[文章编号]1000-8861(2009)02-0210-04
外周血T 淋巴细胞HLA -B27检测辅助诊断强直性脊柱炎
方 宁,章 涛*
,胡锡阶,万卫红,祁 莹,陈代雄
作者单位:遵义医学院附属医院贵州省细胞工程重点实验室收稿日期:2008-01-07;修回日期:2008-09-19基金项目:遵义医学院启动基金(2005-01)
*通信作者:章涛,遵义医学院附属医院贵州省细胞工程重点实验室,贵州遵义563003;T el:0852-*******;E -mail:oceanz t@https://www.doczj.com/doc/735190450.html,
[摘 要] 目的 探讨外周血T 淋巴细胞HLA -B27和血清多项免疫指标检测对强直性脊柱炎(AS)的辅助诊断价值。方法 采用流式细胞仪对52例疑似AS 患者外周血T 淋巴细胞HLA -B27表达进行检测,结合临床资料对AS 作出诊断;对其中37例男性病例血清免疫球蛋白IgG 、IgA 、IgM,补体C3、C4及C 反应蛋白(CRP)含量进行免疫比浊法测定。结果 52例疑似AS 患者共检测出HLA -B27+13例(男性12例,女性1例),确诊AS 患者12人(男性患者11人,女性1人),包括男性HLA -B27+AS 患者9例(最小年龄12岁,最大58岁),男性HLA -B27-AS 2例(分别为20岁和78岁),女性HLA -B27-AS 1例为(9岁),男性AS 确诊患者的HLA -B27阳性率为82%。除补体C3外,男性AS 组IgG 、IgA 、IgM 、C4及CRP 均较男性非AS 组高(P <0.05)。结论 AS 患者体液免疫功能明显活跃,HLA -B27检测有助于临床资料疑似病例的AS 诊断。
[关键词] 强直性脊柱炎;HLA -B27;T 淋巴细胞;免疫功能
[中图分类号] R593.23;R331.1+44;R446.62 [文献标志码] A
Detection of peripheral blood T lymphocytic HLA -B27expression for auxiliary diagnosis of anky -losing spondylitis
FANG Ning,ZHANG Tao,HU Xijie,W AN Weihong,QI Ying,C HEN Daixiong
Key Labo rato ry o f Cell Engineering o f Guizhou Province ,Aff iliated Hospital o f Zunyi Medical College ,Zunyi 563003,China
[Abstract ] Objective To study the value of peripheral blood T lymphocytic HLA -B27expressi on and serum i mmune index levels in auxiliary diagnosis of ankylosing spondylitis (AS).Methods Flow cytometer (FCM)was used to detect the peripheral blood T lymphocytic HLA -B27expression in 52suspected AS cases in which the AS patien ts were diagnosed through clinical date combined with HLA -B27detecting results.Seru m levels of IgG,IgA,IgM ,C4,C3,and C -reactive protein (CRP)were measured by nephelometry in total 37cases of male.Results Among 52suspected AS cases,13samples were detected as HLA -B27posi tive including 12cases of male and 1case female.Through aggregate analysis of clinical date and HLA -B27results,12patients of AS were final diagnosed comprising of 11male and 1female in
which 9cases were HLA -B27+
male AS(12-58year -old),2cases were HLA -B27-male AS(20and 78year -old),and 1case was HLA -B27-female AS (9year -old).The HLA -B27positive rate of male AS patients was 82%.Nephelometry assay performed in total 37cases of male shown that the levels of seru m i mmune globulin (IgG,IgA,IgM ),C4,and CRP in male AS patients were all higher than those of non -AS male cases (P <0.05).Conclusion Detecting of HLA -B27on the surface of peripheral blood T lymphocytes in suspected AS patien ts is helpful for accurately diagnosing of AS patients with active humoral immune function.
