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Afordable CD4 T-Cell Enumeration orResource-Limited Regions: A Status

Report or 2008

F. Mandy, 1* G. Janossy, 2 M. Bergeron, 1 R. Pilon, 1 and S. Faucher 1

1National HIV and Retrovirology Laboratory, Public Health Agency o Canada, Ottawa, Ontario, and InternationalCentre or Inectious Diseases, Winnipeg MB, Canada

2Royal Free and University College Medical School, University College London, United Kingdom and Department oMolecular Medicine, Witwatersrand University, Johannesburg, South Arica

Background: The global struggle with human immunodefciency virus (HIV) and the battle to developaordable CD4 T-cell counting technology are both unulflled goals in 2008. The need or such instru-mentation is more critical now as implementation o antiretroviral therapy (ART) is in progress in many

resource limited regions. Major scaling-up eorts in rural situations are difcult to implement withoutlaboratory inrastructure. CD4 T-cell counting is especially critical when trying to reach individuals withHIV to have them enrolled in ART as soon as they qualiy or treatment based on CD4 count.

Method: This review covers both the chronological evolution and the scientifc milestones o techno-logical development o aordable immunophenotyping. It is more ocused on ow cytometry but doesconsider the potential contribution by digital image cytometry.

Results: Thus ar ow cytometry oered only modest progress toward aordable immunophenotyping. Alist with desirable eatures is oered or side by side comparison. Digital image cytometry has yet toshow its enormous aordable market potential.

Conclusions: It is possible to develop truly aordable, portable ow cytometry but it is not here yet.There are some hopeul signs as there are innovative and practical technical components appearing atregular intervals. However, so ar the technical breakthroughs have been ragmented eorts without anyattempts to consider intercorporate collaboration to optimize critical mass and synergy. The smaller play-ers in the industry have made some progress toward meeting the monumental needs in Arica and Asia.

Digital image cytometry may well be the ultimate winner in the aordable technology race.q 2008 ClinicalCytometry Society

Key terms: human immunodefciency virus-1; antiretroviral therapy; ow cytometry; resource-restrictedconditions; CD41 ; T cell enumeration

How to cite this article: Mandy F, Janossy G, Bergeron M, Pilon R, Faucher S. Afordable CD4 T-Cell Enumerationor Resource-Limited Regions: A Status Report or 2008. Cytometry Part B 2008; 74B (Suppl. 1): S27S39, 2008.

In 2003, the World Health Organization (WHO)responded to the plight o millions o people living withHIV and introduced a strategy called the 3 by 5. Theobjective was to treat 3 million HIV-inected individualsby the end o 2005. The target was not achieved, butthe movement had signicant impact and pioneered abold but only partial ulllment o a commitment. Thedoor to the concept o global implementation o antire-troviral therapy (ART) as the universal standard treat-ment or people who were going to perish with HIV has

Clinical Cytometry supplement (74B, Supplement 1, 2008) titled The

Global Health and Diagnostic (Flow) CytometryBreakthrough in HIV and

Tuberculosis is sponsored by Beckman Coulter, BD Biosciences andCaltag-Medisystems. The contributing authors to this article have

declared the ollowing conficts o nterest: Francis Mandy works as a

part-time consultant or PointCare Technologies as a technical writer and

advisor. The remaining authors have declared no conficts o nterest.

*Correspondence to: Francis Mandy, International Centre or Inec-

tious Diseases, Winnipeg MB, Canada R3B 3PS.

E-mail: [email protected]

Received 20 January 2008; Accepted 1 February 2008Published online 28 February 2008 in Wiley InterScience (www.

interscience.wiley.com).

DOI: 10.1002/cyto.b.20414

Cytometry Part B (Clinical Cytometry) 74B (Suppl. 1):S27S39 (2008)

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been opened (1). The dramatically accelerated imple-mentation o ART combined with persistent global politi-cal pressure has resulted in a two-log cost reduction ofrst-line ART or adults and eventually or children. How-ever, to support ART implementation with essentialmonitoring tests, signifcant cost reductions must also beachieved or essential clinical assays. In the long run,delivery o ART is impossible without sustainable criticalmonitoring tests. Without cost eective CD4 T-cell mea-surement services, reliable patient management is in seri-ous jeopardy in resource-limited regions. It is importantto mention that in situations where CD4 T-cell countingacilities do not exist, this should not hinder the intro-duction o ART to patients with clinical AIDS symptoms(1). However, or quality patient management the criticallaboratory tools include reliable HIV diagnostics such asmeasurements o CD4+ T cells and HIV viral load (VL).

To ensure continuation o scaling-up o ART in areaswith limited resources two strategic targets must beachieved: (i) to reduce the cost o the diagnostic/moni-toring assays; and (ii) to bring testing capacity to areas

where the HIV-inected individuals live, oten in remoterural areas amidst devastating poverty.

In resource-rich countries, HIV-related laboratory tech-nologies have improved over time. Thirty years ago, CD4

T-cell counting was perormed manually with an epi-uorescent microscope. Today, multicolor ow cytome-ters with automated sotware are part o routine HIVimmunophenotyping in most clinical immunology labora-tories. Also in the past decade HIV VL assay sensitivityhas increased dramatically, thanks to improved methodsand introduction o costly automation. However, whenthe monitoring tests are compared with antiretroviralmedication in resource limited countries, neither othese critically needed assays has undergone a dramatic

cost reduction. This situation is especially troubling asCD4 T-cell counting has been around longer than AIDShas (2). In this review some o the technical issues thatgovern aordability are addressed as they are related toCD4- and cellular immunological assays. Most discussionsare ocused on recent developments o the aordable andsustainable immunophenotyping and provide someinsight regarding the possible directions or uture alterna-tive assay developments. There are at least two options,one is to reduce the cost o dedicated CD4 immunophe-notyping machines as or example Cytometry or Lie(C4L) group is attempting. This is a Purdue Universitybased consortium ocused on changing lives through low-cost diagnostics. The second option is to develop robust

ow cytometers designed as exible diagnostic platormscapable o multitasking and delivering much more beyond

T-cell subtype reports. In such cases, the goal is to try toamortize the higher up-ront operational cost withreduced daily labor costs. This is the strategy PointCare

Technology with PointCare NOW has introduced to pene-trate this underserviced rural and urban market.

