCXM (98.1%, P = .6250). WBL FCXM reduced the turnaround time by 50 min compared with that by conventional FCXM. Conclusions. WBL FCXM demonstrated comparable assay performance to that of conventional FCXM. Because this new FCXM protocol is simple and does not compromise assay sensitivity, it has the potential to replace the conventional method in histocompatibility laboratory settings.

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