Background:  Monitoring CD4⁺ and CD8⁺ T-cell counts has become essential in research on blood from healthy individuals and immunodeficient patients such as those with HIV/AIDS. Most procedures for CD4⁺ and CD8⁺ T-cell enumeration require the blood to be < 48 hours old for accurate results. This can often be a problem in resource-poor settings where samples need to be transported long distances for analysis due to a lack of instrumentation or operator availability. It has been reported that Transfix can stabilize blood for CD4⁺ T-cell enumeration for up to 14 days. In this study, we demonstrate the compatibility of aged, Transfix-treated blood with the Guava EasyCD4 and EasyCD8 assays, which use small volumes of blood (10 mL/test) and have previously been found to provide accurate and precise results in multi-site studies compared to predicate methods.

Methods:  Transfix was mixed with EDTA-anti-coagulated whole blood (from HIV⁺, n = 10 and HIV^– donors, n = 5) at a 1:10 ratio and incubated at RT (20 to 25ēC). Transfix-treated samples were analyzed using the EasyCD4 and EasyCD8 assays on Days 0, 2, 15, and 30. CD4⁺ and CD8⁺ T-cell counts were also obtained from fresh blood on day 0. 

Results:  CD4⁺ and CD8⁺ T-cell counts of Transfix-treated samples on all days showed good agreement with counts obtained from fresh blood on day 0. Average CD4⁺ T-cell counts of 0-, 2-, and 15-day-old Transfix-treated blood, when compared with the counts obtained from fresh blood on day 0, gave correlation coefficients of 0.97, 0.98, and 0.99. The average percentage differences were –1.3%, –0.69%, and 0.01%, respectively. Similarly, the average percentage differences for CD8⁺ T-cell counts for Transfix-treated blood on days 0, 2, and 15 were 4.6%, 7.4%, and 5.0%. The staining profiles of the Transfix-treated blood were essentially identical to those at day 0 for fresh blood. With Transfix-treated HIV^– donors, the CD4⁺ T-cell counts were accurate even after 30 days. 

Conclusions:  Transfix-treatment and storage for at least 15 days is compatible with the Guava EasyCD4 and EasyCD8 assays to obtain accurate CD4⁺ and CD8⁺ T-cell counts in HIV⁺ and HIV^– specimens. EasyCD4 and EasyCD8 assays along with Transfix allow analysis of aged blood samples in a low volume, low-cost format with minimal reagent consumption and provides an attractive solution for CD4⁺ and CD8⁺ T-cell determinations in resource-limited settings.

Keywords: CD4 count; Transfix blood stabilizer; aged blood