Immune system monitoring methods

Flow cytometry (cell measurement) is a process used to count, identify, and sort various types of cells. This technique is based on adding monoclonal antibodies (MAb) to a blood sample and running the fluid through a light source, usually a laser beam.

If two antibodies are used, each is bound to a different fluorochrome (coloured dye) and the machine can compare the numbers of cells with different proteins on their surfaces. For example, the ratio between CD4 cells and all white blood cells (leukocytes) can be measured using CD4 and CD45 antibodies, since all white blood cells have CD45 proteins on their surface.

By placing detectors in different places in relation to the light source, different cell types of the same size can be distinguished because they emit light differently i.e. CD4 cells can be distinguished from similarly sized monocytes, which also carry CD4 proteins on their surface. Blood cells can be separated by size, shape, density, and epitope and the relative percentage of each cell subset of interest can be calculated.

Dual platform technology (DPT) refers to a method in which absolute counts are derived from measurements obtained from two instruments – a flow cytometer and haematology analyser (the machine used to produce complete blood counts). It is possible to calculate the ratio between T-cells expressing CD4 and all cells expressing CD45 markers using a flow cytometer and then combine this with absolute counts for white blood cells obtained on a haematology analyser.

Single platform technology (SPT) is a process in which absolute counts of lymphocytes are measured from a single tube by a single instrument. This can be done in several ways.

Some single platform technologies use internal calibrator beads. A known quantity of reference beads are added to a blood sample of known volume and from this process, absolute counts can be calculated.

The volumetric method uses a single reagent and micro-syringe to count cells in a known volume of blood. Some systems also make use of a computer-controlled precision syringe pump for sample transport and volumetric absolute counting, One example of this method is the CyFlow® SL by the German company Partec, which can be used for any kind of cell analysis and absolute volumetric counting. This system is portable (dimensions are 43x16x37 cm.) and can be set up for use in an off-road vehicle, powered by solar panels or a car battery.

In many countries, the cost of CD4 tests is an obstacle to their use and a barrier to treatment.

The systems used in higher-resource settings are not optimal for most other countries because:

• They require relatively fresh blood samples for accurate results and this is a problem if there is not ready, easy, and affordable access to refrigeration and excellent transport links.
• They use over-specified equipment designed for research and for complex full lymphocyte subset analysis that is not required by clinicians who monitor HIV therapy.
• They require stable electricity and pure water.
• They require experienced technicians to set up, maintain, and run.
• For absolute counts, they rely on beads that are costly to buy and require experience to handle.
• They use antibodies whose cost has not been reduced in line with production costs and that must be paid for in foreign currency.

One response is to find ways of delivering these tests less expensively while maintaining or even improving their quality. The other response is to consider alternative measurements of the immune system.

Researchers in South Africa evaluated the performance of single-platform (SP) PanLeucogated (PLG) CD4 methodology in over 50 laboratories, accompanied by a proactive approach to training, internal quality control, and external quality assessment. Two-colour flow cytometry for SP PLG (CD4/CD45) was combined with sample-by-sample bead-count-rate (BCR). This approach was validated versus the conventional predicate SP and dual-platform (DP) 4-colour flow cytometry methods typically used in developed countries. The simpler 2-colour PLG was less expensive, discriminated CD4 T-cells from monocytes without a need for CD3-staining, gave more precise results, offered faster and easier training for users and teachers alike, and accommodated workloads exceeding 3000 tests per laboratory per month.¹

In a small study of just under 50 patients, a microfluidic CD4 cell counting device produced results similar to those obtained from standard flow cytometry. Researchers at Harvard have been studying a device that uses cell affinity chromatography operated under controlled flow to isolate CD4 T-lymphocytes from unprocessed, unlabelled whole blood. CD4 counts are obtained quickly and simply by using an optical microscope.^{2 3}

A non-optical method known as cell lysate impedance has also been described by researchers from Harvard. This electrical method for counting cells is based on the measurement of changes in conductivity of the surrounding medium due to ions released from surface-immobilised cells within a microfluidic channel. When immobilised cells are lysed using a low conductivity, hypotonic media and the resulting impedance change is calculated, using surface patterned electrodes to detect and quantify cell number. The cell lysate impedance spectroscopy method will detect 20 cells/ml and is simple and efficient. The microfluidic device is a single-use cartridge in a hand-held instrument that can be used in point-of-care settings.⁴  

In Malawi, researchers tested a CD4 counting method (Blantyre count) using a CD4 and CD45 antibody combination with reduced blood and reagent volumes. Results were assessed through comparison to TruCount and FACSCount systems and had good agreement with both. The assay is affordable with reagent costs per test of $0.44 USD (£0.22-0.33) for both absolute count and CD4 percentage, and $0.11 for CD4 percentage alone. Noteworthy is that use of the Blantyre count identified 193 patients with WHO clinical stage I or II disease who had CD4 counts of less than 200 cells/mm³ (and would not have been eligible for antiretroviral treatment on a clinical basis alone) and 12 patients with stage III or IV disease with CD4 counts >350 cells/mm³.⁵

The Blantyre method was also tested in a paediatric population and results were compared to those obtained using Panleucogate methods on a Coulter EPICS XL-MCL flow cytometer. Consistent CD4 cell counts and percentage were obtained by the two methods. The Blantyre assay can be performed by local lab technicians at a cost of $0.21 USD or less.⁶

The Guava EasyCD4 assay is another method of measuring CD4 cell counts that uses microcapillary cytometry on a dual platform base. The equipment cost is similar to that for flow cytometry, but individual tests cost less. Preliminary studies have taken place in Uganda and the United States. In Uganda, the positive predictive value was 96% and the negative predictive value was 93%, suggesting that the assay may be used with confidence in resource-limited settings.⁷

Guava Technologies also developed the personal cell analyser (PCA), a single platform flow cytometer featuring microcapillary technology to analyse cells without the use of sheath fluid or beads. It provides easy two-colour detection (1 scatter) for typical cytometry needs. When tested in Thailand, it performed well against both the TruCOUNT and the FACSCount methods. Unfortunately, its use requires a high degree of technical expertise.⁸   

The same group of Thai researchers evaluated the SP flow-rate-based calibration method for determining CD4 cell counts with 2-, 12-, and 16-year-old flow cytometers and results were compared to the bead-based method. The flow-rate approach was reliable in determining CD4 counts on various flow cytometers and offers cost-effective patient monitoring.⁹  

This group also evaluated the revised BD FACSCount CD4 software and reagent system, designed to simultaneously provide CD4 cell percentage and absolute CD4 cell count. Using 149 patient samples, they found correlation and agreement in their results using the new and the standard FACSCount system. The new system allows for lab monitoring of children and adults.¹⁰

In Gambia, an automatic process tool (APT) was developed that is able to define and describe cell populations across large datasets. The APT was developed to identify regulatory T-cells in HIV-positive adults, based on expression of FOXP3. Results from the APT were compared directly to manual analyses performed by five immunologists. The APT tool can process approximately 100 data files per hour and offers objectivity and consistency to data analysis.¹¹ 

One other technique being looked at in Germany is the use of a density-based negative selection method (RosetteSep) that can provide an approximate CD4 count at a cost of around $0.30 USD.¹²