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Related Experiment Video

Updated: Jul 3, 2026

Determination of Immune Cell Identity and Purity Using Epigenetic-Based Quantitative PCR
08:02

Determination of Immune Cell Identity and Purity Using Epigenetic-Based Quantitative PCR

Published on: February 19, 2020

Epigenetic CD4+ T-Cell Quantification from Dried Blood Spots Using a Real-Time Quantitative PCR-Based Assay.

Riffat Munir1, Tracy Sungu1, Denise Lawrie2

  • 1Wits Diagnostic innovation Hub and Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa.

The Journal of Molecular Diagnostics : JMD
|July 1, 2026
PubMed
Summary

An epigenetic quantitative PCR (qPCR) assay using dried blood spots (DBS) shows promise for CD4+ T-cell counting in HIV patients. This molecular method offers a viable alternative to flow cytometry where access is limited.

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Area of Science:

  • Molecular diagnostics
  • Immunology
  • Virology

Background:

  • Conventional flow cytometry for CD4 testing is crucial for advanced HIV disease but faces accessibility challenges.
  • Epigenetic quantitative PCR (qPCR) offers a molecular alternative, potentially adaptable to simplified sample types like dried blood spots (DBS).

Purpose of the Study:

  • To evaluate the analytical performance of an epigenetic CD4+ T-cell qPCR assay using DBS samples.
  • To compare DBS-based epigenetic CD4 counts with reference flow cytometry methods.

Main Methods:

  • An epigenetic CD4+ T-cell qPCR assay (iMune CD4) was used with DNA extracted from DBS samples derived from 150 HIV patients.
  • CD4 counts were compared against flow cytometry (AQUIOS PanLeucogating) using concordance correlation, Bland-Altman analysis, and percentage similarity.
  • Assay repeatability, batch variability, and classification performance at CD4 thresholds were assessed.

Main Results:

  • The DBS-based epigenetic CD4 assay showed good agreement with flow cytometry (concordance correlation coefficient 0.91).
  • Mean bias was -28 cells/μL (-6.9%), with acceptable repeatability (CV 4.0%-11.2%).
  • Sensitivity and specificity at 200 cells/μL were 93.8% and 88.9%, respectively; increased variability noted in larger manual extraction batches.

Conclusions:

  • Epigenetic qPCR-based CD4 quantification from DBS is technically feasible.
  • This method presents a viable alternative to flow cytometry for CD4 testing in resource-limited settings.
  • Further optimization and validation are recommended.