Dry Chemistry Testing: Part 3 of 3
In the developed world, HIV has become more manageable since the early 1980s. It is no longer the automatic death sentence that it once was: for many, it is today a chronic disease managed with anti-retroviral therapy (ART).
Limitations of HIV Detection for the Developing World
Unfortunately, many low-to-middle-income countries (LMICs) miss out on early and effective ART treatments. Sub-Saharan Africa alone accounted for almost 40% of new HIV infections worldwide in 2020[1]. Many people that live with HIV are unaware of their infection until their depleted immune systems can no longer protect them from viral, bacterial, and fungal diseases that those with normal-functioning immune systems deal with easily. To detect and monitor the presence of HIV in humans, the developed world uses two tests.
- Reverse transcriptase polymerase chain reaction (RT-PCR) tests are molecular tests that are used to detect and quantitate the amount of HIV virus in a patient’s blood. However, they cannot tell how much damage has been done to the patient’s immune system. For that assessment, medical professionals need to know how many CD4 T cells are in the patient’s blood.
- Flow cytometry is used to measure how many CD4 T cells are in the patient’s blood. Uninfected patients generally have between 500 and 1,200 CD4 T cells per cubic millimeter of blood in teens and adults. HIV patients have decreased CD4 T-cell counts and various therapies are started or stopped based on this.
Both of these tests are primarily limited to the developed world because they both require expensive equipment, stable electrical power, and trained personnel to operate effectively. Locations in LMICs often lack all three.
An additional problem with absolute CD4 counts is that they vary with age. Young children can have CD4 cell counts as high as 3,000 CD4 cells/mm3. Many cases of HIV in infants and babies are from exposure to the virus from their HIV-infected mothers during pregnancy, at birth, or during nursing. A seemingly normal CD4 count in a young child may actually reflect CD4 depletion due to HIV. One strategy that has been used to normalize CD4 counts is to report them as a ratio of CD4 to total white blood cells. In healthy people of all ages, the percentage of CD4 (CD4%) has a normal range of 25–65%. A CD4% value of 12–15% is roughly equivalent to an absolute CD4 cell count level of 200 CD4/mm3, which is the level at which current guidelines recommend commencement of ART. Delayed or deferred ART is associated with poorer outcomes and premature death.
One of the complications of measuring CD4% is that the CD4 marker protein is also found on white blood cells other than CD4 T cells, albeit in smaller amounts. As CD4 T-cell counts drop in HIV infection, the contribution of these other white blood cells can skew results and make it look like the patient has more CD4 T cells than they actually have. Flow cytometry deals with this problem by measuring other properties of cells (e.g., size and roughness) and ignoring cells that are not T cells.
Simple Dry Chemistry Tests to Detect HIV
PortaScience, Inc. of Monmouth, New Jersey (now a subsidiary of DCN Dx), has developed a dry chemistry card test for the determination of CD4% in finger-stick blood. This test requires only simple equipment and a minimally trained operator. The test is run in two stages.
In the first stage, an unmeasured amount of finger-stick blood is added to a test tube that contains pre-dosed, freeze-dried reagents. One of them is EDTA to keep the blood from clotting. The second is a monoclonal antibody to the CD4 that has been labeled with the enzyme beta-galactosidase (beta-gal). The third reagent is a magnetic particle that has been coated with another monoclonal antibody to a protein called CD15. This CD15 marker is found on monocytes and neutrophils, the white blood cells that also express CD4. After a very brief incubation, the magnetic particles are separated from the supernatant liquid by application of a magnetic field, thus removing the interfering white blood cells. CD4 T cells do not express CD15, so they (and the majority of the remaining white blood cells that are also CD15 negative) remain in the liquid phase.
Next, two small aliquots of the liquid are placed on membranes that have been specially formulated to carry a slight positive charge. White blood cells are positively charged and are retained by the membrane. Red blood cells and unbound anti-CD4 labeled with beta-gal are washed away. One area of the membrane has also been saturated with a substrate that is specific for beta-gal. Another area of the membrane has been saturated with another substrate that specifically reacts with an enzyme called leukocyte esterase that is endogenous to all white blood cells. Color development ensues as the two enzymes react with their substrates. The beta-gal domain produces color in direct proportion to the CD4 T cells present in the depleted liquid, and the leukocyte esterase domain produces color in direct proportion to the white blood cells in the depleted liquid. The signal can be read and quantitated with a digital camera or, ideally, with a cell phone camera.
Cell phone adoption in LMICs has been rapid. It was estimated in 2015 that 94% of adults in those economies have access to cell phones[2]. These devices not only provide a camera to capture the image generated in the test described above—they also provide the connectivity to allow sophisticated image analysis and medical reporting of the results.
Because the readout of this test is a ratio, there is some self-correction that makes the test more robust in the hands of minimally trained operators. Slight variations in the amounts of blood added to the first tube or in the amount of pretreated liquid added to the membranes are cancelled out.
The medical benefit of this information is that ART can be initiated when the CD4% declines to the trigger level of 12–15%. As noted above, ART is key to the survival of HIV patients and is critical in their quality of life. Since many ARTs are self-administered by the patients, compliance with the regimen can also be monitored. CD4% percentages rise with ART. Declining CD4%s do not lie, but patients around the world often do.
This development has focused on a particular type of white blood cell, but in principle this technology could be used to detect and possibly quantitate other cellular targets in a wide variety of biological liquids. PortaScience has also developed tests for white blood cells in human blood and in cow’s milk. Elevated white blood cells in blood and milk are indicative of infection with bacteria and viruses.
Conclusion
If there is a project in your development portfolio that would benefit from a simple dry chemistry card test for a cellular target, consider a confidential conversation with the professionals at PortaScience. The talented scientists and manufacturing experts there can complement your in-house development team. Together, they may be able to arrive at solutions that neither group could develop in insolation. This expanded universe of solutions is why scientists follow the relevant literature and attend scientific conferences. A consultation with PortaScience is a more focused way of gathering information.
Be sure to check out Part 1 and Part 2 of this series on dry chemistry testing.
DCN Dx is a globally recognized contract developer of point-of-care devices based in Carlsbad, California. Since its founding, DCN Dx has committed itself to furthering the rapid diagnostic and point-of-care test market through the continued evolution of technologies and applications. DCN Dx’s cross-functional team of scientists and engineers develop and integrate all aspects of the point-of-care device system, including complex binding reactions, cassettes, sample handling devices, and reader systems. The company assists clients in developing entire rapid diagnostic tests from concept to commercialization.
To learn more about DCN Dx’s services, click here.
[1] https://www.unaids.org/en/resources/fact-sheet
[2] https://www.usglc.org/blog/the-technology-thats-making-a-difference-in-the-developing-world/
