ACS2016 Regulatory T Cell Assay for Immune Thrombocytopenia (24081)
Regulatory T cells (T-Regs) assist with immune homeostasis and self-tolerance, suppressing the immune responses in allergies, diabetes, SLE, ITP and allograft rejection. Their absence can lead to autoimmune disease and thrombocytopenia. A T-Reg assay was established in a clinical laboratory to study Immune Thrombocytopenia (ITP).
EDTA blood samples were separated using Ficoll Paque and cryopreserved for batch testing. The antibody panel included BD Biosciences surface markers CD4-V500, CD25-PECy, CD127-BV421, CD45RA-APC and eBiosciences intra cytoplasmic FoxP3-PE using the Foxp3 / Transcription Factor Staining Buffer Set Kit (eBiosciences). Samples were analysed on a FACSCanto II flow cytometer using FACS-Diva and Flowjo 10.1 software.
A reference range from 30 normal adults (7.7 ± 2.2%) overlapped with the study group of 11 ITP patients (6.6 ± 3.1%). Reduced T-Regs were not seen as a cause of ITP. Serial samples tested up to 28 days post treatment did not show any increase in T-Regs. Naïve CD45RA+ T-Regs however were significantly reduced in pre-treatment ITP (2.25 ± 1.0%) compared to healthy controls (3.54 ± 2.0%; p = 0.012) requiring further study.
Including FoxP3 as a measure of T-Reg function added many hours and complexity to the assay. Delay in sample cryopreservation led to diminished FoxP3 fluorescence and recovery. CD3 FITC was excluded due to interference from the high PMT voltage needed for relatively dim FoxP3 PE. The T-Reg assay is otherwise relatively easy to establish and valuable in a clinical laboratory setting.
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