With the introduction o ART, there is a golden oppor-tunity to assess the minimum need or inrastructure inresource poor locations in order to increase both the

quality and the capacity o basic laboratory services inmost o the disease-burdened rural locations. This kindo orward thinking can go a long way to introduce sus-tainable inrastructure building without compromisingany precious resources committed to HIV. It is becomingevident, especially in Arica, that eective treatment oHIV includes the monitoring o tuberculosis (TB) andmalaria. To provide an eective ront to fght, these threediseases diagnostic activities need to be integrated. Withany public health inrastructure enhancement, most HIV-inected individuals will beneft dramatically. TB will otenpresent itsel as a coinection returning with HIV inec-tion. The demand or low cost, easy-to-use, and reliableCD4 T-cell monitoring devices is enormous but so is arapid test that is reliable or acute malaria.

WHO recommendations call or published scientifcevidence independent o manuacturers to acilitate theeective and objective evaluation o newly introducedaordable CD4 measurement technologies. Paradoxically,the most acute need or such evidence-based evaluationsis in resource poor countries that have the most limited

capacity to conduct expensive method studies. CD4 T-cell numbers in peripheral blood was considered to be areliable test to confrm and monitor immune suppressioneven beore the arrival o AIDS (25). The accurate enu-meration o the absolute numbers o CD4 T-lymphocytesremains the best indicator o HIV disease progression inadults. For a brie period, in the wake o the enthusiasmor the then novel quantitative VL assays, the clinical sig-nifcance o CD4 T-cell enumeration as the best monitor-ing tool or immune status was challenged (6,7). Cur-rently, HIV serology, T-cell subset enumeration, and HIVVL are considered to be essential complementary tests.VL is the best measure o drug efcacy during ART(8,9). Absolute CD4 T-cell counts, combined with VL

remain the best predictor o clinical patient outcome(4,10), including those treated with ART (11,12). CD4

T-cell count is the marker to monitor HIV disease pro-gression, level of mmune suppression, and overallassessment of mmune restoration o patients receivingART (10,13). CD4 T-cell counts remain essential in decid-ing when to start ART (13,14) and when to considermedical interventions to prevent opportunistic inections(15) whereas increase in VL and synchronous reductionin CD4 count are indication to consider altering therapy.CD4 T-cell number trends in specifc group o patientsare also used in epidemiological studies to orecast thepotential cost burden a specifc HIV outbreak will haveon the public health system (16). At this time, drug re-

sistance assays are unavailable or direct patient manage-ment in rural resource poor regions.

The challenge acing the cell-based diagnostics industryis to create a CD4 T-cell counter that is simple to operatein rural settings while being very robust so it is compati-ble with the tropical environment that is known to beextremely hostile to equipment with sensitive electronicsand optics. The assay must also be reproducible at a dra-matically low cost. In 2005, the price or ART in manyparts o Arica and Asia underwent a 50-to 100-old reduc-

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tion (rom $15,000 to $150 per year) albeit this low pricecomes with some qualiying conditions such as fxed or-mulation, specifcations with large confrmed orders, onlyor point-to-point supplies, advance payments, etc. Shoulda cost-reduction model be applied to immunophenotypingsimilar to the one that was implemented to ART delivery,the price o CD4 testing would drop rom the current $4to $50 to under one dollar per test. However, such a lowprice level may not be sustainable especially in rural, lowvolume resource poor regions. The current consensus onCD4 T-cell counting is to aim or a $2 assay or remote ru-ral locations in resource poor regions. Many believe thatthis challenge is an achievable and sustainable target with-out compromising assay quality. The question comes tomind, is ow cytometry necessary or CD4 counting? Dowe really need a ow cytometer or will a low cost digitalimaging device sufce. Howard Shapiro very elegantlyand convincingly argues that ow cytometry is an overkill when all we need to know is how many dual-labeledcells (CD4 and CD3) are in a unit volume o whole blood(17).

NO REVOLUTION JUST GRADUAL EVOLUTION TOWARD MORE

ROBUST CD4 T-CELL IMMUNOPHENOTYPING

It is important to remember that there were no antire-troviral drugs available to combat AIDS in the 1980s.When they did emerge, it was clear that because o pro-hibitive cost, they would not be available in resource lim-ited regions. In Arica, treatment was restricted to latestage secondary opportunistic inections where the moni-toring o CD4 T-cell count is less important. Hence, theimmunophenotyping assay development ocused on thelucrative industrialized world market. By the late 1990s,clinical ow cytometry had steadily evolved rom research-only applications to complex routine immunophenotyping

protocols including polychromatic reagents. The assay pro-cedures were complex to the point that they requiredguidelines and detailed protocols to render them repro-ducible or large multicentric clinical drug trials conductedand still ongoing, such as the NIH/ACTG in the USA (18).

One signifcant protocol switch occurred in some othe leading immunology laboratories in the 1990s. It wasthe elimination o the overt dependency on the duallight scatter strategy (orward vs. side scatter) as the ini-tial step in the immunophenotyping gating protocol dur-ing the execution o any standard CD4 T-cell assay(reviewed in Re. 5). The traditional dual light-scatterprotocol is also reerred to as a homogenous gating strat-egy. As long as the CD4 T-cell counts are reported as a

lymphocyte % (percentage o total lymphocytes), the useo homogeneous dual-light scatter did not pose a signif-cant problem with reproducibility. Nevertheless, suchmorphologic discriminator proved to be incompatiblewith ageing whole blood specimens, once the objectivewas to obtain an absolute CD4 T-cell count. However toreport an absolute count with this traditional method,both the total white blood cell (WBC) count and thelymphocyte dierential count (LD) had to be obtainedrom a second instrument, a hematology workstation

(Fig. 1). This dual assay combination is reerred to as adouble-platorm technology (DPT). In general, the hema-tology instrument results had to be rom a blood sample< 6 h old. I not, the CD4 T-cell absolute count would beunreliable. The mixing o one intrinsic and one extrinsiccell attribute or gating purposes is reerred to as a heter-ogeneous gating strategy. As the traditional approachwas replaced by more robust heterogeneous protocols,

the absolute count problem was only hal solved. Theuse o a combination o uorescent and side scatter asthe primary trigger (CD45/SS), enable a reliable lympho-cyte count determination. However, the total WBC stillremained problematic. To deal with this defciency,some instrument manuacturers added another reagent,a volume-metering bead product, to enable absolutecount to be incorporated into CD4 T-cell immunopheno-typing process. With such an additional reagent, it waspossible to compute cell concentration per unit volumeo blood (panel B on Fig. 2). Known concentrations omicrouorospheres were introduced with each patientspecimen; the absolute count challenge was solved andthe process is known as single-platorm technology (SPT).

