Flow cytometry offers hope for better treatment

Tests provide precise measure of T cells, viral load

A new development in diagnostic technology using flow cytometry to count the amount of virus produced in cells could help clinicians make treatment decisions for HIV-positive patients. Although the approach is available only in research settings, it could eventually augment or supplant plasma viral-load testing, its inventor tells AIDS Alert.

Bruce Patterson, MD, a molecular virologist and assistant professor of medicine at Northeastern University in Boston, has spent the past seven years developing and refining a technique to measure viral particles within a cell while leaving the cell intact — a process known as in-cell viral load. The test, technically called ultrasensitive in situ hybridization, uses flow cytometry to provide an exact measure of how many viral particles a cell is producing. Plasma viral load, the traditional viral-load test now used hand in hand with CD4 count testing, measures only the amount of free virus in the blood.

"This is extremely important because what free viral load doesn’t tell you is which cells are producing the virus, and we know there are many different cells that will produce virus," Patterson says. "It is crucial now to understand where the virus is coming from because of the new research in co-receptors and what they call tropism, which studies what virus bonds to what cells."

As an analogy illustrating the difference between the two measures, Patterson says a cell making virus is like a car factory making cars. If you want to know how many cars the factory is producing, you go to the factory, not the distributor’s lots.

"The best way to look at how the body is producing virus or how much virus the body is producing is to look at the cells that are actually producing the virus, and that is exactly what we can do," he says.

Patterson’s first breakthrough in the area of in-cell viral load was published in 1993 in Science in which he reported how he was able to detect a single copy of HIV within a cell. The technique was difficult, however, and in the intervening years the process has been simplified and shortened. The new process can detect as few as 5 to 10 copes of virus per cell.

The technique uses antibodies and different-colored fluorescent dyes to identify or tag three types of cells with one test. The test, which takes only several hours, can provide a count of one’s CD4 cells as well, he notes.

The test is likely to help explain the discrepancies that can be found between CD4 count alone and a patient’s progression to disease. If you take two patients with identical CD4 counts, there is a good chance that clinical progression will differ between the two. The reason, Patterson says, is that one patient may have 10% of his T cells infected with HIV, while the other may have only 1%. Even when a plasma viral-load test is added, there is critical information lacking, he notes.

"We now use those two markers to determine the status of disease, but the two have nothing to do with one another," he explains. "One is telling you how many T cells there are. The other is telling you how much virus is there [in plasma]. What you don’t know is how many of those T cells are producing virus."

Predicting viral load changes

Based on knowing what type of cells are producing virus and how much virus they are producing, a clinician may change a patient’s treatment because "we know kinetics of the virus are different in different cell types," Patterson says. Moreover, as explained in an upcoming issue of the journal Cytometry, there are now data showing that the technique can predict changes in viral load before they show up in viral-load testing.

The reason, Patterson explains, is that viral-load tests measure virus circulating in the blood, which is a large volume of liquid.

"You can imagine that if there was drug resistance or a person was taken off drug because of intolerance, it would be the cells that would start producing virus prior to enough virus being distributed in the blood so you could detect it," Patterson says. this test can show an increase in viral production prior to an increase in viral load."

Another possible application of the test is decreasing the window period in which infection cannot be detected. This period of up to a month or so exists because it can take that long for antigens to respond and for the virus to distribute from the lymph nodes and into the blood system before it can be detected. Because the test is not restricted to measuring blood, it could possibly detect virus earlier, he adds.

So far, the test has been used for research purposes. However, mass production of test kits for laboratories will begin soon in the United States, Patterson says. As a monitoring test, the technique doesn’t need Food and Drug and Drug Administration approval. Eventually, FDA approval would be sought for insurance reimbursement purposes, he says, adding that the cost of the test would be similar to a viral-load test.

If further studies show that in cell viral-load testing correlates well with plasma viral-load testing, it could possibly supplant the plasma test because the technique also gives a CD4 count reading at the same time. "We could have all the information in one test," he says. "What could be more elegant than that?"