VSVP

Various Options/ Various Solutions

George Janossy, Ilesh Jani & Debbie K. Glencross

Flow cytometry (FCM) is the standard technology for CD4+ cell measurements. FCM is particularly important if a large throughput of samples is required. In the Western countries this technique has remained expensive, especially because large flow cytometers have been used with a costly large reagent panel. As many as 25 different flow cytometric protocols can be used (reviewed by F. Mandy, Report for GAP; 2001). All these protocols provide accurate CD4 cell counts in fresh blood using lyse-no-wash technology. This excellent performance is facilitated by the participation in Quality Assurance programmes.

As might be expected from the varying complexity of the protocols, CD4 cell counts widely vary in costs. These cost variations are unrelated to the precision of the CD4 cell counts [7]. This conclusion has been amply reinforced by the introduction of the PanLeucogating protocol, with the possibility of using relatively inexpensive reagents. This is because this latter system has been documented to increase precision and accuracy despite the lower costs [12].

Recently, the feasibility of designing inexpensive flow cytometers for CD4 T cell counting has been established [18]. Nevertheless, these instruments have not been fully tested and are not yet released for general use. For this reason, here we briefly summarise the existing possibilities. These represent a wide choice. The decisions about selecting one or another system are critically dependent upon the circumstances (in the same way as different types of aeroplanes can be used to race across the Channel by Those Magnificent Men; see adjacent pictures). It is important to emphasize that one system, optimal for research in one country, may not be optimally suited for routine CD4 counts in another setting. Expensive sophisticated flow cytometers can remain idle if the provisions for keeping them in operation are not met or become too expensive due to the need for special arrangements. People have to carefully consider the various options on the basis of their local circumstances.

Here we list seven different technical alternatives and discuss specific features of each technology. You may wonder about the Figures. These are taken from the film ‘Those Magnificent Men and Their Flying Machines’ to show that there are different ways to fly across the Channel – where there is determination there is solution, also for ‘Those MM with Their FACS machines’.

Text Box: 1. Volumetric flow cytometer using CD45 and CD4 primary gating. This option has the highest throughput capacity, up to 400 samples per day, due to the possibility of using fully automated Windows-based software programmes with auto-bio-samplers of high volume [20]. The combination of volumetry and a two-colour protocol with generic reagents on a single-platform contribute to an exceptionally high accuracy at a very low cost. The equipment, Cytoron-absolute made originally by Omron for ORTHO Diagnostics, is no longer available as new from the manufacturers but are currently in use at specialised centres. This technology represents the optimal arrangement for regional centres defining the optimal specifications that the newly designed modern flow cytometers will need to meet [20].

2. The combination of any flow cytometer with any haematology analyser as a double platform using the recently introduced PanLeucogating protocol [12]. This option allows for a relatively high throughput of samples with affordable cost. Only two reagents are needed, namely, CD45 and CD4 used in a two-colour combination. The additional CD45/CD8 double staining in a second tube is an optional extra. Costs can be further reduced by using generic reagents. The accuracy of absolute CD4 counts is superior to the protocols previously used on double platforms because the CD45 gating used here is more robust than the so-called ‘scatter’-gating [12]. Thus samples can travel for longer periods before the quality of immunophenotyping deteriorates. Clearly, this double-platform arrangement is well suited to haematology laboratories where Quality Assurance schemes are in operation for absolute white blood cell counts on the haematology analysers.

3. Any flow cytometers (from BDIS or Coulter-Beckman) with microspheres (beads) to assist single platform operation. This is the arrangement that is most fervently propagated as the optimal system for absolute CD4 T cell enumeration in Western laboratories. Nevertheless, even in the West, the majority of laboratories still use double platform. This single platform arrangement delivers precise CD4 counts but at the expense of a relatively large reagent panel that includes expensive microbeads. Resource-poor laboratories that buy or get flow cytometers (perhaps even at a drastically reduced costs) loaded with an expensive reagent contracts are likely to find this system prohibitively expensive. Further, the clinicians are frequently not very pleased about the increased price wasted on a so-called ‘full lymphocyte subset phenotyping service’ when they require only affordable absolute CD4 counts [11, 12] or, at most, CD4+CD8 counts [20]. Hence the attractiveness of simplifying the staining protocols by utilising fewer reagents (e.g. CD45 plus CD4) during the routine service work even if it relies on a bead-based single platform operation.

4. FACSCount with its specialised reagent supply. A simple, robust version of dedicated flow cytometers, the FACSCount, has remained relatively expensive to run due to the high costs of the specialised reagents needed for its operation. The FACSCount can only process eight samples at a time as a suitable alternative for laboratories with limited sample load.

5. Red diode laser operated flow cytometers. Currently, two commercially available flow cytometers fall under this category, namely the CyFlow and the Luminex100 [18]. The first one is designed for counting cells in clinical laboratories, while the latter one is designed for performing suspension arrays [23]. Importantly, the current position is that both cytometers will need to undergo independent multi-centre validation trials before can be recommended for use in the routine clinical laboratory. The predictable arrival of newly designed, modern flow cytometers with inexpensive light sources will greatly contribute for the introduction of affordable CD4 testing, especially if such instruments operate reliable volumetric absolute counting with double colour immunofluorescence. Interestingly, the first experimental prototypes have proved the concept of red-diode laser instruments [18], and laser light sources emitting other then red colour are also constantly decreasing in price.

6. Systems based on immuno-magnetic selection and image analysis. These are systems based on principles other than flow cytometry. These should maintain the accuracy of flow-based equipment, and perform affordable CD4 assays with a medium throughput. The main advantage of this technology is that the microscope based equipment could be built at costs lower than flow cytometers [21,22]. However, this type of counter is not yet commercially available.

Text Box: 7. Manual methods for CD4 counting. In this category the most promising method is the Dynabead system. In small studies absolute CD4 counts by Dynabeads have shown high correlation to values yielded by flow cytometry. However, these studies did not use appropriate statistics (e.g. Bland-Altman plots) for method comparison and appeared, in some cases, to underestimate CD4 T cell counts. There is, therefore, a clear need to conduct multi-centre studies to consolidate the tentative consensus that recommends manual methods for laboratories with relatively low sample load. This manual method is about 10-14 times less efficient than the volumetric flow cytometer described above [20].

Professional organisations such as the WHO are currently preparing the guidelines for selecting the most appropriate laboratory arrangements for countries that wish to avail themselves with the supply of generic anti-retroviral drugs through the Global AIDS Programme. In addition, there are new trends to extend the usefulness of flow cytometry, with the instruments listed above, to perform multiplexed ELISA-type assays [23] for the differential diagnosis of infectious diseases, including opportunistic infections.

London and Johannesburg, 6^th March 2002

- 2002.