Several new gating combinations were introduced in theearly 1990s (reviewed in re. 19): these included CD45(20), CD3 (21), and CD4 (22,23) with one light scattersuch as orthogonal side scatter. The combination o brightCD45, side scatter, and counting beads is reerred to asCD45-gating with SPT or absolute counting.

US companies manuacturing clinical ow cytometersintroduced FDA approved reagent kits or CD4 T-cellassays to reduce the complexity o both protocol andreagent selection. In HIV clinics, a steady demand

F IG. 1. Dual Platorm Technology (DPT) or CD4 T-cell absolutecounting. Clinical fow cytometers are designed as open fow systems.Traditionally they deliver relative numbers or the percentages o all thecells analyzed. For example, i 10,000 lymphocytes were analyzed andthere were 5000 CD4 T-cells, the fow cytometer will report CD4 T-cells as 50% of ymphocytes. Once the demand switched to absolutecount, the CD4 T-cell percentage of ymphocytes had to be converted.This requires multiplication with two additional parameters rom a he-matology analyzer. To obtain the absolute CD4 T-cell count, the whiteblood cell (WBC) count and the lymphocyte dierential (LD) are takenrom the hematology instrument to be multiplied through with the fowcytometry derived CD4 that is a percentage of ymphocytes. Thismethod is the dual platorm technology or absolute cell counting. There is a variation on the above described conventional DPT. In thecase o the original panleucogating protocol only the WBC count isused rom the hematology analyzer the two other numbers are obtainedrom the fow cytometer (see Fig. 10).

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remained or enumerating CD4 T-cells with these ex-pensive but relatively simple-to-use kits. With the rapidspread o newly introduced lymphoma and leukemiadiagnostics services, the more requent clinical use ofow cytometry shited away rom HIV to cancer diagnos-tics. Thereore, the driving orce behind urther develop-ment o mmunophenotyping protocols in resource rich

countries switched to the new fow cytometry baseddiagnostics o onco/hematological disorders. Where adetailed lymphocyte subset-analysis had to be carriedout or dening T cell subsets, B-lymphocytes, mono-cytes, and NK-cell populations these assays were drivingthe market (18). The complexity o the new assays orwhole blood pathology based oncology, let CD4 T-cellcounting in comparison a relatively simple-to-perormassay. Thereore throughout the next decade, the pursuitof ess expensive alternative instrumentation or CD4

T-cells was not a high priority or the well establishedfow cytometry industry. Equally clear was that selectingprotocols requiring ewer reagents served neither thepriorities of aboratory managers who were equipped

with polychromatic instrumentation nor the reagent mar-keters and manuacturers o the prot oriented fow-cytometry industry.

HISTORICAL DEVELOPMENT OF AFFORDABLE

FLOW CYTOMETRY

Instruments

With the onset o mplementation o 3 3 5 in Arica,the goal was to install instruments as ast as possible in

high throughput laboratories representing the largestArican consumer groups, which were easy to reach andneeded aordable technology. Over the years, interna-tional agencies have learned that such technology instal-lations require the implementation o both internal andexternal quality control programs or CD4 T-cell enumer-ation and technical training on continuous bases in order

to achieve sustainable immunophenotyping, whereinstruments must survive with nominal inrastructureand extreme environmental conditions. Other considera-tions might include intermittent electric power delivery,with requent fuctuations (including spikes that candamage instruments instantly), ambient room tempera-ture can exceed 40 8C by midday, and rerigerator operat-ing temperatures which may reach above 158C. Indeed,the US Army, in collaboration with the Los AlamosNational Laboratory, developed a robust instrument todetect airborne lethal biological weapons in the battle-eld. They produced a compact fow cytometer capableo operating under rugged desert conditions. A commer-cial version o this originally Los Alamos/BioRad instru-

ment, Apogee A40 analyzer rom UK/Italy, was intro-duced to the clinical market or CD4 T-cell countingaround 2004 without much success (24). The rst dedi-cated T-cell subset enumerating instrument, the FACS-Count, was launched by BDIS about 15 years ago (25). Itrepresented a remarkable achievement at a time whenthe other authoritative guidelines still called or 12monoclonal antibodies (MAbs) in 6 tubes using complexdual-color protocols (18). The FACSCount is portableand still sells world-wide including in Sub-Saharan Arica,

F IG. 2. Two Variations on Single Platorm Technology (SPT) or T-cell subset enumeration. As demand increased or absolute counts on a singleinstrument, the SPT, two options became available: (i) The cytometer equipped with built-in syringe system in order to deliver a unit volume o bloodor analysis. This is depicted on panel A on the let hand side. This intrinsic volumetric method does not require additional reagents to obtain absolu tecell counts. (ii) A known volume and concentration o microfuorospheres (or reerence beads) are added to a clinical fow cytometer with each speci-men, as illustrated on panel B. The additional reerence reagents (beads) added to every specimen can considerably increase the per-test costs.

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delivering reliable CD4 T-cell counts to medium-sized ru-ral health centers. This instrument was the frst to countCD4 T-cells using whole blood, no-lyse, with a SPT orabsolute cell count. A single tube is required containingtwo MAbs (CD3 and CD4) and microuorospheres orcounting absolute CD4 T-cells. Currently, the CD3/CD8T-cell assay is an optional kit (25). However, FACSCounthas been expensive to operate, selling initially with a re-agent cost above $20 per test. For over a decade, it wasthe only ow cytometry option available or resourcepoor regions. In the past ew years, the cost per test hasbeen reduced signifcantly in selected resource-poorcountries.

