Cytometry for life

Cytometry for Life (C4L) is a program aimed at lessening the burden of the HIV/AIDS epidemic. The C4L program provides portable battery-powered laboratories that can identify the level of depression of the immune system of an HIV-positive individual to regions of the world lacking this ability. C4L’s portable laboratory is a CD4 T-Lymphocyte counter and is designed to be simple to operate, portable, reliable and function on battery power. C4L was created by J. Paul Robinson and Gary Durack.

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Background

CD4 testing using flow cytometry techniques is a critical component of the AIDS treatment process in terms of identifying candidates to receive life-saving therapy, as well as on-going monitoring of disease progression. Unfortunately, in areas most impacted by HIV/AIDS, such as the African continent, CD4 testing procedures are often plagued by inconvenient time-lags, unavailability of instruments and high-costs. Former UN special envoy for HIV/AIDS in Africa, Stephen Lewis, has been a major proponent of the movement to make CD4 testing “portable” and “easy to use” so as to mitigate the AIDS epidemic in Africa. In May 2006, Lewis issued a challenge to experts in the field of cell analysis at the 2006 International Society for Analytical Cytology (ISAC) Congress, held in Québec, to create a solution to flow cytometers that are “bulky, difficult to use, at about $50,000 each, vastly out of reach of most of the continent”.Medicine October, 2006)

The Science of CD4 testing

CD4 testing is a normal practice in the clinical laboratory, and is accomplished through the use of flow cytometry. Flow cytometry is a standard technique used in virtually every hospital and research institution around the world. While flow cytometry techniques are straightforward, they are not necessarily simple, particularly when applied to clinical practice. A great deal of automated technology is employed to gather measurements, analyze data, and produce reports. Consequently, the cost of performing these tests is relatively high. CD4 testing is most of the time performed by a flow cytometer. In this process, a small tube of blood cells is treated with a reagent which is specific to the CD4 lymphocyte (a type of white blood cell). The reagents used for identification are called monoclonal antibodies (MABs). These antibodies can specifically attach to a certain type of cell. In this case, the antibody will attach to a CD4 T cell. Certain molecules (called fluorochromes) are attached to the antibody to aid in the identification process. When a laser beam is fired at the cells, those cells with the attached molecules emit a signal identifying themselves. This signal is called fluorescence and can easily be measured with sensitive detectors. If hundreds or thousands of cells are forced past the laser very quickly, an accurate count can be obtained of all cells that are present. CD4 cells are measured in this manner. CD is an acronym for “cluster of differentiation,” and that simply means that this molecule can be classified into a well defined group of cells. In this case, it is cluster of differentiation 4, or a CD4 helper T-lymphocyte cell.

Data interpretation

First, the data are collected in the flow cytometer. Each cell is plotted in a histogram display, which is a frequency distribution. In a typical histogram, two peaks appear.The first peak represents cells that are negative, and thus have no specific binding to the probe. The second peak represents cells that are “positive” for that particular marker, or monoclonal antibody (MAB). From histograms, we can determine the percentage of cells positive for this MAB. Using additional monoclonal antibodies it is possible to classify each type of cell (or subset of cells) present in the blood. In the field of cytometry, this is called multiple labeling or multi-color fluorescence.

References

Cytometry for Life official website