A cheap, easily-performed method for determining CD4 cell counts is as accurate as standard flow cytometry, according to a report in the February 1st edition of the Journal of Acquired Immune Deficiency Syndromes. The CD4 Select method yields nearly identical results to standard flow cytometry, requires only the equipment typically used for complete blood count (CBC) analysis, and is much less labour-intensive than other non flow-cytometric methods.
A practical method for regularly monitoring CD4 cell counts has been one of the obstacles to providing better HIV patient care in developing countries. Flow cytometry, the standard method used in developed countries, is too complex and expensive to be practical in resource-limited settings. Various alternative methods have been proposed – see, for example, the aidsmap reports here and here. However, there is no consensus on which method to use, and none have yet been widely adopted. Also, many of these methods are inexpensive but labour-intensive and require specialised training.
Identification of CD4+ lymphocytes (CD4+ T cells) cannot rely solely on identifying the CD4 protein on the cell surface, since monocytes also express CD4 protein and would yield inaccurate results if not eliminated. A team at Chiang Mai University, Thailand has previously reported on their development of a monoclonal antibody (mAb) to the CD4 protein, called MT4, which binds strongly to the CD4 molecule on CD4 lymphocytes (CD4+ T cells) but weakly or not at all to the CD4 on monocytes. Hence, MT4 can be used to tag CD4+ T cells in blood samples with a high degree of specificity.
The Chiang Mai team has now devised a non-flow cytometric laboratory method for accurately counting CD4+ T cells, based on the MT4 mAb. The method takes advantage of the fact that, while flow cytometry is normally used to distinguish different types of lymphocytes (such as CD4 cells), the overall number of lymphocytes can be determined much more simply by an automatic haematoanalyser – the equipment used to assess complete blood counts (CBCs).
The test reagent, CD4 Select, is generated by binding the MT4 mAb to ferrous beads. When the CD4 Select reagent is combined with the blood sample, CD4 cells become bound to the beads and are then magnetically removed from the sample. The percentage of CD4 lymphocytes can then be calculated by subtracting the lymphocyte count in this CD4-depleted sample from the total lymphocyte count in a second, unaltered sample from the same patient; the absolute CD4 lymphocyte count is then derived from the percentage.
This method was compared to standard flow cytometry in blood samples from 100 randomly selected HIV-positive individuals at Maharaj Nakorn Chiang Mai Hospital. Results correlated very closely, to an R-value of 0.932 for percentage, and 0.922 for absolute CD4 cell counts. The CD4 Select method yielded percentages higher than flow cytometry by a mean of 0.06% (95% confidence interval, 9.46% to -9.56%). The researchers note that flow cytometry itself yields slightly variable results, and that the small discrepancies noted "do not necessarily indicate problems with [their] method."
The laboratory research team states that this novel process is "cheap, valid, and simple enough to be conducted locally at small facilities." It is also less time-consuming and labour-intensive than many other alternatives, with a turnaround time of less than one hour, and may represent a viable alternative for assessing CD4 cell counts in resource-limited settings.
Reference:
Srithanaviboonchai K et al. Novel low-cost assay for the monitoring of CD4 counts in HIV-infected individuals. J Acquir Immune Def Syndr 2008;47(2):135-139.