Measuring CD4 and CD8 cell counts: why are they done?

CD4 cell counts for adults are used to:

• Decide when to start treatment.

• Monitor the response to treatment when viral load measurements are unavailable.

• Decide when it is safe to stop prophylaxis against opportunistic infections.

Examples of when to start treatment inlcude:

• Co-trimoxazole when CD4 cell counts are below 500 cells/mm³, according to the World Health Organization's recommendation for Africa.

• Antiretrovirals when CD4 cell counts is at or below 200 cells/mm³ or below 350 cells/mm³. Various protocols have different recommendations, although the WHO recommends 200 cells/mm³ as the threshold for treatment.

- Fluconazole against Cryptococcus when CD4 cell counts are below 100 cells/mm³ (Thailand).

- Clarithromycin against Mycobacterium avium complex when CD4 cell counts are below 100 cells/mm³.

Why are they not done?

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

The systems used in Western hospital laboratories are not optimal for most other countries because:

• they require relatively fresh blood samples for accurate results, which is not a problem when there is easy access to refrigeration and excellent transport links, but becomes a serious problem in other settings

• they use over-specified equipment designed for research and for complex full lymphocyte subset analysis that is not requested by the clinicians who monitor HIV therapy

• they require stable electricity and pure water

• they require experienced technicians to set up and run

• they need a specialised service for maintenance, all of which removes them from the point of care.

• for absolute counts they rely on beads that are costly to buy and need experience to run

• they use antibodies that have not come down in cost in line with production costs, and must be paid for in foreign currency.

Two examples of how this translates into costs per test are as follows.

Conventional flow cytometers, such as FACS-Calibur or Coulter systems, cost US $50,000 or more to set up and about $20 to $30 for each test. The running cost is dominated by the cost of beads needed to provide a full lymphocyte subset analysis. However, these machines can be used to run less expensive protocols.

The FACSCount system a compact modern piece of equipment costs around US $20,000 for the equipment and around US $20 for reagents for each test carried out. This however is a slower system that can only be run with dedicated reagents, which means running costs cannot be reduced without affecting the performance of the test.

One response is to find ways of delivering these tests more cheaply, ideally while maintaining or even improving their quality.

The other response is to consider alternative measurements of the immune system, such as total lymphocyte counts, either on their own or combined with other observations.

Cheaper and more reliable CD4 flow cytometry

A group of laboratory scientists, the Afford CD4 group have shown that cost of CD4 tests can be brought down radically by adopting techniques that are simpler and, in fact, more accurate than those used in most western laboratories. The section that follows is based largely on their ideas (Wilja).

The standard method for CD4 counting is called flow cytometry. Cytometry means 'cell measurement'. This technique is based on adding marked antibodies to the blood sample, then flowing it through a light beam, usually from a laser.

If two antibodies are used, each bound to (conjugated with) a different coloured dye (a fluorochrome), the machine can easily 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. Recent publications from the Afford CD4 group have shown that to measure CD4 counts these two antibodies are entirely sufficient.

Placing detectors in different places in relation to the light source, with a computer program to analyse the results, also allows cells of different shapes and sizes to be counted separately, and at very high speed, because of the different way in which they scatter light.

Combining these two principles allows CD4 T-cells to be counted separately from other larger white blood cells, monocytes, which also have CD4 proteins on their surface (although in smaller amounts).

Absolute counts are harder to obtain and there are three different ways to do so.

The simplest and cheapest is to count cells in a known volume of blood measured with a small syringe, referred to as the volumetric method. In principle, this is the best method, but currently only a few flow cytometers are fitted with the small syringe needed. In this case, it is even possible in principle to work with just one antibody - against CD4 to count CD4 cells or, of course, CD8 to count CD8 cells.

If the cytometer is not fitted with such a syringe, another approach is to add a known quantity of reference beads to a sample of known volume. Absolute counts are then calculated from the number of beads and the number of cells counted in the same sample. This arrangement is expensive because the beads cost $3-6 per test.

Alternatively, it is possible to count 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 for $0.8-$1 on a haematology analyzer (a machine which counts white and red blood cells in a set volume of blood).

At present, the cheapest and most accurate options are provided by two different systems.

The first one is using a single reagent (CD4) on a volumetric flow cytometer. At the moment only a few of these instruments exist and the comparability of results to other systems has to be proven; still this system is the most promising solution for future development.

