Establishment of HLA-B27 cut-off value by flow cytometry and related grey zone sample genotype determination

doi:10.3969/j.issn.1673-8640.2020.01.004

Abstract

Abstract:

Objective To establish the cut-off value of human leukocyte antigen（HLA）-B27 by flow cytometry（FCM）,and to find a solution for related samples in grey zone. Methods Peripheral blood samples of 132 low back and joint pain patients were collected. The expression of HLA-B27 was determined by FCM,and the HLA-B typing was determined by flow reverse sequence specific oligonucleotide（Flow-rSSO）. Results The positive rates of HLA-B27 by FCM and Flow-rSSO were 18.18% and 73.48%,respectively. According to the recommendation rules of FCM HLA-B27 kit,45 samples in grey zone were confirmed by Flow-rSSO,which were all HLA-B27 positive. After the genotype analysis of samples with different d values（the difference between mean fluorescence intensity and the median value of calibrated magnetic beads）,the cut-off values were setted. The d>2 was positive,d≤-7 was negative,and -7<d≤2 was weak positive（grey zone）. The false negative and false positive rates were 0%. The detection rate of HLA-B27 in grey zone was 80.6%. The fluorescence intensity of B27 antigen was not related to the typing of HLA-B27,and there was a relation with the number of HLA-B27. The grey zone samples had HLA-B7 and HLA-B37 genotype. Conclusions Clinical laboratories should establish their own rules for reducing false negative rate and shrink the range of grey zone as far as possible. Grey zone samples usually have special HLA-B genotypes,which can be further determined by Flow-rSSO to improve the accuracy of HLA-B27 determination.

Key words: Human leukocyte antigen-B27, Flow cytometry, Ankylosing spondylitis, Flow reverse sequence specific oligonucleotide

CLC Number:

R446.1

WU Beiying, GU Yanying, FAN Zhenjia, CAI Gang. Establishment of HLA-B27 cut-off value by flow cytometry and related grey zone sample genotype determination[J]. Laboratory Medicine, 2020, 35(1): 15-19.

Figures/Tables 5

References 23

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FCM d值	例数	Flow-rSSO法		范围拟定
FCM d值	例数	HLA-B27/例	其他型别 HLA-B/例	范围拟定
>10	24	24	0	阳性
>5~10	11	11	0	阳性
>0~5	25	25	0	阳性
>-5~0	43	37	6	灰区
>-10~-5	12	0	12	阴性
≤-10	17	0	17	阴性

FCM d值	例数	Flow-rSSO法		范围拟定
FCM d值	例数	HLA-B27/例	其他型别 HLA-B/例	范围拟定
>10	24	24	0	阳性
>5~10	11	11	0	阳性
>0~5	25	25	0	阳性
>-5~0	43	37	6	灰区
>-10~-5	12	0	12	阴性
≤-10	17	0	17	阴性

HLA 基因型	拟定灰区（-5<d≤0）	扩大拟定灰区（-7<d≤2）
B27	37（86.06）	50（80.65）
B07	3（6.98）	4（6.45）
B37	1（2.32）	6（9.68）
B67	1（2.32）	1（1.61）
其他	1（2.32）	1（1.61）
合计	43（100）	62（100）

HLA 基因型	拟定灰区（-5<d≤0）	扩大拟定灰区（-7<d≤2）
B27	37（86.06）	50（80.65）
B07	3（6.98）	4（6.45）
B37	1（2.32）	6（9.68）
B67	1（2.32）	1（1.61）
其他	1（2.32）	1（1.61）
合计	43（100）	62（100）

HLA-B27基因型别	例数	d值
2702:××	2	4.00^#
2704:××^*	55	6.24^#
2705:××	34	2.29^#
2707:××	1	-4.00^#
2715:××	3	4.00^#
2704:2704	2	29.00