A simple enzyme-based method that uses dried blood spots could significantly reduce the cost of CD4 cell counting for developing countries now implementing antiretroviral treatment programmes, according to findings published today in The Lancet.
CD4 counting makes it possible to detect when a patient is at high risk of AIDS-related illnesses, to detect whether a patient without symptoms is responding to treatment, and to detect whether treatment is failing in a patient without symptoms. CD4 counting is thus a very useful tool in the care of HIV-positive people, but it is unaffordable in most resource-limited settings because the technology needed to carry out the tests requires a large initial expenditure to set up a machine called a flow cytometer, which can distinguish CD4 cells from other white blood cells in a sample of blood.
The study, carried out in Zambia by by Prof. Alimuddin Zumla of the Windeyer Institute at University College, London and colleagues at the University Teaching Hospital in Zambia, used dried blood spots collected on filter paper from 42 HIV-positive Zambinas. The blood was analysed using an ELISA test, and the results were compared with the results of conventional flow cytometry, the gold standard for measuring CD4 cell counts in the developed world. The method is novel and would probably allow large quantities of tests to be done quickly; other low cost CD4 counting systems use methods which are labour intensive
The method proved most reliable for CD4 cell counts over 200 cells/mm3, where the average difference was just 13.6 cells/mm3 (correlation coefficient unstated). As the CD4 count measured by flow cytometry fell below 200 cells/mm3, the agreement between the methods declined substantially (54% agreement between the methods). Twenty six of the samples came from patients with CD4 cell counts above 200 cells/mm3, and the authors say that more research is needed in order to understand why much bigger differences were detected in the samples of patients with lower CD4 counts.
Until this phenomenon is understood, the method is likely to have limited applicability in resource-limited settings, where most people with HIV are not diagnosed until they develop symptoms. This group of people are likely to have CD4 counts below 200 cells/mm3. Current guidelines in the developing world recommend treatment for people with minor symptoms or AIDS-related illness where the CD4 cell count has fallen below 200 cells/mm3.
The authors say that they will need to do more work to find out the best method of blood collection, processing and reconstitution. At present, blood is drawn from a pinprick to the finger, blotted onto filter paper, dried, stored and then sent to the central laboratory for processing. In the laboratory, the samples are treated to release CD4 cells, and a testing kit containing antibodies to the human CD4 protein is used to quantify the amount of CD4 cells in the sample.
Underlying weakness of the ELISA CD4 counting method?
Research conducted during the 1990s suggests that even in the optimum conditions, using whole, fresh blood, the TRAx CD4 ELISA test kit performs poorly in comparison with conventional flow cytometry in many patients.
A previous analysis of this technology using whole blood samples rather than dried blood, carried out in the mid-1990s, found that the ELISA-based test performed less well than two other experimental methods, DynaBeads and FACSCount. Whilst those methods showed a corelation of greater than 90% between their results and those of conventional flow cytometry, the TRAx CD4 assay showed a correlation of approximately 60% between its results and those of flow cytometry, and WHO-sponsored researchers felt unable to recommend the method for use in Africa (Lyamuya). However, a larger study carried out in the United States in the early 1990s found a correlation of 87-95% between the results of the TRAx CD4 assay and flow cytometry using whole blood and an HIV-negative control group. Another Baltimore group also found that correlation tended to be lowest in individuals with CD4 cell counts below 200 cells/mm3 (Saah).
Rigourous analysis in comparison with other new methods, such as panluecogating (already adopted in South Africa) will be needed to ensure that this ELISA-based method is worth the considerable effort needed to scale up its use.
Further information on this website
Monitoring where resources are limited - overview of key issue and research on low cost diagnostics.
Lyamuya EF et al. Evaluation of the FACScount, TRAx CD4 and Dynabeads methods for CD4 lymphocyte determination. J Immunol Methods 195(1-2):103-12, 1996.
Mwaba P et al. Use of whole dried blood spots to measure CD4+ lymphocyte counts in HIV-1 infected patients. The Lancet 362: 1459-60, 2003.
Paxton H et al. Comparison of CD4 cell count by a simple enzyme-linked immunosorbent assay using the TRAx CD4 test kit and by flow cytometry and hematology. Clin Diagn Lab Immunol 2 (1): 104–114, 1995.
Saah AJ et al. Helper T-lymphocyte count. TRAx CD4 test kit versus conventional flow cytometry. Arch Pathol Lab Med 121(9):960-2, 1997.