【作者】 李瑞；

【导师】 刘继贤；

【作者基本信息】 苏州大学 ， 儿科学， 2010， 硕士

【摘要】 目的：建立流式细胞术胞内细胞因子(cytokine flow cytometry,CFC)检测法,检测结核病组、潜伏结核感染(latent tuberculosis infection,LTBI)组、非结核病组三组分泌r-干扰素(IFN-γ)的结核杆菌特异性CD4+T淋巴细胞比例。计算各组敏感性及特异性并进行比较；同时将CFC检测法结果与结核菌素试验(即PPD皮试)、结核抗体结果进行对比分析,从而探讨流式细胞术胞内细胞因子检测法检测结核杆菌特异性T细胞—IFN-γ对儿童结核病及潜伏结核感染的诊断价值。方法：连续收集2008年01月～2010年01月苏州大学附属儿童医院门诊及住院部0-14岁疑似结核病患儿86例；潜伏结核感染患儿75例,健康对照组20例。所有入选对象均已接种BCG,并记录详细的临床资料。综合临床资料及相关检查,最终诊断为结核病者41例,其中肺结核病17例,肺外结核病24例；非结核病者45例；潜伏结核感染者75例,进行以下分组：A组：结核病组,41例；A1组：肺结核病组,17例；A2组：肺外结核病组,24例；B组：潜伏结核感染组(LTBI),75例；C组：非结核病组,45例；D组：健康对照组,20例。根据结核杆菌特异的效应T淋巴细胞在特异抗原的刺激下分泌γ-干扰素,检测IFN-γ可以确定体内是否存在对结核杆菌反应的效应T淋巴细胞的原理,结合流式细胞仪(flow cytomeytry,FCM)、胞内细胞因子(intracellular cytokine,ICK)染色法建立流式细胞术胞内细胞因子检测法检测结核菌感染的方法。每组研究对象均进行PPD试验、结核抗体检测以及流式细胞术胞内因子检测(以下简称CFC检测法)并进行全面的临床评估,随访3～6个月。CFC检测法简要步骤：肝素钠抗凝真空管采集入选患儿外周血1ml,取三只流式测定管：①阳性对照管：加入0.2ml肝素钠抗凝全血,加入佛波酯(PMA20ng／ml)、离子酶素(IN01μg／ml)共刺激,加入10μl 0.5mg／mlBrefeldin(BFA)：②刺激管：加入0.2ml肝素钠抗凝全血,加入结核杆菌特异性抗原(ESAT-6+CFP-10各50μl)刺激,加入10μl 0.5mg／ml BFA阻断剂；③未刺激管：加入0.2ml肝素钠抗凝全血,加入10μl 0.5mg／ml BFA阻断剂；5% CO2、37℃,阳性刺激管温箱培养6小时,抗原多肽刺激管培养24小时(温箱,松盖)。然后每管均加入固定剂、破膜剂,并加入荧光标记抗体,上流式细胞仪检测结核杆菌特异性T细胞—IFN-r分泌情况。计算流式检测结果的灵敏度及特异度。并与PPD试验、结核抗体检测结果进行对比分析。结果：1、结核病组及潜伏结核感染组分泌IFN-γ的结核杆菌特异性CD4+T细胞比例均高于健康对照组及非结核感染组(P<0.05)；而结核病组与潜伏结核感染组相比较,非结核感染组与健康对照组相比较,分泌IFN-γ的结核杆菌特异性CD4+T细胞均无明显差异(P>0.05)；2、用CFC法检测,41例结核病患儿90.24%阳性(37／41),9.76%阴性(4／41)；潜伏结核感染患儿85.33%阳性(64／75),14.67%阴性(11／75),结核病组与潜伏结核感染组相比,CFC检测法阳性率无明显差异(P>0.05)；而非结核感染组患儿100%阴性(45／45),阳性率为0；故结核病组及潜伏结核感染组CFC检测法阳性率均高于非结核病组(P<0.05)；健康对照组1例阳性,说明该法特异性为95%(19／20)。3、潜伏结核感染组CFC检测法阳性率为85.33%,与PPD皮试有较好的一致性(85.33%)；而结核抗体检测阳性率为28%,两者相比较,差异有显著统计学意义(P<0.01)。非结核病组PPD假阳性率为40%(18／45),而CFC法假阳性率为0,即对该组来讲,PPD皮试特异性为60%,CFC法特异性为100%,二者比较,差异有统计学意义(P<0.05)。4、结核病组CFC检测法阳性率为90.24%,结核抗体检测阳性率为56.10%,二者相比较,差异有统计学意义(P<0.05)；PPD皮试阳性率为70.73%,二者相比差异有统计学意义(P<0.05)；肺结核病组CFC检测法阳性率为94.12%,肺外结核组为87.5%,二者比较,无明显差异(P>0.05)。结论：1、结核杆菌特异性T细胞—IFN-γ流式检测法敏感性强；且由于ESAT-6、CDP-10两种特异性抗原的应用,特异度高。2、对于潜伏结核感染患儿,结核杆菌特异性T细胞—IFN-γ流式检测法可区分BCG接种反应,故适合我国儿童普遍接种BCG的国情；且所需时间短,一般24小时即可出结果,可避免主观因素对结果的影响,可以快速、准确诊断儿童潜伏结核感染；3、对于结核病患儿,结核杆菌特异性T细胞—IFN-γ流式检测法的敏感性、特异性与PPD皮试、结核抗体检测相比,均高于后者,差异有统计学意义,故该法同样可作为儿童结核病诊断的重要辅助手段。更多还原