The company which appears to be closest to delivering a volumetric flow cytometer is Partec, based in Germany, which has announced a transportable system designed for use in limited-resource settings they have even shown it set up in an off-road vehicle, powered by solar panels. Alternatively, it can be delivered as a compact unit that can be powered from a car battery

A team at the University of Munster, closely associated with the company, has reported promising findings comparing a CyFlow system from Partec with FACS-Calibur from Becton Dickinson. The CyFlow capital equipment cost is less than $20,000 and the cost per test less than $2. The protocol is very simple, with just one reagent and ten minutes incubation at room temperature before the test is run. A multi-centre clinical study, including two centres in limited-resource settings, has recently been completed and publication is due shortly (Greve).

One of the first independent evaluations of Partec's system has recently been reported from Senegal by Senegalese and Belgian researchers (Dieye). This found that while it could give good results which were closely comparable to those from when used by an experienced technician, it was vulnerable to trapped air in the system. It concluded 'Direct volumetric and bead-based single platform measurement on the Cyflow showed good correlations with gold standard cytometers. The precision of volumetric measurements on the Cyflow can be improved by including a time versus fluorescence and a time versus total cell count plot as an additional internal quality control measure.'

Further information is available on Partec's website here

The second idea is to use two machines alongside each other as a dual platform. One of these a haematology analyser counts the number of white blood cells and the other a flow cytometer counts the ratio of CD4 T-cells to all white blood cells, identified using a CD45 antibody. From these two values the absolute number of CD4 cells is calculated. Both the precision and the robustness of this double-platform technology has recently been dramatically improved. When introduced to clinical service in regional laboratories in South Africa and Uganda, the panleucogating protocol has reduced the running expenses of CD4 counting to 20-27% of the previous level of expenditure and the method is easy to run.

A Luminex 100 flow cytometer has been modified, stripping out unnecessary parts, making a system that uses generic antibodies to count CD4, CD8 and CD45, that runs off a 12V car battery and processes results on a laptop computer. The result is all built into one unit - a small wheeled trolley, 68x45cm. As its inventor observes, 'the ultimate cost of such a flow cytometer will be determined by how resolved the global AIDS community is to bring care and treatment to the regions that can least afford it' (Mandy).

A further important point that the CD4 T cell percentages among lymphocytes and the T lymphocyte counts, including both CD4 and CD8 numbers, are clinically important, particularly in children. These parameters are also available, if needed, with the panleucogating protocol when CD8 antibodies are added to the panel.

Low cost generic CD4, CD8 and CD45 monoclonal antibodies are available through the UKs Public Health Laboratory Service which can be joined to different fluorescent dyes for use in these systems. However, these will require further development and rigorous international quality control systems put in place before they can be considered to be viable options in routine diagnosis (see http://www.affordcd4.com/ for further details).

An additional key technology that is being developed and refined at present is a fixative that allows blood samples to be stored and transported over 5 days (or even slightly longer) without loss of accuracy in test results. A prototype for this exists, in the form of a solution called 'Transfix' supplied by the UK Public Health Laboratory Service for purposes of quality assurance sample preparation. This is being developed further by the South African National Health Laboratory Service to enable preserved samples to be run through their testing systems under exactly the same protocols as fresher blood samples (Scott).

A related issue is the problem of transporting the reagents ('labelled antibodies') used to count CD4 and CD8 cells. Chemical stabilisation of these antibodies to make them heat-resistant has now been reported. Fluorochrome (FITC) conjugated antibodies to CD4 and CD8 were unaffected by storage for 4 weeks at 50 degrees Celsius (Beretta).

Recently, a new type of assay has been developed, based on 'microcapillary cytometry'. This method is used by the Guava EasyCD4 testing equipment. Each CD4 cell count test using this method is around 25 times cheaper than using flow cytometry, and requires less blood. However, a study comparing the two methods found that they gave almost identical results across a range of CD4 cell counts^[1].

Microscope-based CD4 counting systems

There appear to be at least three main alternative systems which use microscopes to count CD4+ cells in the blood; some can also be used for CD8+ cells. Two of these are manual and labour intensive so they will be difficult to scale up to match expanding access to antiretrovirals. Their value has still to be fully determined, but is likely to be in supporting small-scale programmes, and to supplement and extend access to CD4+ and CD8+ counting in places or at times when other options are not available. The third uses automated image analysis but exists only in a prototype at present.

