HLA-B27 Antigen Detection With Flow Cytometry For Ankylosing Spondylitis Diagnostics

Ankylosing spondylitis(AS)is a chronic inflammatory disease.Because early symptoms of AS are similar to general joint pain,it often causes misdiagnosis and missed diagnosis,resulting in the inability to be effectively controlled and severely leading to disability.Based on the strong relationship between ankylosing spondylitis and human leukocyte antigen HLA-B27,the expression of HLA-B27 antigen in human peripheral blood T lymphocyte population was detected by flow cytometry,and a qualitative method to detect HLA-B27 negative or positive was established,which could assist the clinical diagnosis of ankylosing spondylitis.Firstly,HLA-B27 FITC,CD3 PerCP and HLA-B7 monoclonal antibodies from different suppliers were evaluated.Standard cell lines and clinical HLA-B27 positive expression samples were used to determine the appropriate concentration of each antibody used.The optimal concentrations were HLA-B27 FITC 0.08?g/Test,CD3 PerCP 0.125?g/Test and HLA-B7 0.25?g/Test,respectively.The resolution and fluorescence efficiency could meet the requirements for clinical diagnosis.Secondly,three monoclonal antibodies were used to form a combined reagent,and the method was established to qualitatively detect HLA-B27 with flow cytometry.Compared with the RT-PCR method,a threshold range for qualitative judgment of HLA-B27 was set using the samples of clinically-known HLA-B7 negative and positive expression.It was found that there was a clear distinction on the fluorescence intensity between HLA-B27 negative and positive samples.Moreover,the threshold value was selected by ROC curvesis with the sensitivity and specificity greater than 0.97.The positive judgment threshold was 9138,and the negative threshold was 6801.In order to eliminate the differences between the instruments,standard flow microspheres were used to calibrate different flow cytometers,and the results after the modification reached 100%consistency.Finally,the reliability of new method established in the present work was verified with compliance,precision,linear range,and analytical specificity.The coincidence rate between new method and the RT-PCR method was found to be 100%,and the intra-assay precision and the inter-assay precision coefficient of variation were less than 5%,When the cell concentration of clinical sample was in the range of 0.5×103?40×103/?L,new method could still maintain the accuracy.The effects of interfering substances in serum were investigated.It was found that there were no interference cross-effects when rheumatoid factor?29.0 IU/mL,cholesterol ? 272 mg/dL,bilirubin?7.35 mg/dL,prolactin?27.8 ng/L,progesterone?30.0 ng/ml,human chorionic gonadotropin?139 IU/L,thyroid stimulating hormone ?36.4 mIU/L,.In this thesis,suitable monoclonal antibodies were screened and flow cytometry was used as a detection platform for qualitative detection of HLA-B27 antigen.The reliability of new method was verified,which would be useful for clinical screening of ankylosing spondylitis.