Specifications of qPCR based epigenetic immune cell quantification
Author:
Schildknecht Konstantin1, Samans Björn1, Gussmann Jasmin1, Baron Udo1, Raschke Eva1, Babel Nina2, Oppatt Julia1, Gellhaus Katharina3, Rossello Araceli1, Janack Isabell1, Olek Sven1ORCID
Affiliation:
1. Ivana Türbachova Laboratory for Epigenetics, Epiontis, Precision for Medicine GmbH , Berlin , Germany 2. Marienhospital Herne, Klinik I für Innere Medizin, Centrum für Translationale Medizin , Herne , Germany 3. Bundesinstitut für Risikobewertung , Berlin , Germany
Abstract
Abstract
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.
Publisher
Walter de Gruyter GmbH
Subject
Biochemistry (medical),Clinical Biochemistry,General Medicine
Reference35 articles.
1. Apoil, PA, Puissant-Lubrano, B, Congy-Jolivet, N, Peres, M, Tkaczuk, J, Roubinet, F, et al.. Reference values for T, B and NK human lymphocyte subpopulations in adults. Data Brief 2017;12:400–4. https://doi.org/10.1016/j.dib.2017.04.019. 2. Thompson, ME, Stephen, IR, Wood, R. Harmonized guidelines for single-laboratory validation of methods of analysis. Pure Appl Chem. Chimie pure et appliquée 2002;74:835–55. https://doi.org/10.1351/pac200274050835. 3. Ma, H, Bell, KN, Loker, RN. qPCR and qRT-PCR analysis: regulatory points to consider when conducting biodistribution and vector shedding studies. Mol Ther Methods Clin Dev 2021;20:152–68. https://doi.org/10.1016/j.omtm.2020.11.007. 4. Mazzara, MS, Christian, Delobel, P, Broll, H, Damant, A, Paoletti, C, et al.. Definition of minimal performance requirements for analytical methods for GMO testing. In: JRC Sci Techn Rep. Luxembourg: European Network of GMO Laboratories (ENGL); 2008. 5. Yi, JS, Rosa-Bray, M, Staats, J, Zakroysky, P, Chan, C, Russo, MA, et al.. Establishment of normative ranges of the healthy human immune system with comprehensive polychromatic flow cytometry profiling. PLoS One 2019;14:e0225512. https://doi.org/10.1371/journal.pone.0225512.
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