Related Experiment Video
Updated: Jul 3, 2026
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
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.
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.