Another approach to aordability was developed byGuava Technologies. They redesigned the ow cell, theheart o the hydrodynamic uidic system o a ow cy-tometer. This innovative alteration eliminated the needor sheath uid that is traditionally essential to achievecell alignment in the ow cell (26). The elimination osheath uid with microcapillary ow was an importantbreakthrough or regions such as Sub-Saharan Arica

where clean water is a precious commodity. Conven-tional ow technology guzzles up liters o expensivefltered-water. Further advantage o this microcapillarysystem is the signifcant reduction in reagent-volume pertest. The corresponding reduction o specimen volumehelps with minimizing biohazard discharge volume, andit is also a welcome innovation or pediatric applications. The Guava gating strategy is similar to the FACScount,both technologies use a variation on T-gating protocol(Figs. 3A and 3B). Currently, the Guava Easy CD4 tech-nology provides the lowest reagent cost per test, but theGuava PCA instrument cost is higher compared to theFACSCount. The integration of ntrinsic volumetric solu-tion in ow cytometry is a signifcant engineering

advancement in the direction o aordable absolute CD4 T-cell count. It eliminates the use o costly uorescentmicrospheres with each specimen. Previously, OrthoDiagnostics with the Cytoron-absolute had already intro-duced a motorized syringe system or volumetric deliv-ery or clinical application (22,27) but this instrumentwas not developed or resource-poor settings and it didnot survive. Variants of ntrinsic volumetric technologywere frst incorporated into aordable systems by PartecGmbH (28) and later by Guava Technology (26); seepanel A on Figure 5. The Guava PCA uses a syringe sys-tem whereas in the case o Partec, the volume measure-ment is achieved with a dual linear sequential air/liquidsensing device integrated with a motorized syringe eed-

back drive system. The least expensive Partec CD4counting instrument, the CyFlow Counter (28), has thisvolumetric eature packaged together with no-lysetechnology. The most recent robust simple-to-use arrivalon the market, PointCare NOW, rom PointCare Technol-ogy, also uses a syringe system or absolute count (29).

The next major innovation was to switch to reeze-dried and low-heat dried reagents to eliminate the excru-ciating cost burden o cold-chain, transportation/delivery,and on-site rerigerated storage. This strategy was

recently introduced by PointCare Technology (29) andReaMetrix o India (30), respectively. PointCare NOW isan automated ow cytometer that operates with colloi-

dal gold-labeled reagents. This approach eliminates thecomplexity o using expensive vibration resistant mountsor uorescence signal detection flters and most o thepotential optical alignment problems. It detects scatteredlight at our dierent angles all at once. Such a compactoptical bench design is quite energy-efcient, thus a sig-nifcant inroad toward aordability has been achievedwith the LED powered optics (Fig. 4). With the Point-Care NOW system, sample handling is ully automatedwith all manual pipetting eliminated. This is a greatadvantage since pipettes in Arica and Asia are rarely cali-brated. Once the vacutainer tube containing the speci-men is placed in the designated compartment, the com-plete analysis cycle is carried out without any urther

manual intervention. This approach comes a long way toulfll the ideal low-throughput rural requirement. Thecurrent lack o compatibility with available internationalexternal quality control schemes is a problem rom labo-ratory quality management point o view. This instru-ment is modestly priced, but the reagent costs per testare relatively high. The higher cost is in part is justifed asthe instrument provides a our-part hematology dieren-tial count, a hemoglobin count and both an absolute anda lymphocyte percent CD4 T-cell enumeration or about

F IG. 3. T-Gating methods A and B. Method A: The Guava Easy CD4protocol is represented on the right side in Panels A1 and A2. OnPanel A1, a manually gated heterogeneous histogram (SS/CD3) repre-sents T-cells as they are separated rom monocytes (M), other lympho-cytes (L), NK cells (NK) and granulocytes (G). The gated CD3 1 T-lym-phocytes are all in the open square. Panel A2 illustrates the second

manually gated homogeneous (CD3/CD4) bivariate histogram where thedouble labeled T-lymphocytes (CD3 1 /CD41 ) are in an open circle .Method B: The FACSCount protocol is represented on Panel B1 andB2 on the let side. Panel B1 illustrates the sotware controlled homo-geneous dual fuorescent histogram (CD3/CD4) in the histogram onlythe dual labeled cells are in the gate. Panel B2 is a cartoon to illus-trate how the automated gating sotware can be compared to the tradi-tional bivariate histogram. CD4 T-cells are a subset identied asCD31 /CD41 they are in the open square. The reerence beads whichare used to assist with SPT absolute counting are identied in bothPanels. [Color gure can be viewed in the online issue, which is availableat www.interscience.wiley.com.]



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$10 in under 8 min. It will take some time beore themarket place will be able to assess the cost-efectivenesso providing such a comprehensive clinical data setwhile the patient is still with the health-care giver (i.e.,at the point o care).

There are three combinations where ow cytometryunctions are mixed with other technologies (Fig. 6). Wealready discussed the situation (panel B in Fig. 6) when

a ow cytometry and hematology analysis are combinedas is the case with PointCare NOW. A hematology ana-lyzer can be modied to count stained cell nuclei suchas the Symex PocH-100i (panel A in Fig. 5). It can read

the nal results rom a Dynal manual kit or CD4 count-

ing. Yet another approach is a hybrid- or bioanalyzerow cytometry (panel C in Fig. 6). Such instruments arecapable o analyzing both cell- and bead-based assays.With hybrid ow cytometers, it is important to think interms o amortization o nstrument cost over severalyears. They are a relatively new class o cytometers onthe market. There are three instruments o this type: theLuminex 100 (Luminex Corp., the Guava 96 PCA (GuavaTechnology), and the FACSArray (BDIS). All are equippedwith built-in microtiter plate sample-handling to process96 assays nonstop. These hybrid ow cytometers canalso be reerred to as hybrid ow application platorms(HyFAP). The capacity o these platorms can beenhanced to perorm a variety o mmunoassays and mo-

lecular probe assays (3134). In the uture, it is probablythe speed o sotware development that will determinethe extent o success that this technology will have. It ispossible that the broad application potential will justiythe relatively high cost o such platorm. With HyFAP,cells can be processed to provide CD4 T-cell counts(23), and bead-bound immune-based assays (replacementassays or various ELISA) simultaneously (31,34). A easi-bility study on the use o HyFAP or HIV serology testinghas been reported or the detection o circulating anti-bodies rom resh and dried blood spots (DBS) (33).Others have used this multitasking platorm or thedetection o vaccine-preventable inectious diseases inchildren (32). ReaMetrix is also developing a HyFAP plat-

orm system which will reach the Indian domestic mar-ket in 2008. The multitasking concept is the same; theywill provide an instrument that will ofer a whole panelo diagnostic assays that are required. To summarize,new alternative ow cytometers have arrived. Theyinclude instruments using microcapillaries and the non-uorescent reagents. A ow chart (Fig. 7) places thesevarious key eatures where they appear related to owsignal detection and cell propulsion methods availableon afordable cytometers.

