Simplified cytometry for routine monitoring of infectious diseases

Abstract

The interacting epidemics of HIV/AIDS, tuberculosis (TB) and malaria in resource-poor areas of the world have created a critical need for rapid, simple, affordable apparatus and tests that will permit patients with these diseases to be promptly diagnosed and properly managed.

As documented in the current issue of Clinical Cytometry, complex flow cytometric analyses used in affluent countries for CD4+ T cell counting in HIV/AIDS have been simplified, introduced, and quality assessed in resource-restricted countries of Africa and the Caribbean, where simple gating protocols such as panleucogating now provide accurate and precise CD4+ T cell counts on a large scale. CD4/CD8 ratios in infants may replace more expensive molecular tests for HIV infection; simplified flow cytometry is also compatible with HIV viral load-associated lymphocyte activation tests and with antigen-specific cellular immune response assays that rapidly diagnose active TB in both HIV-negative and HIV-TB co-infected individuals.

In addition, it is becoming evident that smaller, much less expensive fluorescence imaging cytometers can be used for CD4 counting, immunophenotyping, and hematology and for other applications such as diagnosis and drug-susceptibility testing of TB and diagnosis of malaria. With the gradual, organized expansion of the much-needed diagnostic networks in the underprivileged countries, the most cost-effective apparatus may be one capable of performing tests for all the three diseases mentioned. The most sustainable systems will be those that can be assembled and maintained, to the greatest extent possible, in the countries where they will be used. © 2008 Clinical Cytometry Society

The April 15, 2002 issue of Clinical Cytometry (1, 2) described the state of T-cell subset counting 20 years after AIDS was first reported. By that time, it had become clear that the “new” epidemic in the United States and other affluent countries was related to a much larger, but largely overlooked epidemic in Africa and other resource-poor areas of the world, where the disease has had a devastating impact on public health.

This issue of Clinical Cytometry again addresses the unmet needs of resource-poor areas, touching on simplified, sustainable, and affordable cytometric approaches to both CD4 counting and monitoring of patients while on anti-retroviral therapy (ART) as well as to diagnosis of other epidemic infectious diseases, notably tuberculosis (TB) (3, 4) and malaria (4).

HIV/AIDS—A CYTOMETRICALLY DEFINED DISEASE

When the disease we now know as HIV/AIDS was described in 1981 (5), fluorescence flow cytometers with the sensitivity necessary to measure immunofluorescence had been available for less than a decade, and the monoclonal antibodies used to demonstrate a profound loss of what are now called CD4-positive T lymphocytes had just reached the market (6, 7). The absolute count of CD4+ T cells in peripheral blood was quickly identified as a valuable prognostic measurement in HIV-infected patients (8), and this led many clinical institutions to acquire flow cytometers, particularly after relatively small, user-friendly benchtop instruments with personal computer-based data analysis systems became available in the mid-1980s.

CD4 counting itself had progressed beyond the single-parameter, dual-platform measurements to three- and four-color analysis with relatively sophisticated gating strategies (9), and the need for systematic quality assessment had been perceived and met, at least to some extent, in both developed and developing countries (10, 11).

CD4 COUNTING IN RESOURCE-POOR ENVIRONMENTS: LESSONS LEARNED

During the past decade, “high-end” research flow cytometers using as many as five excitation beams to measure fluorescence from 17 or more fluorochromes (12) have become commercially successful despite their high prices, and highly complex multiplexing methods (13) have amplified capabilities for analysis of both structure and function of cells. Clinical applications such as leukemia/lymphoma immunophenotyping and minimal residual leukemia detection are now being explored using 6- and 8-color protocols (14). Such sophistication comes at a price; more and more costly reagents are required, and quality assurance (15) and data analysis (16) procedures are more complex and time-consuming, and therefore harder to introduce into large numbers of laboratories.

For CD4 counting, however, there is ample evidence that more is not necessarily better. Current guidelines (9, 17) acknowledge that “primary CD4 gating”, using a single CD4 fluorescence-labeled antibody with lymphoid side scatter display (18), is a workable protocol. These guidelines favor the use of the combination of CD4-CD45 two-color fluorescence and side scatter to establish particularly robust gates: first for all leucocytes and then for strongly CD45+, CD4+ lymphocytes. This latter practice has been followed both by those using flow cytometers with three- or four- (or more) color measurement capability and, as “PanLeucogating,” by others doing analyses using less sophisticated instruments with only CD45 and CD4 antibodies (18–21). Indeed, the CD4 guidelines of the US Centers for Disease Control, compiled in 2003 (17) have already left the door open for simpler approaches, noting that “… several alternative methods are available that require fewer reagents and involve more cost-effective gating algorithms … As published validation data accumulate from multi-site studies … these potentially more cost-effective options will be considered as alternative or substitute methods.” A current consensus clinical standard document referred to as CLSI H42-A2 has also endorsed the “PanLeucogating” method (22). We believe that the publications from the different countries in the current issue of Clinical Cytometry have gone a long way to establish this claim (19–21).