The two systems which are commercially available now are:

• DynaBeads (Dynal Biotech)

• Cytospheres (Beckman Coulter)

The DynaBead (magnetic beads) method uses two different sets of beads. One type (CD14 beads) binds to monocytes and is used to remove them. The second type (CD4 beads) is then used for counting CD4+ T cells. These CD4+ cells are stained with acridine orange to make the cell nuclei visible for counting under a fluorescent microscope. During the procedure several steps of centrifugation and washing are involved that render this method far more error-prone than the lyse-no-wash method used in flow cytometry.

A modified DynaBeads system (using a different stain for the cells) can be used with a light microscope, which is an even less expensive piece of equipment - it can use a mirror and daylight as a light source.

The Cytosphere system (Beckman Coulter) has advantages for haematology technicians who are familiar with the shapes of cells. Here the monocytes are not removed but appear different under the microscope, so the bead-covered CD4+ T cells can be counted. Unfortunately, when researchers in Uganda compared the results to those from conventional flow cytometry, they found a tendency to overestimate CD4 counts (Karcher). Comparing 71 paired tests on blood samples taken from people with HIV before and during ARV treatment, the median values were 343 (manual) compared to 258 (flow cytometry). 'Different treatment indications would have resulted in 14% of patients if the cut-off was set at 350 cells/microlitre and in 22% if the cut-off was set at 200 cells/microlitre.'

Two further non-flow systems, TRAx CD4 and Zymmune, have been withdrawn from the market following takeovers of the companies that first introduced them.

It has been demonstrated that it may be possible to automate a version of the non-flow counting systems, by replacing visual counting with computerised counting. The recently designed EasyCount system uses no moving parts but counts cells in a known volume of blood in a small chamber. The CD4+ cells, coated with magnetic CD4 antibody, are lifted to viewing level and counted by image analysis. The accuracy of CD4 counting and how precisely CD4+ T cells and monocytes are separated in this system are still unknown.

In comparative evaluations performed by the French ANRS, DynaBeads gave the best results, and in a subsequent multicentre evaluation in Senegal, Burkina Faso and Cote dIvoire it has given results that are directly comparable with established flow cytometry systems. The cost of a fluorescent microscope (around US $2,000) is far less than any current flow cytometer and reagent costs (US $4-$5 per test) about the same as panleucogating and much lower than the fully priced mainstream commercial flow systems. Technicians could be trained to operate the tests in three days with no prior experience (Diagbouga).

Two alternative methods - DynaBeads and Coulter Cytospheres - were evaluated by Bergeron at Health Canada's National HIV Immunology Laboratory, as part of a programme of international assistance. Of the two, they found that DynaBeads gave more reproducible results and was simpler to operate, took less time, but was somewhat more expensive. The mean values for both methods matched the results obtained with a flow cytometer (Bergeron).

DynaBeads, Capcellia ELISA tests and automated FACScount systems were compared on 115 blood samples taken in Lagos, Nigeria. DynaBeads emerged as the cheapest (US $4) and most cost-effective option; equivalent to Capcellia but less than half the price (Audu).

A newer system which is still under development is the 'SemiBio Ligand Catcher'. This basically uses a treated microscope slide to which CD4 cells adhere and can be made visible (Cao). Currently a manual, light microscope based method. However, image analysis software is being developed to automate it with the inclusion of a digital camera on the microscope. Cost per sample is given as US $15 - $20, which is not that attractive, despite the low capital cost for the system. Getting results in less than an hour from a whole blood sample does sound good, although the inclusion of washing stages suggests that technicians will need careful training to operate it.

The way ahead

These recent developments illustrate three important points.

First, improved flow cytometry with low running costs is already in full operation in some leading centres in Africa. Consequently, the main conclusion about the feasibility of further improvements by using simple volumetric cytometers also has considerable scientific credibility.

Second, these areas of modern technology currently receive almost no industrial attention and support. This is an exceptionally short-sighted attitude because flow cytometry, with the use of similar instruments, is already making inroads into microbiology in applications ranging from the differential diagnosis of infectious diseases to food safety and checking water quality. It would appear eminently sensible to make the effort to adapt flow cytometry to meet its full potential, as a system that should be far more robust, portable, and therefore usable directly in clinics rather than merely in the rarefied atmosphere of the Western research laboratory. This is a case of market failure which may justify public sector investment to correct.

