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Simplified Dynabeads Method Using Light Microscopy for Enumerating TCD4+ Lymphocytes in Resource-Limited Settings

Background: We demonstrated the feasibility of implanting the Dynabeads method for CD4+ T lymphocyte enumeration in resource-poor settings (ANRS 1226 study). However, as this technique requires a fluorescence microscope which is not usually available in these settings, WHO has encouraged to simplify the method allowing TCD4+ lymphocyte counting under a light microscope.

Methods: TCD4+ lymphocytes enumeration was assessed using Dynabeads after staining cells nuclei with nonfluorescent dyes and readings under light microscope (DLM). A total of 305 triple of values of CD4 cells counts were generated by both Dynabeads method using a light microscopy (DLM), Dynabeads method using a fluorescent microscope (DFM) and the single-platform flow cytometry technique (FCM). The accuracy of DLM was analyzed using 4 fresh blood samples showing 200, 400, 500 and 1000 cells/μl in FCM respectively. Correlations have been studied between the 3 methods. The DLM was then evaluated for its ability to correctly segregate absolute TCD4+ lymphocyte values at the thresholds of 200 cells/μl and 350 cells/μl.

Findings: Cells nuclei staining with Sternheimer-Malbin, Turck1, and Giemsa allows TCD4+ lymphocytes enumeration using DLM. FCM has shown the greatest standard deviations and amplitudes. The reproducibility of DLM was better than FCM. The correlation coefficient between FCM and DFM was 0.975 and it was 0.973, 0.972 and 0.969 with DLM using Sternheimer-Malbin, Turck1 and Giemsa, respectively. The ability of DLM to correctly segregate TCD4+ lymphocyte values at the threshold of 200 cells/μl and 350 cells/μl was good.

Conclusion: Reliable TCD4+ enumeration can be obtained with DLM. These results will contribute in resourcelimited- settings to further reduce the cost of TCD4+ lymphocytes counting and make it more widely available in peripheral laboratories and even in central laboratories that face problems with maintenance and stock-out of reagents for flow cytometers.


Serge Diagbouga, Dézemon Zingué, Eloi Bahembera, Antoinette Kaboré, Hervé Hien, Adama Ouiminga and Laurent Caignault

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