F IG. 4. PointCare cell distribution with our diferent light scatter.Colloidal gold labeling technique to count CD4 T cells withPointCareNOW. This gating technique is based on identiying leukocytes includ-ing monocytes, neutrophils and lymphocytes on a histogram with or-ward scatter (FS) on the x axis and Axial Light Loss (ALL) on the y axis. The lymphoid scatter on T-helper cells is altered by colloidgold labeled CD4 antibodies. They bind to CD4 1 T cells (see heavyblack-rimmed circle). The CD4 negative lymphocytes include B, NKand CD8 T-cells. The colloidal gold tagged CD4 1 T-cell are pulledaway rom the monocytes. With this bivariate histograms both plateletsand neutrophils are visualized. [Color gure can be viewed in theonline issue, which is available at www.interscience.wiley.com.]

F IG. 6. Mixed Flow Cytometry. Currently there are three ways to com-bine ow cytometry with other technologies to obtain CD4 T-cell meas-urements: ( A) Two models o bench-top Sysmex hematology analyzercan be used to read stained cell nuclei to support the Dynal manualCD4 T-cell enumerating kit; ( B) The PointCare NOW instrument, a owcytometer designed to operate both as a hematology analyzer and CD4 T-cell counter simultaneously; and ( C) Hybrid ow cytometers designedwith various capacities to perorm automated analysis with both cell-and bead-based assays.

F IG. 5. Intrinsic Single Platorms on Some Afordable Flow Cytome-ters. Several instrument manuactures concluded that it is possible toofer volumetric solution with lower costs per test. This solution isavailable as a built-in syringe, electrode interace or a combination oboth. The syringe driven option is utilized by Guava, PointCare and allthe hybrid instruments. Partec CyFlow Counter uses the dual, syringeand electrode, volumetric option.

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REAGENT AND NOVEL GATING STRATEGY DEVELOPMENT

One can also consider the innovative diversity that isevolving in terms o gating strategies, hence reagentselection. Efcient reagent selection can go a long waytoward aordability (35). Various novel gating strategieshave led to the cost eective gating strategies throughthe reduced sizes and volumes o the reagent panels.One international organization, the Clinton Foundation,looked at the various new aordable options and has

accepted some o them. The Clinton Foundation negoti-ated CD4 T-cell counting assay prices around $4 per test(35), eliminating CD8 T-cell counting with the FACS-Count and other systems.

CD3 T-cell Gating

The extrinsic attribute o T-cells, the expression oCD3 molecules as a T-cell linage specifc marker, wasintroduced in combination with CD4, a unction specifcmarker (21). This T-gating combination was frst imple-mented on the FACSCount (25). In this case, it is a com-bination o green and red colors that defne the location

o the orange colored CD4 T-cell (Fig. 3A). The red CD3and the green CD4 MAbs provide a mixed orange coloror the dual stained CD4 T-cell population. This CD4 T-cell count strategy requires the total o three reagents,two MAbs (CD3 and CD4 MAbs) and counting beads oreach specimen. It also includes a ree auto-isotype con-trol eature (Fig. 8). With this method, there is no needto lyse the red cells as only uorescent cells areobserved by the optical system; hence there is a urtherreduction in reagents.

Primary CD4 Gating

By 1999, progress dictated a six-tube, 12 MAb proto-col or CD4 T-cell counting. Most guidelines recom-mended processing specimen within 6 h o phlebotomy(18). Sherman et al. introduced the concept o simpliy-ing CD4 T-cell counting (36). First, the primary CD4 gat-ing using a single-reagent, CD4 MAb, was developed(22,23). By combining a unction specifc marker CD4

with side scatter, CD4 T-cells could be resolved (Fig. 9).

F IG. 7. Flow Signal Detection and Cell Propulsion Methods. Thisfow chart illustrates some o the most promising evolutionary develop-ments in fow cytometry technology which may have impact on acceler-ating aordability o CD4 T-cell measurements in resource limitedregions. Fluorescence is no longer the only option available or immu-nophenotyping as indicated in Panel 7B. The use o nonfuorescenttagging method gives the option to eliminate cold-chain, rerigerationand light sensitivity issues. The other innovation with potentially signi-icant impact is the alternative cell propulsion technologies listed inboth 7A and 7B. The utility o acoustic ocusing to deliver aordableinstruments is unknown at this point. However there are two optionsavailable to replace hydrodynamic ocusing. Guava technologies hasintroduced l -capillary system and on Panel A. The sweep fow technol-ogy introduced by PointCare on Pane 7B also utilized less water com-pared to hydrodynamic ocusing.

F IG. 8. A Built-in Advantage with Heterogeneous Gating Technology.Using a lineage marker as part o a heterogeneous gating (SS/CD3)strategy has some additional benets. When implementing a T-gatingprotocol such as one used by Guava, the desired cluster of abeledT-cells pull away rom the cells that are not CD3 positive. The non- T-cell population serves as a built-in automatic isotype control. It isknown that it is unnecessary to add a reagent to CD4 T-cell immuno-phenotyping, however it is reely available. In some o the most hostilerural destinations this protocol provides an assurance that the reagentsand specimen survived the trip to the laboratory in good condition. In

the cartoon a strong separation between T-cell gate and other cells isvisible. Monocytes, B cells ands NK-cells exhibit some nonspecicbinding reaction as they are some distance rom the y axis, see thedark horizontal arrows. However this low level staining does not jeop-ardize the rapid identication o the positive T-cell population.