[Key w ords ] ankylosing spondylitis;human leukocyte antigen B27;T lymphocyte;immune function
Brewerton 等[1]
于1973年首次发现人类白细胞抗原(human leukocyte antigen -B27,HLA -B27)与强直性脊柱炎(ankylosing spondylitis,AS)之间存在密切关联,此后的研究表明HLA -B27与银屑病性关节炎、Reiter 综合征、前眼色素层炎和溃疡性结肠炎等也存
在不同程度的相关性[2]
。由于AS 临床症状与许多疾病相似而难以确诊,因此HLA -B27的检测已成为
临床诊断和鉴别诊断AS 的重要指标[3]
。本研究采用流式细胞仪检测外周血T 淋巴细胞HLA -B27,并
结合临床资料对52例疑似AS 病例作出了最终诊
断,同时对确诊后的男性AS 患者血清多项免疫指标进行了检测,籍以观察AS 患者血清免疫功能变化特点,为今后AS 的临床诊断提供参考。
1 材料与方法
1.1 研究对象 本院骨科和免疫内分泌科疑似AS 住院患者52例,其中男性37例,女性15例,年龄在9~78岁之间;临床主要表现为腰痛,X 光片显示骶髂关节面密度增高、关节间隙模糊等关节炎征象,部分病例腰段脊柱旁韧带钙化,呈/竹节样0改变。1.2 试剂 HLA -B27检测试剂盒购自美国BD 公司;免疫球蛋白(I gG 、IgA 、Ig M)、补体(C3、C4)检测试剂为上海科华公司产品;C 反应蛋白(CRP)检测试剂
#
210#
购自德国DiaSys 公司。1.3 HLA -B27检测及结果判断 肝素抗凝真空管采集外周血2mL,应用美国BD 公司FACSCalibur 流式细胞仪和HLA -B27专用分析软件,对52例疑似患者外周血T 淋巴细胞HLA -B27抗原表达进行检测分析。操作步骤为:取30L L 试剂A 加入到12m m @75mm 的塑料试管底部,加入90L L 充分混匀的新鲜抗
凝全血(确保W BC 的浓度在3.5@103~9.4@103L L -1
之间),低速混匀,室温下避光静置15~20min;每管加入2mL 1@浓度的溶血素,低速混匀,室温下避光静置10~12min;1500r P min 离心5min,弃上清液,加入2mL 含0.1%叠氮钠PBS 洗1次;1500r P min 离心5min,弃上清,加0.3mL 含1%多聚甲醛的PBS 固定液,低速混匀后置4e 冰箱,24h 内上机检测。每个样品采集的细胞数不少于20000个,以CD3(T 淋巴细胞)设门并检测其HLA -B27的平均荧光强度值(MFI),大于本批HLA -B27试剂盒设定界值147者为阳性,否则报告为阴性。
1.4 疑似患者AS 诊断标准 参照1984年修订的
纽约诊断标准[4]
,结合上述HLA -B27检测结果对HLA -B27阳性和阴性AS 做出诊断。
1.5 血清免疫指标测定 参照试剂盒说明制备样
品,采用免疫比浊法用日本Olympus AU 2700型全自动生化分析仪对上述37例男性受试者免疫球蛋白IgG 、IgA 、IgM,补体C3、C4及CRP 进行检测分析。1.6 统计学分析 血清免疫指标检测结果以 x ?s 表示,用SPSS13.0软件包对实验数据进行t 检验,P <0.05为差异有显著性。
2 结果
2.1 外周血T 淋巴细胞HLA -B27检测及AS 诊断结果 结果如图1、表1所示。52例疑似AS 病例共
检测出HLA -B27+
13例(男性12例,女性1例),参照1984年修订的纽约诊断标准并结合HLA -B27检测结果共确诊AS 患者12人(男性患者11人,女性
1人),包括男性HLA -B27+
AS 患者9例(最小年龄
12岁,最大58岁),男性HLA -B27-AS 2例(分别为
20岁和78岁),女性HLA -B27-AS 1例为(9岁),其中,男性AS 确诊患者的HLA -B27阳性率为82%(9P
11);3例男性HLA -B27+
非AS 患者最终分别诊断为血清阴性关节炎(21岁)、幼年型类风湿性关节炎(13岁)和银屑性关节炎(45岁),女性唯一1例HLA -B27+
(53岁)
确诊为银屑病性关节炎。
a ) negative samples;
b ) positive samples.
图1 外周血T 淋巴细胞HLA -B27表达的流式细胞仪检测图
Fig 1 Analysis of HLA -B27expression on peripheral blood T lymphocytes by Flow cytometry
表1 52例外周血T 淋巴细胞HLA -B27表达检测及AS 诊断结果 Tab 1 Detection of peripheral blood T lymphocytic HLA -B27expression and AS diagnosis in 52suspected cases group n mean age B27+
nu mber
AS subjects number
B27+P B27-AS subjects and mean age
B27+AS subjects
mean age B27-AS subjects
mean age male 3741.61211934.6249.0female 1541.3110-19.0zusammen
52
-13
12
9
-3
-
2.2 男性AS 患者血清免疫指标检测结果 37例男性病例血清多项免疫指标检测结果见表2。除补体C3外,男性AS 组IgG 、IgA 、I gM 、C4及C RP 均较男性非AS 组高(P <0.05)。
#
211#
表211例男性AS患者血清免疫指标检测结果
Tab2Serum im mune globulin,C3,C4and CRP levels in11AS cases of male
group n IgG P(g#L-1)IgA P(g#L-1)IgM P(g#L-1)C3P(g#L-1)C4P(g#L-1)CRP P(mg#L-1) AS male1126.55?9.08 4.80?1.26 2.33?0.17 1.41?0.490.75?0.2713.30?5.21 non-AS male2611.61?2.30a 1.66?0.70a 1.62?0.44b 1.13?0.19c0.26?0.05a 2.48?1.84a reference value8~160.7~3.30.5~2.20.8~1.60.1~0.40.1~8.0
a)P<0.01,b)P<0.05,c)P>0.05,vs AS male group.
