Discussion
DuraClone B27, a unitized, room temperature reagent for HLA B27 screening in whole blood on Flow cytometers.
Neema Jayadas1, Dharna Kachroo1, Sridhar Ramanathan2
Beckman Coulter India Pvt. Ltd, Bangalore Development Centre, Bangalore, India
Background:
Flow cytometric HLA B27 screening to test patients for auto immune diseases have gained precedence over classical methods like lymphotoxicity tests in general diagnostic laboratories, as it is rapid, and cost effective. HLA B27 testing is often prescribed because of its association with Ankylosing Spondylitis and other inflammatory diseases. . Current HLA-B27 flow cytometric tests have complex workflows, involving manual pipetting steps and running of positive controls to determine HLA B27 expression. Along with this, is the hassle of cross reactivity with the B7 antigen and the interpretation of the data output by experienced technicians and/or doctors.
Methods:
Beckman Coulter’s DuraClone B27 is a room temperature stable unitized dried down reagent that is compatible with multiple Beckman Coulter and Beckton Dickenson flow cytometers. A simple cut off Intensity value called the ‘Determinant’ helps type a sample as positive or not positive for HLA B27 expression. Multi channel fluorescent beads present in the unitized reagent tube are standardize the PMT voltages across platforms and thus aids in a robust read out of the result. A 1000 sample data set validates the determinant value. The reagent comprises two clones of the anti HLA B27 antibody tagged to a fluorescent dye along with labeled anti CD3 antibody for specific selection of HLA B27 expression on T-Lymphocytes. The combined use of two clones of HLA B27 antibody (ABCm3 and FD705) in the reagent greatly increases the specificity of the reagent while the unlabeled anti B7 antibody in the reagent minimizes cross reaction between the HLA B27 antibody and B7 antigens. A simple stain-lyse-no wash protocol enables an easy and rapid workflow.
Results:
Reagent compatibility across multiple platforms has been demonstrated across 90 blinded clinical whole blood samples with a correlation of greater than 97% on the median intensities of the HLA-B27 expression levels. Furthermore, HLA B27 expression in clinical samples from using DuraClone B27 on a flow cytometer showed to have more than 95% specificity and sensitivity with respect to PCR across more than 190 samples.
Conclusion:
DuraClone B27 is a robust, convenient and cost effective solution for HLA B27 typing in whole blood samples across multiple flow cytometric platforms, showing high sensitivity and specificity.
