Ann Lab Med.  2013 May;33(3):174-183. 10.3343/alm.2013.33.3.174.

Flow Cytometric Human Leukocyte Antigen-B27 Typing with Stored Samples for Batch Testing

• Seo BY
• Won DI

• KMID: 1781322
• DOI: http://doi.org/10.3343/alm.2013.33.3.174

Abstract

BACKGROUND
Flow cytometry (FC) HLA-B27 typing is still used extensively for the diagnosis of spondyloarthropathies. If patient blood samples are stored for a prolonged duration, this testing can be performed in a batch manner, and in-house cellular controls could easily be procured. In this study, we investigated various methods of storing patient blood samples.
METHODS
We compared four storage methods: three methods of analyzing lymphocytes (whole blood stored at room temperature, frozen mononuclear cells, and frozen white blood cells [WBCs] after lysing red blood cells [RBCs]), and one method using frozen platelets (FPLT). We used three ratios associated with mean fluorescence intensities (MFI) for HLAB27 assignment: the B27 MFI ratio (sample/control) for HLA-B27 fluorescein-5-isothiocyanate (FITC); the B7 MFI ratio for HLA-B7 phycoerythrin (PE); and the ratio of these two ratios, B7/B27 ratio.
RESULTS
Comparing the B27 MFI ratios of each storage method for the HLA-B27+ samples and the B7/B27 ratios for the HLA-B7+ samples revealed that FPLT was the best of the four methods. FPLT had a sensitivity of 100% and a specificity of 99.3% for HLA-B27 assignment in DNA-typed samples (N=164) when the two criteria, namely, B27 MFI ratio >4.0 and B7/B27 ratio <1.5, were used.
CONCLUSIONS
The FPLT method was found to offer a simple, economical, and accurate method of FC HLA-B27 typing by using stored patient samples. If stored samples are used, this method has the potential to replace the standard FC typing method when used in combination with a complementary DNA-based method.

Keyword

HLA-B27 typing; Flow cytometry; Sample storage; Frozen platelets

MeSH Terms

Blood Platelets/metabolism
Erythrocytes/metabolism
*Flow Cytometry
Freezing
HLA-B27 Antigen/*blood
HLA-B7 Antigen/blood
Histocompatibility Testing
Humans
Leukocytes, Mononuclear/metabolism
Real-Time Polymerase Chain Reaction
Spondylarthropathies/diagnosis
Temperature
HLA-B27 Antigen
HLA-B7 Antigen