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

doi:10.3969/j.issn.1671-2587.2026.04.012

JOURNAL OF CLINICAL TRANSFUSION AND LABORATORY MEDICINE ›› 2026, Vol. 28 ›› Issue (4): 539-545.DOI: 10.3969/j.issn.1671-2587.2026.04.012

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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

Abstract

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

CLC Number:

R446.6

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

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