Diagnosis Value Of Antigen-specific-T Cells IFN-gamma By Flow Cytometry In Children With Tuberculosis

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.