Thirdly, non-flow techniques including microscopic methods, especially if they can be combined with fast image analytical systems, will remain exceptionally attractive for small laboratories.

An interesting variation on the microscope-based approach involves the use of microchip manufacturing to miniaturise an antibody-based cell-capture system. The microchip could then be 'read' using a modified digital camera. This could even lead to 'point of care' testing, with small blood samples taken by a healthcare worker and results becoming available on the same day. A prototype system is being developed by a team led by Dr Bill Rodriguez at Harvard University and has been demonstrated in field trials in Botswana (Graham).

In principle, this system could be dramatically cheaper than other alternatives, not least because only very small quantities of reagents are needed. In practice, this may depend on successful negotiation of a potential minefield of patent rights - although this may be less of a problem for the system's use in limited resource settings than in wealthier markets such as the USA. The accuracy may not precisely match that of flow cytometry, but provided flow cytometry is available as a back-up and for quality assurance, this may not matter when set against the immediate usefulness of a cheap test that can identify people who should definitely be on treatment.

A preliminary report has appeared, of a method of measuring CD4 protein levels in whole dried blood spots (Mwaba).The technique uses an antibody 'sandwich' to capture and detect CD4 proteins in the sample. As it stands, it doesn't give a close match to CD4 T-lymphocytes measured by flow cytometry, and the discrepancy (an overestimation of CD4 T-cells) increases as the actual CD4 count declines. One possible reason for this is that the method counts CD4 expressed by monocytes as well as that on T-cells. Flow cytometry and methods such as DynaBeads separate out the monocytes either because of their different shape (FC) or using antibodies to proteins expressed by monocytes but not T-cells (DB). While it might be very helpful if reliable CD4 counts could be done from dried blood spots, this clearly has not yet happened.

Other strategies for reducing CD4 costs

Dr Praphan Phanuphak, director of the HIV-NAT centre at Chulalongkorn University, near Bangkok, Thailand, argues that it is a mistake to rely on total lymphocyte counts in place of CD4 on the basis that the former costs less. There are several ways of reducing the cost of CD4 counts even with existing technology. He argues that even when these make the results less accurate, they are still far closer to proper CD4 counts than relying on total lymphocyte counts.

Among these strategies are:

• using smaller blood samples, and smaller quantities of the reagents to test them with.

• using Dynabeads (see above) to take CD4 cells out of part of a test sample, and running two total lymphocyte counts, one on the unaltered sample and one on the sample with CD4 cells extracted. Comparing the two results then gives an estimate of the number of CD4 cells without any manual counting of cells.

A small-scale preliminary study in Thailand used blood samples from 55 HIV negative and 56 HIV positive donors. This showed that reducing the quantity of reagents to either a half or a quarter of those normally used, with a sample size of half that recommended by manufacturers, is still able to give reasonably accurate results for HIV positive blood samples. As the authors point out, larger-scale tests would be essential before this is put into clinical practice (Nookhai).

Total lymphocyte counts

If a haematology analyser is available, capable of accurate white blood cell counts, this can be used as the basis for CD4 counting, as discussed under that heading.

Total lymphocyte counts can be used as a surrogate for CD4 counts, when combined with clinical evaluation. Specifically, WHO recommends that in someone who has a TLC of less than 1200/mm³ and symptoms of HIV-related disease, ARV treatment should be considered as if they had a CD4 count of 200 or less. This is a sensible recommendation to deal with situations where antiretroviral treatment should urgently be provided and where CD4 counts are not available.

However, total lymphocyte counts are a very poor and misleading substitute for CD4 counts in the pre-symptomatic phase of HIV disease, especially when CD8 counts are elevated alongside reduced CD4 counts. In this group, antiretroviral treatment is likely to be of greatest value if it can be started at or before the time when CD4 counts go below 200, regardless of the CD8 count. Consequently, the introduction of CD4 counts for clinical decision-making remains an important aim.

A total lymphocyte count (TLC) - which includes CD4 and CD8 cell numbers -- can be done more cheaply than CD4 counts, on a haematology analyzer which is more readily available than a flow cytometer. A white blood cell count (WBC) is even easier. So could one of these be used as a substitute for a CD4 count?

