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

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

3. 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 !!!

4. 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 March 2014

5. 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

6. 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

7. 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

8. Flow citometry interpretation
Granulocytes
Monocytes
Lymphocytes
CD 3+ Ly

9. Results 1:

10. Results 2:

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

12. 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

13. 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