流式荧光法PRA检测中降低非特异性本底的组合预处理研究*

doi:10.3969/j.issn.1671-2587.2026.04.012

临床输血与检验 ›› 2026, Vol. 28 ›› Issue (4): 539-545.DOI: 10.3969/j.issn.1671-2587.2026.04.012

流式荧光法PRA检测中降低非特异性本底的组合预处理研究^*

王璐璐, 陈利达, 芦宏凯, 刘希曦, 郭伟洁, 高欣, 曹永彤

中日友好医院输血科,北京 100029

收稿日期:2026-07-08 出版日期:2026-08-20 发布日期:2026-08-26
通讯作者: 曹永彤,主要从事器官移植排斥标志物研究,（E-mail）caoyongtong@sina.com。
作者简介:王璐璐,主要从事移植免疫学和器官移植配型研究,（E-mail）luluhappy_1004@163.com。
基金资助:
^*本课题受中央高水平医院临床业务费项目（No.2025-NHLHCRF-XKJS-B-06）资助

A Pre-treatment Strategy for Reducing Non-specific Background in Flow Fluorometric PRA Testing

WANG Lulu, CHEN Lida, LU Hongkai, LIU Xixi, GUO Weijie, GAO Xin, CAO Yongtong

Department of Blood Transfusion,China-Japan Hospital, Beijing 100029

Received:2026-07-08 Online:2026-08-20 Published:2026-08-26

摘要/Abstract

摘要： 目的评估冻融-高速离心-EDTA组合预处理方法在降低流式荧光法群体反应性抗体（PRA）检测非特异性背景中的有效性和安全性。方法回顾性分析2025年6月—2025年9月于临床送检的232例PRA检测标本。以至少一个阴性质控微珠荧光强度超出本批次试剂盒推荐范围定义为高本底。对高本底标本依次进行冻融（-30 ℃冷冻12 h,20 ℃复融）、高速离心（13 000×g,15 min）及EDTA（终浓度2.3 mmol/L,孵育10 min）处理。为验证EDTA的增量效果,选取前31例高本底标本进行配对分析（方法一：无EDTA;方法二：含EDTA）,后续28例仅采用方法二处理。另取10例本底正常标本验证方法安全性。结果 232例标本经常规高速离心预处理后,173例（74.6%）阴性质控正常,59例（25.4%）为高本底。59例高本底标本经方法二（含EDTA）处理后,22例（37.29%）完全正常化,22例（37.29%）部分改善,15例（25.42%）反应增强。前31例配对对比显示：方法二（含EDTA）改善率（74.19%）较方法一（64.52%）呈升高趋势,但差异无统计学意义,且使3例标本分类提升。10例本底正常标本经方法二处理后,各质控MFI均无显著变化（P>0.05）。结论冻融-高速离心-EDTA组合预处理方法可有效降低部分PRA检测高本底标本的高本底,其中EDTA是关键步骤且对正常标本无显著干扰。

关键词: 群体反应性抗体, 流式荧光法, 阴性质控, 标本预处理, 质量控制

Abstract: Objective To evaluate the efficacy and safety of a combined "freeze-thaw, high-speed centrifugation, and EDTA" pre-treatment method in reducing non-specific high background in flow fluorometric panel reactive antibody (PRA) testing. Methods A retrospective analysis was performed on 232 PRA testing specimens clinically submitted from June 2025 to September 2025. High background was defined as the fluorescence intensity of at least one negative control bead exceeding the manufacturer's recommended range. High background Samples were sequentially treated by freeze-thaw (-30 ℃ for 12 h, thawed at 20 ℃), high-speed centrifugation (13 000×g, 15 min), and EDTA (final concentration 2.3 mmol/L, incubated for 10 min). To verify the incremental value of EDTA, a paired analysis was performed on the first 31 high-background samples using two methods: Method1 (without EDTA) and Method2 (with EDTA). The remaining 28 specimens were treated with Method2 only. Additionally, 10 normal-background specimens were selected to verify the safety of the method. Results After routine high-speed centrifugation, 173 of the 232 specimens (74.6%) had normal negative control MFI, and 59 (25.4%) still exhibited high background. After treatment with Method2 (with EDTA), 22 specimens (37.29%) achieved complete normalization, 22 (37.29%) showed partial improvement, and 15 (25.42%) showed enhanced reactivity. Paired analysis of the first 31 samples indicated that Paired comparison of the first 31 specimens showed that Method2 yielded a higher effective improvement rate (74.19%) than Method1 (64.52%), with 3 samples improvement of the classification. Furthermore, the mean fluorescence intensities (MFIs) of all quality control markers in the 10 normal-background samples showed no significant changes after treatment (P>0.05), demonstrating good safety. Conclusion The combined "freeze-thaw, high-speed centrifugation, and EDTA" pre-treatment method effectively reduces high-background in PRA testing. EDTA is a crucial step in this protocol, and the method shows no significant interference with normal-background specimens.

Key words: Panel reactive antibody, Flow cytometry, Negative control, Specimen pre-treatment, Quality control

中图分类号:

R446.6

王璐璐, 陈利达, 芦宏凯, 刘希曦, 郭伟洁, 高欣, 曹永彤. 流式荧光法PRA检测中降低非特异性本底的组合预处理研究^*[J]. 临床输血与检验, 2026, 28(4): 539-545.

WANG Lulu, CHEN Lida, LU Hongkai, LIU Xixi, GUO Weijie, GAO Xin, CAO Yongtong. A Pre-treatment Strategy for Reducing Non-specific Background in Flow Fluorometric PRA Testing[J]. JOURNAL OF CLINICAL TRANSFUSION AND LABORATORY MEDICINE, 2026, 28(4): 539-545.

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流式荧光法PRA检测中降低非特异性本底的组合预处理研究^*

A Pre-treatment Strategy for Reducing Non-specific Background in Flow Fluorometric PRA Testing

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