Though the debate over treatment access at the First South African Conference on AIDS dominated the news headlines, the conference also yielded some important clinical information. Advances in diagnostics were presented during an oral session on Wednesday morning. There were two reports on new lab tests that may dramatically improve and speed the diagnosis and appropriate response to active TB (click here to read the article). Two other presentations were more concerned about cutting costs, one by offering a potential lower cost alternative to viral load testing (click here to read the article). The other report was on the PanLeucogating CD4 test, which has made CD4 cell testing much more widely available in South Africa because it is two-thirds less expensive than the standard CD4 test (which can cost anywhere from $20-40).
The standard CD4 cell test is pricey because it is an overly complicated procedure performed on a rather expensive piece of equipment called a flow cytometer (which essentially means cell counter). The flow cytometer can evaluate various characteristics of cells at once, including their size and the distinguishing proteins on their surface.
In order for the machine to “see” a cell, a blood sample is injected into a fluid suspension that streams the cells, single file, past a laser (or lasers) and then light detectors record data about the cells. The distinguishing proteins on the surface of the cells, such as a CD4 cell receptor, can be detected if special chemicals, ‘tagged’ with fluorescent dyes, have been added to the sample. The dyed chemicals are monoclonal antibodies that bind to specific receptors. These monoclonal antibodies aren’t free and thus add significantly to the cost of the procedure. Furthermore, they are overused in the standard CD4 test to detect features, such as size, which the machine can already easily ‘see.’
PanLeucogating is cheaper because it leaves out the redundant use of monoclonal antibodies. It uses only two, one for the CD45 receptor, to isolate leucocytes (white blood cells), and one for the CD4 receptor, which isolates the CD4 cells. This gives the ratio of CD4 cells to leucocytes in the sample. This ratio must be referenced to the absolute white cell count to calculate the absolute CD4 cell count. The absolute white blood count can be measured, simply and cheaply (for less than a $1) by using a haematology analyser. This approach is not only less expensive, it is usually more accurate than the standard CD4 cell test.
During the diagnostics session, Dr. Leslie Scott of South Africa’s National Health Laboratory Services and University of the Witwatersrand described an attempt to even further reduce the cost of PanLeucogating by substituting a dye, thiazole orange, for the CD45 antibody. Thiazole orange can distinguish between white cells from red cells by their nucleic acid content. Furthermore, thiazole orange is readily available, cheap (cents per 100 tests compared to Rands for each test), and stable — it can be stored on the shelf at room temperature.
Her team compared the similarity of results on 94 clinical specimens tested by using both CD45/CD4 and thiazole orange/CD4. Latex beads were added to the samples in order to test how comparable white blood cell measurements taken by the flow cytometre, using either thiazole orange or CD45, were to those measured by the haematology analyser. (The addition of latex beads to a blood sample lets a flow cytometre calculate absolute cell counts but the beads are too expensive to be used routinely for this purpose in the developing world.)
According to a percentage similarity statistical model, thiazole orange was effective in isolating white blood cells (CV=6.3%) in the samples. However, it was potentially less reliable than CD45 (CV=4.7%) when it was compared to the white cell count from a haematology analyser. Although, there was overall agreement in CD4 cell counts achieved by thiazole orange and CD45 (CV=8.8), in some samples, counts were found to be significantly less reliable.
To find out why, Scott’s team performed an additional analysis looking at the samples individually. They found that the thiazole orange tests were very reliable in 26 samples that were less than 24 hours old but not for the samples in the study that were several days old, (and some were over eight days old).
“One of the advantages of PanLeucogating [with CD45/CD4 antibodies]”, said Dr. Scott, “is that it makes delayed sample testing possible—up to six days old—because the lineage specific marks are robust.” But even though thiazole orange “can reduce test costs to less than US $2-3 per test, it cannot be used for delayed testing.” Dr. Scott concluded, “Thus, it is not a solution for the developing world.” She noted that, ironically, thiazole orange could be used to reduce the cost of CD4 tests in the developed world.
Delayed sample testing is common in the developing world. Even in the most developed parts of South Africa, which has a better courier and transportation infrastructure than much of the rest of Africa, diagnostic facilities frequently receive aged samples. According to a poster presentation also at the conference, by Denise Lawrie of South Africa’s National Health Laboratory Service, the average delay from collection to time of CD4 cell testing is approximately 24 hours at Johannesburg Hospital, which serves as the reference laboratory for state hospitals and clinics in Gauteng Province.
Nevertheless, even though many believe PanLeucogating is still too expensive, it has improved access to CD4 tests dramatically in South Africa. Lawrie reported that since the introduction of CD45/CD4 PanLeucogating in May of 2002, there has been a greater than 100% increase in requests for CD4 cell tests at the hospital, and requests from some facilities have increased by more than 300%. Furthermore, the number of samples rejected due to age-related stability problems has also decreased dramatically, to less than 1% of the total samples tested.
Further information on this website
Monitoring where resources are limited - viral load and CD4 counting
Scott L et al. Cost effective CD4 monitoring by nucleic acid assisted
PanLeucogating. First South African AIDS Conference, Durban, abstract T1-S5-A26, 2003.
Lawrie D et al. The effect of the introduction of CD45-assisted PanLeucogating technique on the volume of samples tested for CD4 counts over a 1 year period in the National Health Laboratory Service. First South African AIDS Conference, Durban, abstract, T1-P30, 2003.