F IG. 9. Primary CD4 gating. The simplest immunophenotyping proto-col or CD4 T-cell counts utilizes a single CD4 antibody with hetero-geneous histogram: CD4 intensity (CD4 FL) on the x axis and sidescatter (SS) on y axis. This combination discriminates betweenstrongly CD41 lymphocytes T(h), CD4-negative lymphocytes (T(s), B,NK), monocytes (M) larger cells with lower fuorescent CD4 intensityand granulocytes (G). Primary CD4 gating works optimally in adultswhen volumetric fow cytometers directly count the numbers o CD4positive lymphocytes present in a unit volume o resh blood. The sidescatter (SS) is essential to resolve CD4 brightly fuorescing lympho-cytes rom the CD4 dimly fuorescing monocytes that are not alwaysmuch larger then a lymphocyte.



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This simple gating strategy was also introduced on thePartec CyFlow SL (37,38) and later on the Partec CyFlowCounter but here without side scatter (28). Morerecently, a second parameter was again added to theCyFlow Counter so it gates much the same way as theprimary CD4 gating protocol. In adults, the absoluteCD4 T-cell counts obtained by primary CD4 gating onvolumetric cytometers are generally reliable withoutusing additional reagents (22,23,37,38). However, thesemethods rely on clear identifcation and separation oCD4 T-cells rom monocyte population which in somesituations can be problematic unless both the CD4 andside scatter parameters are used.

CD45 Fluorescence-Based Panleucogating

Next, the pan-leuco- gating strategy was developedas a DPT or CD4 T-cell measurement using conventionalow cytometers in high throughput hospitals wherethere is a hematology analyzer near by. This simplifedprotocol designed to work on any ull-eatured clinical

ow cytometer (20), eliminated the need or countingbeads without compromising CD4 T-cell count quality(39,40) using only two MAbs, CD45 and CD4 (39). Theoriginal panleucogating method is a DPT where not onebut two o the three numeric elements required to gen-erate the absolute count (Fig. 1) are acquired with aow cytometer. The components generated by owcytometry on a heterogeneous histogram (SS/CD45) arethe lymphocyte dierential (LD) and CD4 T-lymphocytepercentage. The operator draws a large gate (Gate A)that includes all the CD45-tagged leucocytes (Fig. 10A).

The second gate (Gate B) also in Figure 10A includesonly the bright CD45 stained cells, the lymphocytes. It isthe content o Gate A that is sent to the second hetero-geneous histogram (SS/CD4) to determine the actualCD4 positive events (Fig. 10B). With this strategyonly the total WBC count is obtained rom a hematologyanalyzer. The CD4 T-cell absolute count is generated bymultiplying the two elements rom the ow cytometerwith the one rom the hematology analyzer. This strategyworks well on samples that have been drawn up to 35days earlier (39,41). A version o CD45-assisted gatingstrategy is now modifed or use on clinical ow cytome-ters with SPT and included in most international guide-lines published in the past 5 years (4144). The abovepanleucogating method was urther modifed by Glen-cross et al. to incorporate inexpensive counting beads(45). There is also a multilaboratory study reported roma North American trial in this issue (46).

AFFORDABLE CD4 T-CELL COUNTING REMAINS

AN ORPHAN TECHNOLOGICAL CHALLENGE

The blame or slow evolution cannot be placed juston myopic vision and greed associated with the health-care industry o resource-rich countries. Companies withpublicly traded shares must make proft and they are notin a position to become charitable organizations. Thereis, however, a general lack o vision and commitment on

a global political level. Jerey Sachs, a leading globaleconomist, published a book called The End o Pov-erty. He makes a compelling case that elimination oglobal poverty is an economic possibility in our time.

There was historical evidence that pipeting was themajor error source in absolute cell counting. This is whythe entire hematology industry switched to hands-ocap piercing technology several decades ago. Suchrestrictions were not imposed on the ow cytometryindustry when it became obvious that absolute CD4 T-cell counts were needed. Yet the cap piercing technol-

ogy eliminates all manual pipeting errors and signif-cantly reduces biohazard exposure associated with inec-tious specimens. Although it was recognized 20 yearsago that it was important to provide an accurate abso-lute count, until recently, only three companies haddeveloped an intrinsic volumetric solution (which alsokeeps this test more aordable). Most instruments tothis day require the addition o costly beads to eachspecimen. While the pharmaceutical industry was devel-oping powerul antiretroviral drugs, immunologist in theresearch laboratories gained considerable understandingregarding interaction between unctional, linage, andactivation markers. Cell-surace receptors such as theCD4 are unctional markers. CD4 positive cells can

belong to various lineages including the T-helper cellsubset and monocytes. Other groups developed betterunderstanding o how to preserve receptors or antigenicafnity while reezing cells and lyophilizing monoclonalantibodies. Powerul water-cooled lasers have given wayto air-cooled units with more modest but still signifcantenergy requirements. The latter development was ol-lowed by expensive but compact solid-phase laser mod-ules, and then very low power consuming and aordableLEDs. We have learned that the two most critical compo-

F IG. 10. Panleucogating Method. This robust method is based oninitial gating with CD45 antibody - in combinations with CD4 antibody.The frst step is to gate on all CD45 leucocytes (Gate A on Panel 10A),a large gate that includes all positive CD45 leucocytes including lym-pho-, mono- and granulocytes. The absolute counts or cells includedin Gate A are quantifed with a WBC count rom a haematology ana-lyser. Next, CD4 T-lymphocytes are counted among the leucocytes asshown in the smaller rectangular Gate B on Panel 10A. Finally, thebright CD45 lymphocytes Gate B are also identifed to calculate the

CD4 T-cells as percentage o ymphocytes as they are gated on Panel10B in the open square.

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This simple gating strategy was also introduced on thePartec CyFlow SL (37,38) and later on the Partec CyFlowCounter but here without side scatter (28). Morerecently, a second parameter was again added to theCyFlow Counter so it gates much the same way as theprimary CD4 gating protocol. In adults, the absoluteCD4 T-cell counts obtained by primary CD4 gating onvolumetric cytometers are generally reliable withoutusing additional reagents (22,23,37,38). However, thesemethods rely on clear identifcation and separation oCD4 T-cells rom monocyte population which in somesituations can be problematic unless both the CD4 andside scatter parameters are used.

