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Related Concept Videos

Real Time RT-PCR02:57

Real Time RT-PCR

Real-time reverse transcription-polymerase chain reaction, or Real-time RT-PCR, is an analytical tool used to determine the expression level of target genes. The method involves converting mRNA to complementary DNA with the help of an enzyme known as reverse transcriptase, followed by the PCR amplification of the cDNA. These two processes can be performed simultaneously in a single tube or separately as a two-step reaction.
The real-time quantification of the number of amplified products is...

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Epigenetic Regulation of Cardiac Differentiation of Embryonic Stem Cells and Tissues
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Specifications of qPCR based epigenetic immune cell quantification.

Konstantin Schildknecht1, Björn Samans1, Jasmin Gussmann1

  • 1Ivana Türbachova Laboratory for Epigenetics, Epiontis, Precision for Medicine GmbH, Berlin, Germany.

Clinical Chemistry and Laboratory Medicine
|November 20, 2023
PubMed
Summary

Epigenetic quantitative PCR (qPCR) offers a standardized, stable, and precise method for immune cell counting, outperforming traditional flow cytometry in immune monitoring for diagnostics and clinical trials. This technique enables reliable individual reference intervals, detecting significant changes with approximately 50% variation.

Keywords:
epigeneticsimmune monitoringqPCR validation

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Area of Science:

  • Immunology
  • Molecular Biology
  • Clinical Diagnostics

Background:

  • Immune monitoring is crucial for immunocompromised patients, but current methods like flow cytometry face standardization challenges and logistical demands.
  • Limited sample availability necessitates population-based reference intervals, hindering personalized diagnostics.
  • Epigenetic quantitative PCR (qPCR) emerges as a viable alternative with broad applicability and minimal logistical requirements.

Purpose of the Study:

  • To characterize the analytical performance specifications (APS) of epigenetic qPCR assays for quantifying immune cell populations (CD3+, CD4+, CD8+ T cells, B cells, NK cells).
  • To evaluate the suitability of epigenetic qPCR for immune monitoring in clinical trials and diagnostics, considering regulatory requirements.
  • To assess the diagnostic utility of individual reference intervals derived from epigenetic qPCR data.

Main Methods:

  • Five epigenetic qPCR-based assays were developed and validated against regulatory guidelines for analytical performance, including bias, variability, linearity, ruggedness, and sample stability.
  • The validated assays were applied to capillary blood samples from 25 healthy donors over 28 days.
  • Index of Individuality (IoI) and Reference Change Values (RCVs) were calculated to assess the diagnostic potential of individual reference intervals.

Main Results:

  • Epigenetic qPCR assays met all defined analytical performance specifications, demonstrating high precision, stability, and reliability.
  • The assays successfully quantified CD3+, CD4+, CD8+ T cells, B cells, and NK cells.
  • Analysis of IoI indicated advantages of individual reference intervals over population-based ones. RCVs suggested that approximately 50% changes in cell counts signify clinically relevant alterations.

Conclusions:

  • Epigenetic cell counting using qPCR provides a precise and stable method for immune monitoring.
  • The technique's performance and the derived RCVs support its use in clinical trials and diagnostic applications.
  • Epigenetic qPCR offers a promising tool for advancing personalized immune monitoring and diagnostics.