A San Francisco study compared three values: CD4 count, TLC, and 'absolute lymphocyte count' (excluding large lymphocytes) for a random sample of 2057 US patients, 86% male. Using values of ALC under 1750 or TLC less than 1900 cells per mm³ came the closest to matching the CD4 threshold of 350 cells. Lower values had a similar relationship to a CD4 threshold of 200 cells. The sensitivity and specificity of the two alternative tests was similar and between 71 and 75% (Jacobson).

A North American paediatric study has been reported, based on bloods taken before 1996 and before the introduction of HAART. Low TLC and white blood cell counts (WBC) were associated with higher death rates - though the Pearson correlation coefficient* - r - was only 0.49 between CD4 and TLC and was even lower (0.32) between CD4 and WBC (Mofenson).

*Correlation coefficients measure how closely one value is related to another: 0.0 is completely unrelated and 1.0 (or 1.0) is a simple straight-line connection.

Similarly, an Italian paediatric study looked at total lymphocyte count in infants and young children born to mothers with HIV and came to the conclusion that a total lymphocyte count below 2000 was an excellent surrogate for CDC criteria for PCP prophylaxis in children (Ebo).

However, these results cannot be transferred to other populations, with a different nutritional status and exposed to a range of other diseases, without more research.

Stored blood samples from the US Multicenter AIDS Cohort Study (MACS) were analysed and it was observed that in a population of HIV positive men, TLC and haemoglobin levels (another easily measured blood parameter) can define a population at high risk of developing AIDS. Both values were generally stable until CD4 counts fell to around 350, after which TLC would drop by more than 31% per year and haemoglobin by more than 11% per year. Men whose TLC and haemoglobin fell sharply went on to develop AIDS, on average, just over a year after these declines began. A sudden drop in TLC and haemoglobin could have been taken as an indication for starting antiretroviral therapy in this population (Gange).

As with the previous study, these results would need to be tested further in resource-limited settings before they could be applied there. In particular, their relevance to women, especially pregnant women, has to be questioned. Although haemoglobin levels do correlate with survival of people with HIV in Africa (especially of women who have given birth), the appropriate response may be very different in a female population suffering chronic iron deficiency and exposed to malaria, compared to a North American male population. The authors also observe that further work is needed to identify the frequency of testing best suited to identifying people in need of treatment.

One recent Indian study did claim that total lymphocyte counts correlated well with CD4 counts, but it was absurdly small - the total sample was: "11 normal healthy individuals and five HIV-infected patients who were classified as symptomatics"! (Srihari).

Another Indian study, from Pune (Mane), recruited 749 patients and carried out a full sensitivity, specificity and predictive power analysis for TLC < 1200 to predict CD4 < 200. It found that if TLC was under 1200, there was only a 68% probability that the CD4 count would be under 200. Only if age, gender and haemoglobin levels were also taken into account could it become at all reasonable.

In Nairobi, Kenya, a substantial study found TLC to be a poor substitute for CD4 counts. "The overall correlation coefficient between the total lymphocyte counts and the absolute CD4+ cells counts among the 230 HIV-1 infected clients was low (Rho=0.66). When we further analysed the correlation coefficient of total lymphocyte counts by CD4+ cells counts categorised by HIV-1 disease stage among the HIV infected clients, no obvious trend was detected. In addition, the sensitivity and specificity of the TLC at different cut-off points above and below the 1200/mm3 recommended by WHO with an absolute CD4+ cell count of 200/mm3 was unsatisfactory." The researchers conclude that effort would be better put into making proper CD4 counts more affordable, and that ARV provision can and should be expanded regardless of access to these tests (Kimani).

A smaller Nigerian study compared the performance of TLC with CD4 measurement using the manual DynaBeads system for 66 individuals with HIV, 10 of whom commenced HAART during the study. Although it found a highly significant correlation between the two, it concluded that TLC was a poor substitute (Akinola).