Several relatively simple flow cytometers, generally smaller and less expensive than typical benchtop systems, are now in use in the field for CD4 counting. The oldest and most established of these systems is the FACSCount (BD Biosystems, San Jose, CA) (23), which makes two-parameter fluorescence measurements of cells stained with antibodies to CD3 and CD4, yielding an accurate and precise count of CD4+ T cells. The system can also be used to count CD8+ T cells, and a stain combination allowing total lymphocyte counts [and, therefore, the lymphocyte percentages needed for accurate diagnosis in children under 5 (24)] to be determined is now being tested. Erythrocytes are not lysed in the FACSCount procedure; beads present in the reagent kits are used to derive volumetric counts. Partec (Münster, Germany) has offered a variety of CyFlow instruments, the first of which measured only fluorescence from antibody to CD4; however, CD4 gating appears more accurate in the CyFlow SL system, in which both side scatter and fluorescence measurements are used (25). The CyFlow features volumetric sample delivery; counting beads are therefore not needed. Guava Technologies (Hayward, CA) produces the EasyCD4 system, a compact flow cytometer that measures forward scatter and fluorescence (26). The fluidics system does not require sheath fluid, and sample delivery is volumetric; this reduces reagent costs. PointCare Technologies (Marlborough, MA) has developed flow cytometers that detect colloidal gold-labeled CD4 antibody binding to cells using only light scattering measurements, making the optical system and detectors simpler, more robust, and less expensive than those in fluorescence-based instruments. Clinical trials have been done, but results have not been published.

It is established that making CD4 counts available should prolong lives (27, 28), and the World Health Organization, in connection with its “3 by 5” program, stresses the need to do so (29). It is also established (10, 11, 19, 20, 30) that laboratory professionals in resource-poor areas can deliver high volumes of properly quality-controlled flow cytometric CD4 counts in an organized cost-effective manner equivalent (or superior) to the performance of laboratories operating in better-endowed countries.

Even the simplest flow cytometers, however, may require more support and infrastructure than is readily available outside regional centers in many resource-poor countries (31–33). At present, manual CD4 counting techniques (34), requiring relatively well-trained technicians to process samples and then perform counts using microscopes and hemocytometers, are in use in Africa, Haiti, and elsewhere. Reagent costs for these time-consuming methods are, in some cases, higher than those for flow cytometry; accuracy and precision are lower, and quality assurance remains a problem. Regrettably, these manual CD4 counting techniques also fail when faced with the huge increase in the demand for CD4 counts and when the continued monitoring of patients who are already on anti-retroviral therapy is requested (19). Health care reform in general (35) and increasing the number of trained health professionals in particular (36) are not problems that those of us who develop and use cytometric methods can solve, although we can, should, and do participate in efforts to define the qualities of the optimal systems to use and to improve distribution of reagents and apparatus and support and maintenance of the latter (32, 33, 37, 38).

A NEW CYTOMETRY—THE USES OF ADVERSITY

It does, however, behoove us to focus our attention on what cytometry can, does, and does not do in the area of infectious disease management in both resource-poor and affluent countries. Cytometry, in environments in which it is affordable, has displaced manual methods for detecting and counting various types of cells primarily because instruments can analyze larger sample volumes than can practically be examined by microscopists and because the combination of specific reagents and precise quantitative measurements allows more accurate cell identifications to be made. CD4 counting and TB and malaria diagnosis from slides (4) all represent instances in which it is difficult, if not impossible, for even an infallible human observer to devote as much time to a specimen as would be required to produce acceptably accurate and precise results. For CD4 counting, cytometry, in the form of flow cytometry, at present, is universally acknowledged as the “gold standard” method. Manual methods, whether based on microscopy or immunochemistry, are acknowledged as stopgap or even “second class” alternatives. For diagnosis of malaria and of TB and other bacterial infections, however, cytometry, if considered at all, is almost universally and shortsightedly dismissed as too expensive, even in affluent countries.