3讨论
HLA是人体内最为复杂的遗传多态性系统, HLA的主要功能是参与抗原的处理与呈递,参与免疫应答的遗传控制及T细胞的活化,在免疫调节中起重要的作用[5]。AS是一种主要累及中轴关节的慢性炎症性疾病,常以腰背痛和外周关节炎为首发症状,多发生于青壮年,男女比例约为6.4B1[6]。群体研究表明,AS患者在不同人群和种族中都与HLA-B27存在相关性,且AS的群体发病率与人群的HLA-B27阳性率呈正相关,中国人群的HLA-B27阳性率约为8%,AS发病率为0.3%[7]。
HLA-B27基因属于MHC-?类基因,人外周血有核细胞上均可表达HLA-B27,尤以淋巴细胞表面表达丰度最高。超过90%的强直性脊椎炎患者其HLA-B27抗原表达为阳性。由于强直性脊椎炎患者的临床症状与许多疾病相似而难以确诊,因此HLA-B27的检测已成为临床诊断和鉴别诊断强直性脊椎炎的重要指标[8]。与微量细胞毒法和PCR检测方法比较,流式细胞仪检测HLA-B27的表达无须分离淋巴细胞,且操作简单,其检测灵敏度可达100%,特异性为97.4%,不同仪器型号及实验室之间的检测重复性高,已成为国际上通用的最佳方法[9]。本文选择检测T淋巴细胞B27的表达能最好地满足实验的敏感性和特异性,若用FSC-SCC设门淋巴细胞,可能混入其它细胞(如单核细胞或细胞碎片,不易与淋巴细胞区分),导致检测结果不准确[10]。当样本HLA-B27抗原的平均荧光强度大于或等于标准品道值则判定为HLA-B27阳性,反之则为HLA-B27阴性。
本组52例疑似AS病例共检出HLA-B27+13例(男性12例,女性1例),结合临床资料和X线检查共确诊AS患者12人(男性患者11人,女性1人),包括男性HLA-B27+AS患者9例(最小年龄12岁,最大58岁),男性HLA-B27-AS2例(分别为20岁和78岁),女性HLA-B27-AS1例为(9岁),其男性AS 确诊患者的HLA-B27阳性率为82%,略低于文献报道的比例,可能与本组资料样本数较少有关。从本组资料可见,HLA-B27+病例和确诊的AS患者绝大部分系男性,男女AS患病比例为4.4B1,男性HLA-B27+AS患者平均患病年龄远低于男性HLA-B27-AS患者,这表明HLA-B27+AS的患病时间明显提前。本研究同时对最终确诊的11例男性AS患者血清多项免疫指标进行检测,除补体C3外,其血清IgG、I gA、IgM、C4及CRP水平均较男性非AS患者组高,这表明,AS患者体液免疫处于活跃状态,支持免疫因素参与AS发病的观点[11]。
研究表明,除AS与HLA-B27存在密切关联外,其它一些疾病与HLA-B27抗原的表达也有着或多或少的相关性。譬如,Reiters综合症的HLA-B27阳性率为70%~90%,关节炎性银屑病的HLA-B27阳性率为50%~60%,葡萄膜炎的HLA-B27阳性率为40%~50%,溃疡性结肠炎伴有关节病的HLA-B27阳性率为5%~10%等,故而可将此类疾病统称为/B27疾病0[12]。本组资料3例男性HLA-B27+非AS 患者最终分别诊断为血清阴性关节炎(21岁)、幼年型类风湿性关节炎(13岁)和银屑性关节炎(45岁),女性唯一1例HLA-B27+(53岁)确诊为银屑病性关节炎,说明HLA-B27的检测对上述非AS疾病也具有一定的辅助诊断价值。
虽然HLA-B27与AS高度相关,但因HLA-B27阳性群体中仅有不到5%的个体最终发展成AS,故不能将HLA-B27检测作为随机人群AS的筛选指标[13]。只有在受检者临床症状、体症及X线检查疑为AS时,HLA-B27检测为阳性则可明显支持AS的临床诊断。由于AS具有较强的家族遗传倾向,其亲属中发病率比一般人高出近50倍,因此,应当对AS 患者一级亲属做HLA-B27抗原检测,这对AS早期诊断、延缓发病乃至预防发病均有重要意义。
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213
#