4-Color Flow Cytometric Cytotoxicity Crossmatch Using Whole Blood Lysis

IF 0.8 4区医学 Q4 IMMUNOLOGY

Transplantation proceedings Pub Date : 2025-03-15 DOI:10.1016/j.transproceed.2025.02.034

Dong Il Won , Jeong-Hoon Lim , Jang-Hee Cho , Chan-Duck Kim , Seung Huh

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引用次数: 0

Abstract

Background

The conventional lymphocyte crossmatch (XM) assay necessitates mononuclear cell isolation. In our previous work, we introduced a novel 3-laser, 4-color flow cytometric (FC) XM protocol utilizing whole blood lysis (WBL) and CD45 fluorescence-triggered acquisition to detect human leukocyte antigen (HLA) antibody binding. Building on this, we aimed to adapt these advancements for complement-dependent cytotoxicity (CDC) XM using FC (FCCDC).

Methods

A total of 164 donor/recipient pairs undergoing transplantation were stratified into 2 groups based on donor-specific HLA alloantibody presence: DSA-positive (DSA+, n = 73) and DSA-negative (DSA−, n = 91). The DSA− group was further subdivided by ABO compatibility into ABO-incompatible (ABOi, n = 52) and ABO-compatible (n = 39) subgroups. Protocol optimization for the WBL FCCDC with CD45 V500-C was conducted using a FACSLyric cytometer (BD Biosciences). T and B cell indices were calculated as delta (test minus control) percentages of dead cells (Δ%DC). WBL FCCDC results were compared with those of conventional FCCDC in each group.

Results

WBL FCCDC showed no significant quantitative difference from conventional FCCDC. In the DSA+ group, B cell Δ%DC values were 28.00 ± 27.14 and 19.16 ± 27.74 for WBL FCCDC and conventional FCCDC, respectively (P = .0777). No ABO antibody interference was observed in the ABOi subgroup. Qualitatively, B cell WBL FCCDC sensitivity in the DSA+ group was 69.9%, comparable to conventional FCCDC sensitivity (65.8%, P = .5078). Additionally, WBL FCCDC reduced the turnaround time by 50 minutes relative to conventional FCCDC.

Conclusions

WBL FCCDC achieved performance equivalent to conventional FCCDC, similar to WBL FCXM. The absence of adverse effects from the lysis step supports its integration into XM assays. Given its simplicity and maintained sensitivity, the WBL FCCDC protocol presents a viable alternative to conventional methods in histocompatibility laboratories.

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全血溶解四色流式细胞术细胞毒性交叉配伍。

背景：传统的淋巴细胞交叉配型（XM）检测需要分离单个核细胞。在我们之前的工作中，我们介绍了一种新的3激光，4色流式细胞术（FC） XM方案，利用全血溶解（WBL）和CD45荧光触发采集来检测人类白细胞抗原（HLA）抗体结合。在此基础上，我们的目标是利用FC （FCCDC）将这些进展应用于补体依赖性细胞毒性（CDC） XM。方法：164对接受移植的供体/受体按供体特异性HLA同种抗体水平分为2组：DSA阳性（DSA+, n = 73）和DSA阴性（DSA-, n = 91）。根据ABO相容性将DSA-组进一步细分为ABO不相容亚组（ABOi, n = 52）和ABO相容亚组（n = 39）。使用facslric细胞仪（BD Biosciences）对CD45 V500-C的WBL FCCDC进行方案优化。T和B细胞指数以死亡细胞的Δ（试验减去对照）百分比（Δ%DC）计算。比较各组WBL FCCDC结果与常规FCCDC结果。结果：WBL FCCDC与常规FCCDC在数量上无显著差异。DSA+组WBL FCCDC和常规FCCDC的B细胞Δ%DC值分别为28.00±27.14和19.16±27.74 （P = .0777）。ABOi亚组未见ABO抗体干扰。定性上，DSA+组B细胞WBL FCCDC敏感性为69.9%，与常规FCCDC敏感性（65.8%,P = .5078）相当。此外，与传统FCCDC相比，WBL FCCDC的周转时间缩短了50分钟。结论：WBL FCCDC的性能与传统FCCDC相当，与WBL FCXM相似。裂解步骤没有副作用，支持其集成到XM分析中。由于其简单性和保持的敏感性，WBL FCCDC方案在组织相容性实验室中提供了一种可行的替代方法。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Transplantation proceedings 医学-免疫学

CiteScore

1.70

自引率

0.00%

发文量

502

审稿时长

60 days

期刊介绍： Transplantation Proceedings publishes several different categories of manuscripts, all of which undergo extensive peer review by recognized authorities in the field prior to their acceptance for publication. The first type of manuscripts consists of sets of papers providing an in-depth expression of the current state of the art in various rapidly developing components of world transplantation biology and medicine. These manuscripts emanate from congresses of the affiliated transplantation societies, from Symposia sponsored by the Societies, as well as special Conferences and Workshops covering related topics. Transplantation Proceedings also publishes several special sections including publication of Clinical Transplantation Proceedings, being rapid original contributions of preclinical and clinical experiences. These manuscripts undergo review by members of the Editorial Board. Original basic or clinical science articles, clinical trials and case studies can be submitted to the journal?s open access companion title Transplantation Reports.