CD45 Fluorescence-Based Panleucogating

Next, the pan-leuco- gating strategy was developedas a DPT or CD4 T-cell measurement using conventionalow cytometers in high throughput hospitals wherethere is a hematology analyzer near by. This simplifedprotocol designed to work on any ull-eatured clinical

ow cytometer (20), eliminated the need or countingbeads without compromising CD4 T-cell count quality(39,40) using only two MAbs, CD45 and CD4 (39). Theoriginal panleucogating method is a DPT where not onebut two o the three numeric elements required to gen-erate the absolute count (Fig. 1) are acquired with aow cytometer. The components generated by owcytometry on a heterogeneous histogram (SS/CD45) arethe lymphocyte dierential (LD) and CD4 T-lymphocytepercentage. The operator draws a large gate (Gate A)that includes all the CD45-tagged leucocytes (Fig. 10A).

The second gate (Gate B) also in Figure 10A includesonly the bright CD45 stained cells, the lymphocytes. It isthe content o Gate A that is sent to the second hetero-geneous histogram (SS/CD4) to determine the actualCD4 positive events (Fig. 10B). With this strategyonly the total WBC count is obtained rom a hematologyanalyzer. The CD4 T-cell absolute count is generated bymultiplying the two elements rom the ow cytometerwith the one rom the hematology analyzer. This strategyworks well on samples that have been drawn up to 35days earlier (39,41). A version o CD45-assisted gatingstrategy is now modifed or use on clinical ow cytome-ters with SPT and included in most international guide-lines published in the past 5 years (4144). The abovepanleucogating method was urther modifed by Glen-cross et al. to incorporate inexpensive counting beads(45). There is also a multilaboratory study reported roma North American trial in this issue (46).

AFFORDABLE CD4 T-CELL COUNTING REMAINS

AN ORPHAN TECHNOLOGICAL CHALLENGE

The blame or slow evolution cannot be placed juston myopic vision and greed associated with the health-care industry o resource-rich countries. Companies withpublicly traded shares must make proft and they are notin a position to become charitable organizations. Thereis, however, a general lack o vision and commitment on

a global political level. Jerey Sachs, a leading globaleconomist, published a book called The End o Pov-erty. He makes a compelling case that elimination oglobal poverty is an economic possibility in our time.

There was historical evidence that pipeting was themajor error source in absolute cell counting. This is whythe entire hematology industry switched to hands-ocap piercing technology several decades ago. Suchrestrictions were not imposed on the ow cytometryindustry when it became obvious that absolute CD4 T-cell counts were needed. Yet the cap piercing technol-

ogy eliminates all manual pipeting errors and signif-cantly reduces biohazard exposure associated with inec-tious specimens. Although it was recognized 20 yearsago that it was important to provide an accurate abso-lute count, until recently, only three companies haddeveloped an intrinsic volumetric solution (which alsokeeps this test more aordable). Most instruments tothis day require the addition o costly beads to eachspecimen. While the pharmaceutical industry was devel-oping powerul antiretroviral drugs, immunologist in theresearch laboratories gained considerable understandingregarding interaction between unctional, linage, andactivation markers. Cell-surace receptors such as theCD4 are unctional markers. CD4 positive cells can

belong to various lineages including the T-helper cellsubset and monocytes. Other groups developed betterunderstanding o how to preserve receptors or antigenicafnity while reezing cells and lyophilizing monoclonalantibodies. Powerul water-cooled lasers have given wayto air-cooled units with more modest but still signifcantenergy requirements. The latter development was ol-lowed by expensive but compact solid-phase laser mod-ules, and then very low power consuming and aordableLEDs. We have learned that the two most critical compo-

F IG. 10. Panleucogating Method. This robust method is based oninitial gating with CD45 antibody - in combinations with CD4 antibody.The frst step is to gate on all CD45 leucocytes (Gate A on Panel 10A),a large gate that includes all positive CD45 leucocytes including lym-pho-, mono- and granulocytes. The absolute counts or cells includedin Gate A are quantifed with a WBC count rom a haematology ana-lyser. Next, CD4 T-lymphocytes are counted among the leucocytes asshown in the smaller rectangular Gate B on Panel 10A. Finally, thebright CD45 lymphocytes Gate B are also identifed to calculate the

CD4 T-cells as percentage o ymphocytes as they are gated on Panel10B in the open square.

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ment. The accuracy o the projected test volume permonth will have a signifcant impact and probably willbe a key actor to guide decision making. See Table 1 orthe breakdown o eatures listed or various aordablesystems. When looking at long-term cost, an examplerom each o the three categories of ssues can beselected or illustrative purposes. One can enquire about

the savings that are associated with reagents that have ayear long shel-lie without any rerigeration or cold-chain transportation requirements. Similarly, in the caseo cost per test, the saving can be assessed i the systemrequires only one or two MAbs but without any addi-tional demand or lysing solutions or or ow countingbeads; in such a case, the per-test costs over the yearswill be dramatically lower. A fnal example is rom thelabor cost containment and quality management section.In a case where the manuacturer might be able to pro-

vide a suitable rugged transport container or a robustinstrument in order to regularly circulate between threeor our villages on a weekly basis, the amortization costo the system drops to below 30% per village. Many spe-cifc eatures o service (Table 1), needs to be confrmedwith local suppliers, keeping in mind that services maychange with time. Fluctuation in price is also likely to

occur or the prices of nstruments and reagents; there-ore, they are not included in this comparison.

ISSUES RELATED TO TECHNOLOGY TRANSFER AND

SUSTAINABLE QUALITY LABORATORY MANAGEMENT

Biosaety issues are oten neglected in resource limitedlocations. They must be considered when establishinglaboratory space, equipment lists and operating budgets.Laboratory managers can be held legally responsibleor not implementing, accommodating, and enorcing

Table 1Guide for Selecting an Appropriate CD4 1 T-cell Counting System a

CategoriesApogee

A40FACSCount

BDISGuava

PCAPartec

CyFlow C.PointCare

NOWSysmex

Poch-100i

Environmental energy issues (E)E-1 Use of ow energy laser or LED 1 1E-2 Low water consumption 111

E-3 No cold-chain & or need or rerigeration 1E-4 Impervious to day-light exposure 1 1E-5 Nominal waste collection 11E-6 Protection against power fuctuationsE-7 Operates without ACE-8 Eliminated stand-alone computer 1 1E-9 Battery/solar power ree or an option 1 1