【Abstract】 Objects:To measure the proportion of tuberculosis-antigen-specific CD4+T cells excreted cellular interferon-gamma by flow cytometry (CFC) in tuberculosis group, latent tuberculosis group, non-tuberculosis group. Statistic the positivity and specificity of each group, and then compared them. Meanwhile comparing the results of cytokine flow cytometry test, tuberculin skin test and tuberculosis antibody test to discuss the diagnosis value of antigen-specific-T cells IFN-gamma by flow cytometry in children with tuberculosis.Methods:Total of 86 consecutive cases of children aged from 0-14 years who were being assessed for suspected tuberculosis,75 cases for latent tuberculosis,20 cases for healthy control were enrolled in this study from patients’ room or outpatient in Children’s Hospital Affiliated Soochow University during 2008.01 to 2010.01.everyone had BCG vaccination history,and received full clinical assessment.at last,final diagnosis of 41cases were tuberculosis, including 17 pulmonary tuberculosis,24 extrapulmonary tuberculosis.45 cases were not tuberculosis infection.we divide all the children into:group A:tuberculosis,41cases. Including group A1:pulmonary tuberculosis,17cases; group A2: extrapulmonary tuberculosis,24cases; group B:latent tuberculosis infection,75 cases (short for LTBI); group C:non-tuberculosis,45cases; group D:healthy control,20cases. All of them received CFC test, PPD test and tuberculosis-antibody test. According the stimulation of tuberculosis specific antigen ESAT-6, CFP-10, the specific T lymphocytes in whole blood will excrete cellular interferon-gamma, combined the flow cytometry and intracellular cytokine, we create cytokine flow cytometry (CFC).the brief procedures are as following:1ml of blood was collected in Na heparin tubes.samples were divided into three 0.2-ml aliquots. One was incubated with ESAT-6 and CFP-10,one with PMA and ION,the final aliquot served as a non-stimulated control.co-stimulatory antibodies were added to all the aliquots.the blood was incubated at 37℃for several hours in the presence of the Golgi transport inhibitor brefeldin A added during the last 6h. Then fixed, washed, stained the blood for flow cytometry evaluation.at last,statistic the positivity and the specificity of each group,and then compared them.meanwhile comparing the results of cytokine flow cytometry test, tuberculin skin test and tuberculosis antibody test.Results:1、whether in groupA or groupB, the proportion of tuberculosis-antigen-specific CD4+T cells excreted cellular interferon-gamma are higher than groupD (P<0.05),while there is no obvious statistical difference between groupD and groupC(P>0.05);2、By CFC,the positivity is 90.24%(37/41), negativity is 9.76%(4/41)in groupA; the positivity is 85.33%(64/75), negativity is 14.67%(11/75) in groupB; the positivity is 13.33%(6/45), negativity is 86.67%(39/45) in groupC. So the positivity in groupA and groupB is higher than groupC (P<0.05). While there is no statistical difference between groupA and groupB (P>0.05).3、The positivity is 85.33% of CFC in groupB, has a good concordance with PPD test. While the positivity is only 28% of tuberculosis antibody test in this group, compared with the former, has significantly statistical difference (P<0.01).4、In groupA, the positivity is 90.24% by CFC, compared with the positivity 50.10% of tuberculosis antibody test, there is statistical difference between them (P<0.05).the positivity is 70.73% by PPD test,compared with CFC’s positivity, has statistical difference(P<0.05).but the positivity between groupA1 and groupA2 has no statistical difference(P>0.05).Conclusins:1、the method of antigen-specific-T cells IFN-gamma measured by flow cytometry has higher sensitivity,and because of the tuberculosis-specific-antigen ESAT-6,CFP-10,has better specificity.2、The identification by flow cytometry of antigen-specific T lymphocytes upon antigen stimulation of whole blood has a better positive predictive value than PPD test, because this test enables the identification of BCG vaccination. And could represent a further tool in the diagnosis of LTBI.3、As to tuberculosis, this test has a better positive predictive value than PPD test and tuberculosis antibody test. Because it has higher sensitivity and specificity. And also could be a important tool in the diagnosis of tuberculosis in future.更多还原