An analytical cytometer, as opposed to a cell sorter, can be thought of as a box into which cells are put and from which numbers are retrieved. As long as the right numbers emerge, the user should not be overly concerned about what is in the box; however, to keep it working in an unfriendly environment with minimal maintenance, it is desirable to minimize its size, complexity (number of total and moving parts), energy consumption, and cost. This minimalist ideal is rarely achieved, or even approached, in cytometers designed by the affluent for the affluent, but we submit that the philosophy is essential in developing systems for use in resource-poor areas. It is also desirable for the user to have to do as little as possible to as few cells as possible between the time at which the cells are collected from a patient or other source and the time at which they are introduced into the apparatus, and for any reagents used to be stable over a wide range of temperature and humidity (37). It is, finally, desirable to locate as much as possible of the capacity for manufacturing both the apparatus and reagents in or near the countries in which they will be used (37, 38).

THE WIDENING SCOPE OF SIMPLE CYTOMETRIC DIAGNOSTICS

CD4 counts, however affordable, are of little or no benefit to the approximately four million people uninfected by HIV who die of malaria and TB each year, or to the substantial number of children who die of bacterial sepsis because they are misdiagnosed on clinical grounds as having malaria and thus fail to receive antibiotic therapy (31). It thus makes sense to extend the mission of affordable cytometry well beyond the CD4 count. A cytometer that measures fluorescence in two to four spectral regions (with the possible use of side scatter) is usable not only for a wide range of immunophenotyping and hematologic applications, but also for a wide range of biochemical and serological assays using “suspension arrays” of multiplex labeled microbeads (39, 40). The CD45 pan-leukocyte antibody can readily be used to provide absolute total white blood cell (WBC) counts and WBC differentials (40). CD4/CD8 ratios in babies born to HIV-seropositive mothers can potentially replace more expensive HIV-1 DNA and reverse transcriptase polymerase chain reaction analysis (24). Resuscitating previously well described lymphocyte activation tests, combining CD8/CD38 staining with routine CD4/CD45 analysis can revolutionize and streamline the monitoring of patients on ART (41). Minimal residual disease in childhood leukemia can be detected 19 days after the start of therapy (40). Finally, in the differential diagnosis of active tuberculosis (TB), flow cytometric assays for the interferon-gamma (IFN-γ) synthetic capacity of CD4+ T cells show significant advantages over the ELISA (e.g. Quantiferon-Gold) and the enzyme-linked immunospot (ELISpot) tests, particularly in patients who are co-infected with HIV and TB (3, 40, 42).

Although one would not expect a flow cytometric “dream machine” (38) capable of making all the measurements described above to cost significantly more than the apparatus now designed for use in resource-poor areas; there is a distinct possibility that one or more substantially smaller and less expensive instruments could accomplish the same range of tasks. The minimalist approach to instrument and procedure development also pays additional dividends; less operator training and maintenance are required, and quality control, without which no laboratory can adequately function, is simplified.

Measuring cells one at a time, as is done in a flow cytometer, requires intense light sources, usually lasers or arc lamps, and multiple sensitive detectors, usually photomultiplier tubes, which increase the cost and complexity of the apparatus. Laser scanning cytometers and automated fluorescence microscopes, which incorporate precision mechanical assemblies for stage motion and focusing, are similarly complex and expensive. It is, however, possible to build a much simpler, cheaper cytometer by using a high-intensity light-emitting diode (LED), costing only a few dollars, to illuminate a relatively large area of a slide or other static substrate and a digital camera chip, costing a few tens of dollars, to make low-resolution images of all of the cells in a specimen at once (43, 44). Such an apparatus can readily detect low-level fluorescence signals in the range expected from cells stained with fluorescent antibodies, as well as the substantially stronger signals associated with cells stained with nucleic acid stains, fluorescent redox or membrane potential probes, etc.

Approaches to CD4 counting using similar simple fluorescence imaging systems have been described elsewhere in the literature (45–48); the penultimate paper in this issue (4) provides evidence that a single such instrument could be used for diagnosis and drug susceptibility testing of TB and for diagnosis of malaria. As is noted there, “the apparatus is no more complex than a cellular phone and, indeed, utilizes components similar to those used in many such phones, i.e., a digital camera chip, a blue LED, and a microprocessor with wireless communications capability.” It should neither take long nor cost much to determine whether such devices do indeed meet the desperate needs that have motivated their design.

If and when these simple instruments are made and provided to a wide range of laboratories worldwide, we may well ask how we could have done without them for so long (4, 33, 37), as we now do regarding mobile phone networks. The publications in this special issue clearly emphasize the need for learned societies and healthcare professionals in both resource-poor and affluent countries to adopt a proactive and pragmatic approach to diagnostics development. Those of us who have been involved with CD4 counting from the early days remember the AIDS activists who kept us focused and honest; too many of them did not live long enough to benefit from the therapeutic and diagnostic advances we are now trying to make available to as many HIV/AIDS patients as possible. Getting the job done properly may now require individual and collective activism on our part.