Cost issues (C)C-1 Minimal antibody numbers used in small volumes 111C-2 Simultaneous CD4 absolute and CD4% 1 1 1 1C-3 Method without lysing reagent 1 1C-4 Method without counting beads 11111C-5 Patient/control data space onboard 1 1 1C-6 Minimal daily quality control cost 111C-7 Cost/availability o preventive/service contract 1 1 1 1 1C-8 Internet/satellite/GMS compatibility 1C-9 Compatibility with EQAP (NEQAS,QASI) 1 1 1 1

C-10 Maximum daily sample throughputLabor-related issues (L)L-1 Elimination o manual gating 11L-2 Manual pipetting is avoided 1L-3 Onboard automated data management 1L-4 Availability o continuous-trainingL-5 Portability: is transport case available 111L-6 Small bench-top oot-print 111L-7 Barcode reader is available or an option 1L-8 High skill-level or operator is eliminated 1L-9: Additional capacity o hematology 1 1

a This supplement to Clinical Cytometry includes publications which describe how to run conventional clinical fow cytometersthat have originally been designed or afuent countries in challenging economical settings (45,55). Both the protocols o primaryCD4 gating (Fig. 9) and CD45/CD4 gating (Fig. 10) are well suited to run on 4-colour fow cytometers marketed by dierent compa-nies [45,55,67]. In this Table, however, only the instruments that are potentially capable o running with inerior inrastructure inrural or semi-rural conditions are included. The criteria shown in this Table are complementary to the other parameters such as thecost o the instrument, precision, accuracy and reproducibility, critical issues that are oten available rom observations by External

Quality Assessment protocols. Importantly, the cost o the instrument may not be the most signicant actor when all other issuesactored into the total package. A prudent manager will study the operational budget adjusted or the specic requirements o his/her laboratory as well as the costs per tests with the three areas o cost containment: (i) environmental and energy managementissues (E- issues in Part 1 above), (ii) options available or containment o cost per test (C-issues in Part 2 above), and (iii)labor cost containment (L-issues in Part 3 above). The projected test volume per week/month/year will help to guide decisionmaking about sustainable service costs and will serve well or negotiations. The plus signs assignments are incomplete as not alldata has been available at the time this table was generated.

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proper saety procedures in the laboratory. In mostresource-rich countries, external quality assessment pro-grams (EQAP) or CD4 T-cell enumeration are mandatoryand are in place (5154). These protocols involve ship-ping stressed challenge specimens at regular intervals.Internationals agencies such as WHO, Global AIDS Pro-gram (GAP), the Clinton Foundation, among others, havelearned that technology transer workshops can acceler-ate the efective installation and acceptance o externalquality assessment programs (EQAP) or resource poorregions. These topics are discussed in more detail byGlencross in this issue (55).

CONCLUSIONS

During the past 5 years, ow-based cellular tests haveimproved and are more robust but they are still too ex-pensive. Kestens et al. validated a primary CD4 gatingprotocol in Cambodia (56). Looking orward, there aretwo major strategies that may bring about sustainabilityand afordability where it is needed the most. First, morehybrid instruments might be introduced either as point

o care or at regional laboratory level that will loweroverall cost or a large variety o assays beyond CD4

T-cell enumeration. A new truly portable and afordablededicated user-riendly ow cytometer such as theCytometry For Lie (C4L) could take care o most o theART related CD4 monitoring needs o rural nursing sta-tions. Flow cytometers will also have to deliver, in theuture, versatility through efective implementation oexible sotware-driven hybrid instrument platorms.

They must be capable o a wide range multiplexed assaypanels covering both ELISA-type bead- and cell-basedassays (3134,57) including, or example, antiretroviraldrug toxicity assays. The multiplexing platorm approacho the uture can promote the one-stop-shopping con-

cept or the divergent serological, immunological, hema-tological test market. With the new generation o owcytometry platorms that show considerable versatility,alternative approaches to PCR amplied plasma viralRNA as a surrogate VL tests could be introduced. Cell-associated markers such as CD38 expression levels onCD8 T-cells (58,59) have also been proposed as anafordable alternative or monitoring ART in HIV-inectedpatients. Cell-associated viral RNA (60,61) and morerecently, related ow-cytometric changes in monocytes(62) are being studied as candidate assays or viral reser-voir monitoring by ow cytometry. Isothermal amplica-tion o viral RNA combined with bead-based ow detec-tion assays (63) may also be exploited in the uture as

an afordable and simpler alternative to PCR ampliednucleic acid assays. HyFAP will probably be in demandparticularly in resource poor regions because o themore economical operation and servicing o a limitednumber o aboratory instruments where the objective isto provide a wide spectrum o clinical assays, perormedautomatically using a single platorm. Assay combina-tions are user selected as required. It is a bold cost efec-tive approach to consolidate clinical laboratory testingrequirements with relatively low volume in remote loca-

tions with stable patient populations. By contrast, inresource rich countries the multiunctional HyFAP canonly be efectively considered at the time when the vari-ous dedicated instruments may become obsolete. Conse-quently, a situation might arise where the Northernhemisphere will ollow the example o the Southernhemisphere much the same way as North America iscatching up to 82% o the world with the switch oGlobal System or Mobile (GSM) phone communicationsystem. A question was posed in the introduction,whether a ow or nonow cytometry system will takethe lead to reach the badly neglected rural market inresource poor regions. At rural decentralized zonesafordable technologies with minimal moving parts andno requirement or air-conditioning or sheath uid,remain an almost irresistible option or counting CD4 T-cells. Greve, Tibbe, and Li, rom the University o

Twente, built several portable compact image analyzercytometers that have not yet reached the market (64).Shapiro is working on an image analyzer where CD4 T-cells are recorded by a digital camera much like the light

is captured by digital imaging telescopes rom stars. Thepixels ofthe uorescing cells are analyzed with a pro-cess he reers to as cellular astronomy (65). There isalso a commercial instrument that has been in the pipe-line or 6 years using similar concepts, which has yet toreach the market. Cheng et al. are developing a microui-dic nonow device that does not require labeling o theCD4 antibody, however at this point it is not a robusttechnology (66). Based on what is currently available andhow little success nonow instrumentation has had soar, it is more likely that the ow-based technology willbe the rst to make signicant impact on the rural mar-kets o Arica and Asiabut the proponents o owcytometry may not have the last word, say, in another 5

years.

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p-0025 a affordable cd4 tcell enumeration for resource limited regions a status report for 2008

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