Flow citometry or molecular biology for HLA-B27 detection in
seronegative spondylarthropaties ?
Flow cytometry or molecular biology for HLA-B27 detection in
seronegative spondylarthropaties ?
Author: Student Armean Iulia, 4th grade, GMCoordinator: Prof.
Dr. Minodora DobreanuBackround:Simptomps : lumbosacral pain >3
months
pain and stiffness of the thoracic regiondecreased thoracic
expansiondecreased lumbar mobility
Backround:Ankylosing spondilytisChronic inflammatory
diseaseAxial skeletonHLA B27Flow 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 2014Material s and methods 1:Type of study :
retrospectiveSample: 98 persons with ages between 14 and 80
yearsFlow 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
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
Flow citometry technique
6Anti CD3(phycoerythrin)Anti HLA-B27(fluorescein)Material s and
methods 2:Direct marking (the fluorochrome is attached to the
monoclonal antibody
T Lymphocyte CD3HLA-B27
LymphocytesMonocytesGranulocytesCD 3+ LyFlow citometry
interpretationResults 1:Results 2:
LymphocytesMonocytesGranulocytesCD 3+ Ly
Patient2 (HLA-B27+)Patient1 (HLA-B27-)Patient 3 (HLA-B27
!!)Results 3:
Results 4: HLA B39 is one molecule which can shade HLA B27
resulting false negative reactionsAlso it can create a picture of
false expression of HLA B07 resulting a false HLA B27 positive
reaction HLA B13 phenotype shows no interference
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
conclusions:
