More than 1,000 children worldwide become infected with HIV each day, most as a result of mother-to-child transmission of the virus. Without timely evaluation by medical professionals and access to antiretroviral treatment, half of them will die before their second birthday.1
Resource-limited regions of the world such as sub-Saharan Africa, Eastern Europe, and Central Asia have been particularly hard hit. In 2009, approximately 370,000 children in sub-Saharan Africa were newly infected with HIV. This represents more than 90% of all new HIV infections in children worldwide.2From 2001 to 2008, HIV infection rates in infants and children in Eastern Europe and Central Asia increased by 23%.2Contrast these statistics with those in the United States, where fewer than 200 children a year are infected with HIV.2
Florence, an HIV-positive woman, holds her new baby just four hours after delivery in sub-Saharan Africa.
Photo courtesy of Elizabeth Glaser Pediatric AIDS Foundation/James Pursey.
Despite very challenging conditions presented by these regions, aggressive education, access to antiretroviral drugs, and the availability of new portable technology are combining to make a significant difference for the youngest victims of this disease.
Monitoring infected patients
Although antiretroviral treatments have become more accessible to patients in developing countries, the ability to effectively assess patients, determine when treatment should be started, and monitor the immune system once treatment is initiated are also critical to success. PCR-based viral load tests, which provide a quantitative measure of HIV nucleic acid in a patient’s blood, are commonly used to assess and monitor patients in developed countries.
“While it’s certainly best to monitor patients with viral load tests, these tests are very expensive, and the equipment is often not available in resource-limited regions,” notes Suzanne Willard, PhD, an HIV nurse-clinician who worked at the Elizabeth Glaser Pediatric AIDS Foundation, an organization active in regions of the world that have been deeply impacted by HIV and AIDS. The Foundation seeks to prevent pediatric HIV infection and to eradicate pediatric AIDS through research, advocacy, and prevention and treatment programs.
Where viral load testing is impractical, another method for tracking the progression of the disease is to count CD4 T-cells. The number and percentage of those T cells in human blood will decrease with the progression of the immune deficiency virus.
Flow cytometry
Flow cytometry is typically used to measure CD4 cell levels in blood samples. Flow cytometry measures and analyzes multiple physical and chemical characteristics of cells as they flow in a fluid stream, single file, through a laser beam. The technology can be used for counting and sorting cells based on their individual characteristics and for assessing cell viability. Flow cytometry can also be used for complex studies of immune function, apoptosis (programmed cell death), cancer, stem cells, and to support drug discovery.
Unfortunately, traditional flow cytometry systems used to measure CD4 levels are also expensive, complex to operate, and typically located in the core labs of larger hospitals—often far from patients living in rural communities in resource-poor countries. These instruments require highly-trained personnel to run tests and interpret results. In addition, these systems are not well-suited to pediatric patients because they require a relatively large sample of blood.
Without access to tests that allow them to properly assess and monitor patients, clinicians in developing countries are often forced to blindly prescribe treatment and rely on patient observation to monitor the efficacy of that treatment.
“You really need point-of-care CD4 testing,” notes Stephen Moore, MD, who has devoted time to training physicians in a number of countries, including Kenya. “You shouldn’t have to send the sample out and then wait a few days to get a report back. When the test is available at the point-of-care, you can make a more timely decision, and that improves the quality of care.”
Benchtop flow cytometers
The availability of easy-to-use benchtop flow cytometers is a great asset utilized in counting and monitoring T cells for pediatric patients in places like sub-Saharan Africa. The Elizabeth Glaser Pediatric AIDS Foundation has been supporting the use of portable flow cytometers throughout their programs in Africa for a number of years. A CD4 baseline is recorded before a patient starts on antiretroviral treatment and then on a regular basis afterwards to assess the effectiveness of the treatment. “This process is helping clinicians improve the quality of care,” says Willard.
The Foundation also helps facilities track whether patients have had a CD4 count within the last six months as a standard-of-care indicator. “Once an instrument is in place in these communities, those numbers go up,” Willard notes.
Paired with all-in-one cell analysis kits, these instruments are enabling clinicians and laboratory staff to leverage the power of flow cytometry for CD4 counts and CD4 percentage measurements, regardless of skill level or access to a core facility. Because these systems require smaller sample volumes, generate less waste, have lower operating costs, and are easier to set up and run than traditional flow cytometers, they are especially well-suited for rural settings and pediatric patients.