In Yaounde, Cameroon, researchers evaluated TLC in isolation from clinical symptoms and found only a weak correlation (r = 0.41, p=0.05) between TLC and CD4 in a population of 149 HIV-1 positive adults. Taking a TLC below 2000 as a cut-off value identified 84.6% of people with CD4 counts below 100, and 76.3% of people with CD4 counts below 200. However, it had very poor specificity (42.3% and 46.7% respectively) and a low predictive value. Taking a TLC below 1000 as the value improved the specificity (in other words, most really did have low CD4 counts), but reduced the sensitivity (missed many with low CD4 counts). They concluded that TLCs 'are of limited value in predicting CD4 counts and should not be substituted for CD4 counts in this population. Nevertheless, cheaper and less sophisticated methods of determining CD4 counts should be evaluated and made available for use in this population' (Mbanya).

At Somerset Hospital, Cape Town, 266 patients treated with HAART were tested using CD4 lymphocyte counts, total lymphocyte counts and viral load measurements at baseline, then weeks 4, 8, 12 and 48. A significant correlation was seen between TLC and CD4, at r = 0.61 - which is still not very close. At a 'group level' there was also a negative correlation between TLC and viral load changes, i.e. TLC rose as viral load went down. However, in both cases it is not clear that TLC can safely be used to advise individuals with HIV on whether their treatment is continuing to work or should be changed (Badri).

155 HIV positive people in Ethiopia were followed over 4.5 years by researchers. 27 had died by August 2001, with sufficient information to be able to analyse predictive markers. Simple markers such as anaemia (using international definitions adjusted for sex and altitude in this highland population), total lymphocyte count below 1500 per mm³, body mass index below 18.5 kg/m²* and HIV-related symptoms (WHO disease stages III and IV) were 'independent predictors of short-term survival'. During the 4.5 years of the study, 121 HIV positive participants would have been treated if the criterion of one simple marker being present (anaemia, total lymphocytes under 1500, or HIV disease stages III-IV) had been used, compared to 127 following international guidelines (CD4 counts under 350, viral load over 55,000, HIV disease stages III-IV). 114 people would have met both sets of criteria, of whom 91 (80%) would have started treatment at the same time, regardless of which set of criteria had been used. The value of these simple markers for monitoring response to therapy remains to be explored - obviously, anaemia would not be appropriate if AZT were included in treatment regimens used (Mekonnen).

* Body mass index = weight in kg divided by square of height in metres

The need for quality assurance

Whatever system is adopted, it is crucial that external quality control is put in place to ensure that the test results from individual laboratories are equivalent.

The aim of quality assurance is to ensure that doctors and patients are getting the same information from lab tests, no matter which lab they use or the methods used there.

At its simplest, quality assurance can be a matter of one laboratory distributing a number of samples to a network of other laboratories, which then report their measurements for each of the samples. They can then be told whether their results agree with those from other laboratories in the network. If they do, then all is probably well. If they do not, this can prompt an investigation of training for staff, the state of equipment, supply and storage of reagents, or other matters which may need attention.

In extreme cases, doctors and patients can be told to run additional tests to confirm that patients are getting the correct treatment.

Within Brazil, the national quality assessment programme for CD4 counting uses EDTA-stabilised whole blood samples, distributed across a network of 74 labs in 25 Brazilian states. A pilot study by 12 labs showed that the samples remained fit for testing up to two days after being drawn, and three national surveys have now taken place in 1999, 2000 and 2001. Each used four anonymous samples, one of which is a duplicate, and the results obtained by individual labs can be compared to the results obtained throughout the network (Kallas).

This principle can be applied equally well at an international level, among reference laboratories, or at a national or district level, where a proportion of patients may have their blood tested at a reference laboratory as well as it being tested locally, to provide a check on the reliability of local testing services. It can and should operate across sectors, to include private sector laboratories as well as those run by public health authorities. It becomes particularly important in multi-centre clinical trials, to ensure that test results which are relied on as indicators of treatment efficacy mean the same thing in different centres, which may have different patient populations.

One of the requirements is that usable samples can be transported to the different laboratories involved in the scheme. This is easy for systems based on testing dried blood spots, relatively straightforward for frozen samples, but much harder when tests require undamaged cells in blood or plasma. Systems based on transferring fresh blood samples at high speed by courier cannot easily be extended internationally or to remote areas with poor transport links.

Thus, international CD4 testing quality assurance systems are being developed, based on new systems for stabilizing fresh blood samples (Bergeron). The QASI programme encompasses 156 labs in 47 countries, including Australia, Canada, the USA, and now Brazil (Barcellos).

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