Specifications of qPCR based epigenetic immune cell quantification.

OBJECTIVES: Immune monitoring is an important aspect in diagnostics and clinical trials for patients with compromised immune systems. Flow cytometry is the standard method for immune cell counting but faces limitations. Best practice guidelines are available, but lack of standardization complicates compliance with e.g., in vitro diagnostic regulations. Limited sample availability forces immune monitoring to predominantly use population-based reference intervals. Epigenetic qPCR has evolved as alternative with broad applicability and low logistical demands. Analytical performance specifications (APS) have been defined for qPCR in several regulated fields including testing of genetically modified organisms or vector-shedding. METHODS: APS were characterized using five epigenetic qPCR-based assays quantifying CD3+, CD4+, CD8+ T, B and NK cells in light of regulatory requirements. RESULTS: Epigenetic qPCR meets all specifications including bias, variability, linearity, ruggedness and sample stability as suggested by pertinent guidelines and regulations. The assays were subsequently applied to capillary blood from 25 normal donors over a 28-day period. Index of individuality (IoI) and reference change values were determined to evaluate potential diagnostic gains of individual reference intervals. Analysis of the IoI suggests benefits for individual over population-based references. Reference change values (RCVs) show that changes of approx. Fifty percent from prior measurement are suggestive for clinically relevant changes in any of the 5 cell types. CONCLUSIONS: The demonstrated precision, long-term stability and obtained RCVs render epigenetic cell counting a promising tool for immune monitoring in clinical trials and diagnosis.