流式细胞术检测HLA-B27阴阳性判读标准的建立及灰区样本的基因型分析

doi:10.3969/j.issn.1673-8640.2020.01.004

摘要/Abstract

摘要：

目的建立流式细胞术（FCM）检测人类白细胞抗原（HLA）-B27的阴阳性判读标准,同时探讨灰区样本的特性及应对措施。方法采用FCM检测132例腰背关节痛患者外周血HLA-B27的表达,以流式反向序列特异性寡核苷酸探针（Flow-rSSO）法检测患者HLA-B位点的分型。结果 FCM和Flow-rSSO法检测HLA-B27的阳性率分别为18.18%和73.48%。根据FCM HLA-B27试剂盒推荐的判读规则,灰区样本45例,经Flow-rSSO法验证后,HLA-B27全部为阳性。通过对不同d值（荧光强度的均值与校准磁珠中位值的差值）范围样本的基因型分析设定判读规则,即d>2均判读为阳性,d≤-7为阴性,-7<d≤2为弱阳性（灰区）,假阴性率和假阳性率均为0%,灰区样本HLA-B27检出率为80.6%。B27抗原荧光强度与HLA-B27的分型类别无关,而与带有HLA-B27的个数有关,灰区样本通常带有HLA-B7和HLA-B37基因型。结论实验室应拟定自己的HLA-B27阴阳性判断规则,在保证真阳性率的同时,降低假阴性率,并且将灰区范围尽量缩小。灰区样本通常具有特殊的HLA-B型别,可通过Flow-rSSO法来进一步检测,以提高HLA-B27的检出准确度。

关键词: 人类白细胞抗原-B27, 流式细胞术, 强直性脊柱炎, 流式反向序列特异性寡核苷酸探针

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

中图分类号:

R446.1

吴蓓颖, 顾燕英, 范臻佳, 蔡刚. 流式细胞术检测HLA-B27阴阳性判读标准的建立及灰区样本的基因型分析[J]. 检验医学, 2020, 35(1): 15-19.

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.

图/表 5

参考文献 23

[1]	肖林林,王璐璐,赵卫卫,等. ROC曲线在HLA-B27界值设置中的应用[J]. 检验医学,2016,31(4):338-339.
[2]	金士正,程曦,李桢,等. Flow-rSSO与PCR-SSP方法在骨髓库HLA基因分型中的应用比较[J]. 江西医学检验,2006,24(4):309-311.
[3]	FELDTKELLER E,KHAN M A,VAN DER HEIJDE D,et al. Age at disease onset and diagnosis delay in HLA-B27 negative vs. positive patients with ankylosing spondylitis[J]. Rheumatol Int,2003,23(2):61-66.
[4]	BROWN M,WORDSWORTH P.Genotyping HLA-B27 in spondyloarthropathies[J]. J Rheumatol,1998,25(4):820-821.
[5]	BREWERTON D A,HART F D,NICHOLLS A,et al.Ankylosing spondylitis and HL-A27[J]. Lancet,1973,1(7809):904-907.
[6]	SCHLOSSTEIN L,TERASAKI P I,BLUESTONE R,et al.High association of an HL-A antigen,W27,with ankylosing spondylitis[J]. N Engl J Med,1973,288(14):704-706.
[7]	VAN DER HEIJDE D,RAMIRO S,LANDEWÉ R R,et al. 2016 Update of the ASAS-EULAR management recommendations for axial spondyloarthritis[J]. Ann Rheum Dis,2017,76(6):978-991.
[8]	NEUMÜLLER J,SCHWARTZ D W,DAUBER E,et al. Evaluation of four monoclonal antibodies against HLA-B27 for their reliability in HLA-B27 typing with flow cytometry (FC):comparison with the classic microlymphocytotoxic test (MLCT)[J]. Cytometry,1996,26(3):209-215.
[9]	SEIPP M T,ERALI M,WIES R L,et al.HLA-B27 typing:evaluation of an allele-specific PCR melting assay and two flow cytometric antigen assays[J]. Cytometry B Clin Cytom,2005,63(1):10-15.
[10]	LEVERING W H,WIND H,SINTNICOLAAS K,et al.Flow cytometric HLA-B27 screening:cross-reactivity patterns of commercially available anti-HLA-B27 monoclonal antibodies with other HLA-B antigens[J]. Cytometry B Clin Cytom,2003,54(1):28-38.
[11]	SAS D F,SAS M J,JOHNSON K R,et al.Junctions between lens fiber cells are labeled with a monoclonal antibody shown to be specific for MP26[J]. J Cell Biol,1985,100(1):216-225.
[12]	ROELANDSE-KOOP E A,BUISMAN B,VAN HANNEN E J,et al. Rapid HLA-B27 screening with real-time TaqMan PCR:a clinical validation in the Dutch population[J]. Clin Chem Lab Med,2011,49(12):1979-1985.
[13]	AFSHAN N,BASHIR M,TIPU H N,et al.Optimization of an in-house PCR method for the detection of HLA-B*27 alleles[J]. Biomed Rep,2018,8(4):385-390.
[14]	ZENG Y,HITI A,MORANVILLE S,et al.Human HLA-B27 typing using the BDTM HLA-B27 kit on the BD FACSViaTM system:A multicenter study[J]. Cytometry B Clin Cytom,2018,94(5):651-657.
[15]	KHAN M A.An update on the genetic polymorphism of HLA-B*27 with 213 alleles encompassing 160 subtypes(and still counting)[J]. Curr Rheumatol Rep,2017,19(2):9.
[16]	LIU Y,JIANG L,CAI Q,et al.Predominant association of HLA-B*2704 with ankylosing spondylitis in Chinese Han patients[J]. Tissue Antigens,2010,75(1):61-64.
[17]	GONZALEZ-ROCES S,ALVAREZ M V,GONZALEZ S,et al.HLA-B27 polymorphism and worldwide susceptibility to ankylosing spondylitis[J]. Tissue Antigens,1997,49(2):116-123.
[18]	FULLER A A,RODEY G E,PARHAM P,et al.Epitope map of the HLA-B7 CREG using affinity-purified human alloantibody probes[J]. Hum Immunol,1990,28(3):306-325.
[19]	LEVERING W H,WIND H,HOOIJKAAS H,et al.Flow cytometric screening for HLA-B27 on peripheral blood lymphocytes[J]. J Biol Regul Homeost Agents,2003,17(3):241-246.
[20]	LEVERING W H,VAN DEN BEEMD R,TE MARVELDE J G,et al. External quality assessment of flow cytometric HLA-B27 typing[J]. Cytometry,2000,42(2):95-105.
[21]	LINGENFELTER B,FULLER T C,HARTUNG L,et al.HLA-B27 screening by flow cytometry[J]. Cytometry,1995,22(2):146-149.
[22]	HOFFMANN J J,JANSSEN W C.HLA-B27 phenotyping with flow cytometry:further improvement by multiple monoclonal antibodies[J]. Clin Chem,1997,43(10):1975-1981.
[23]	COATES E,DARKE C.Routine HLA-B27 typing by flow cytometry:differentiation of the products of HLA-B2702,B2705 and B*2708[J]. Eur J Immunogenet,1998,25(1):29-37.

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

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