Larger flow cytometry systems require 300 μL of blood, which is a significant amount for an infant. However, portable benchtop systems typically require only 10 μL. The smaller sample requirement helps to avoid spreading contamination and reduces the patient discomfort from a blood draw that requires a greater amount of patient blood specimen.
Absolute CD4 counts in children undergo variation with age and development; as a result, this value cannot be used in isolation to monitor young patients. An accepted indicator for this patient population is CD4 percentage, which is absolute CD4 counts expressed as a percentage of total lymphocytes. Traditional flow cytometers require use of separate reagents and measurements for absolute and percentage counts. Some benchtop systems employ a single reagent to measure both from a single patient sample.
“In developing countries, where the poverty is tremendous, there just isn’t enough money to buy $100,000 machines,” notes Moore. “Before having the benchtop system, we treated patients by observing clinical symptoms.”
Meeting the challenge
Even with the availability of benchtop technology, however, access to the equipment remains a challenge for clinicians and patients in these locations. Organizations like the Elizabeth Glaser Pediatric AIDS Foundation and the Foundation for Orphaned, Abandoned and Disabled African Children (FOADAC) and individuals like Dr. Moore are facilitating access.
FOADAC recently coordinated the donation of a benchtop flow cytometer system to Nkambe District Hospital. This hospital serves several hundred thousand residents, many of whom survive on subsistence farming. Before the arrival of the portable system, the majority of CD4 counters in the Northwest Region of the nation of Cameroon resided in the major city of Bamenda. Transporting blood samples from rural towns to Bamenda—a full day’s trip on unpaved roads—proved to be too much of an obstacle. In the absence of quantitative CD4 test results, doctors had to prescribe treatment without this critical information.
“Prior to the benchtop flow cytometry system being placed at the hospital in Nkambe, it could take weeks for patients in such geographically isolated places to receive results from Bamenda,” says Tony Ndifor, PhD, of Johnson & Johnson Pharmaceutical Research and Development and a Cameroonian-born U.S. resident who facilitated the donation. “It was not uncommon for patients to worsen or die while waiting for results.” Ndifor anticipates the instrument will help local epidemiologists monitor treatment and control outcomes in the region. Local access to the benchtop system results in greater availability of information concerning CD4 enumeration and CD4 percentage measurements for patients.
A promising outlook
Significant advances are being made in the fight to stop the spread of pediatric HIV and AIDS in developing countries. Forward momentum will be sustained through ongoing education, greater access to antiretroviral therapies, and availability of innovative technologies that are well-suited to these challenging environments. Progress is tangible and can be measured in unexpected, deeply gratifying ways, as described by Dr. Willard. “Ten years ago when I came to Africa for the first time, I saw streets lined by coffin makers. Now, I don’t see coffin makers. I see people building furniture.”
How it works: benchtop flow technology
Flow cytometers, once the province of specialists in core labs, have developed into increasingly portable, but still powerful, instruments. Portable systems leverage a patented, micro-capillary technology that eliminates complicated fluidics, reduces waste, and minimizes maintenance-all features essential for use of the instruments in developing areas.
In traditional flow cytometers, the sheath fluid required to transport the sample past the lasers results in a large volume of liquid waste. The micro-capillary flow cell minimizes waste-reducing it from liters a day to just a few milliliters. This in turn reduces biohazardous waste disposal requirements and associated costs.
Moving sheath fluid through a flow cytometry system requires pumps and other fluidics. Eliminating this hardware gives benchtop systems a smaller footprint. As the name implies, these systems can fit on even a crowded lab bench.
Additional features of the flow cell contribute to the system’s ease of use. It is self-aligning, which ensures proper placement. It is also user-replaceable; that is, it can be removed at any time for cleaning and maintenance, reducing the need for downtime for service visits.
Jim Mulry is the worldwide Clinical Development Manager with EMD Millipore, a division of Merck KGaA of Darmstadt, Germany. Jim brings more than 30 years senior management experience and success in global diagnostics sales and marketing. EMD Millipore is maker of the guava Auto CD4/CD4% benchtop flow cytometry system.
