Interference of high levels of bilirubin on lymphocyte subset determination in peripheral blood by flow cytometry and its elimination methods

doi:10.3969/j.issn.1673-8640.2022.12.013

Abstract

Abstract:

Objective To investigate the interference of high levels of bilirubin on lymphocyte subset determination in peripheral blood by flow cytometry（FCM） and its elimination methods. Methods Totally，51 patients with serum total bilirubin（TB） >200 μmol/L and undergoing lymphocyte subset determination were enrolled，which included 6 cases of elevated direct bilirubin（DBil）（DBil group） and 45 cases of elevated indirect bilirubin（IBil）（IBil group）. The non-jaundiced samples from 6 healthy children（control samples）were collected，and plasmas with high DBil were added to the control samples to prepare for interference samples. Paired difference experiments were used to analyze the interference of bilirubin on lymphocyte subsets determined by FCM and to assess the dose of bilirubin interference. The effects of washing-staining-lysis method and staining-lysis-washing method on bilirubin interference elimination were assessed. Results In elevated IBil group，lymphocyte subset determination was not interfered. But in elevated DBil group，the fluorescence expressions of fluorescein isothiocyanate（FITC）-labeled CD3^- cells and CD3⁺ cells were not clearly demarcated. For interference samples treated by staining-lysis method，the fluorescence expressions of FITC-labeled CD3^- cells and CD3⁺ cells were also not clearly demarcated. When DBil≤150 μmol/L，no effect on FCM was found. When DBil≥200 μmol/L，the interference was greater. Such interference could be eliminated to the greatest extent by both washing-staining-lysis method and staining-lysis-washing method. Staining-lysis-washing method was more operable，and no significant difference was found in lymphocyte subsets between interference samples and control samples（P>0.05），and the percentages of CD3⁺，CD3⁺CD8⁺，CD3⁺CD4⁺，CD3^-CD16/56⁺，CD3^-CD19⁺ cells and CD4/CD8 ratios between the 2 samples were positively correlated（r=0.977，0.994，0.950，0.941，0.996 and 0.965，P<0.05）. Conclusions High level of DBil can interfere with the fluorescence expressions of FITC-labeled CD3^- cells and CD3⁺ cells in lymphocyte subset determination in peripheral blood by FCM. Staining-lysis-washing method can eliminate such interference to the greatest extent.

Key words: Bilirubin, Flow cytometry, Lymphocyte subset, Interference, Elimination method

CLC Number:

R446.1

XUE Yan, XU Li, DANG Liheng, WANG Chao, CUI Yaqiong, WANG Ping, WANG Ning, ZHANG Xinjie, LIU Yang. Interference of high levels of bilirubin on lymphocyte subset determination in peripheral blood by flow cytometry and its elimination methods[J]. Laboratory Medicine, 2022, 37(12): 1169-1173.

Figures/Tables 7

References 8

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样本	例数	CD3⁺细胞/%	CD3⁺CD8⁺细胞/%	CD3⁺CD4⁺细胞/%	CD3^-CD16⁺/56⁺细胞/%	CD3^-CD19⁺细胞/%	CD4/CD8比值
对照样本	6	64.4±7.4	26.5±12.5	33.6±5.5	23.4±10.7	10.6±4.2	1.54±0.71
干扰样本	6	65.0±7.7	27.2±11.5	33.3±4.4	24.3±10.6	10.0±4.7	1.43±0.60
t值		-0.807	-1.090	0.363	0.782	0.293	1.355
P值		0.457	0.325	0.732	0.470	0.781	0.233

样本	例数	CD3⁺细胞/%	CD3⁺CD8⁺细胞/%	CD3⁺CD4⁺细胞/%	CD3^-CD16⁺/56⁺细胞/%	CD3^-CD19⁺细胞/%	CD4/CD8比值
对照样本	6	64.4±7.4	26.5±12.5	33.6±5.5	23.4±10.7	10.6±4.2	1.54±0.71
干扰样本	6	65.0±7.7	27.2±11.5	33.3±4.4	24.3±10.6	10.0±4.7	1.43±0.60
t值		-0.807	-1.090	0.363	0.782	0.293	1.355
P值		0.457	0.325	0.732	0.470	0.781	0.233

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