Flow cytometry or molecular biology for HLA-B27 detection in seronegative spondylarthropaties ? Author: Student Armean Iulia, 4th grade, GM Coordinator: Prof. Dr. Minodora Dobreanu

Backround: Simptomps : lumbosacral pain >3 months pain and stiffness of the thoracic region Simptomps : decreased thoracic expansion decreased lumbar mobility

Ankylosing spondilytis Backround: Chronic inflammatory disease Ankylosing spondilytis HLA B27 Axial skeleton Flow cytometry or molecular biology Interference of specific monoclonal antibody HLA B27 with other related phenotypes !!!

Purpose: This study is meant to emphasize the interference problem of anti HLA-B27 Ab used in flow cytometric tests for patients diagnosed in Emergency Hospital of Tg. Mures during May 2010- March 2014

Material s and methods 1: Type of study : retrospective Sample: 98 persons with ages between 14 and 80 years Flow cytometry tests with a BD FACS Calibur analyser, using BD HLA-B27 kit For the uncertain results, HLA-SSP(sequence specific primers) and HLA-SSO(sequence specific oligonucleotides) typing techniques were used to clarify genotype

Flow citometry technique Sample Preparation + "staining" Equipment setings- PMT voltage adjustment, the adjustment of spectral overlap (optimization). The acquisition of the unknown samples The Analysis of the obtained data

Material s and methods 2: Direct marking (the fluorochrome is attached to the monoclonal antibody CD3 HLA-B27 Anti CD3(phycoerythrin) Anti HLA-B27(fluorescein) T Lymphocyte

Flow citometry interpretation Granulocytes Monocytes Lymphocytes CD 3+ Ly

Results 1:

Results 2:

Results 3: Granulocytes Monocytes Lymphocytes CD 3+ Ly Patient 3 (HLA-B27 !!) Patient1 (HLA-B27-) Patient2 (HLA-B27+)

HLA B13 phenotype shows no interference Results 4: HLA B39 is one molecule which can shade HLA B27 resulting false negative reactions Also it can create a picture of false expression of HLA B07 resulting a false HLA B27 positive reaction HLA B13 phenotype shows no interference

conclusions: Flow cytometry remains the preferred test in screening for the presence of the HLA-B27 molecule 85% of cases are diagnosed by this technique Anti-HLA B27 antibodies are prone to binding to various cross-reactive HLA-B molecules All the unclear situations should be solved by molecular HLA typing techniques