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RESEARCH: Calman A MacLennan, Michael K P Liu, Sarah A White, Joep J G van Oosterhout, Felanji Simukonda, Joseph Bwanali, Michael J Moore, Eduard E Zijlstra, Mark T Drayson, and Malcolm E Molyneux Diagnostic accuracy and clinical utility of a simplified low cost method of counting CD4 cells with flow cytometry in Malawi: diagnostic accuracy study BMJ 2007; 335: 190 [Abstract] [Full text]

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CD4 counts and HIV therapy: flow cytometry is not the answer

Michel Erlewyn-Lajeunesse   (30 July 2007)

Consolidation of affordable flow cytometric PanLeucogating testing protocols to ensure sustainable reliable CD4 monitoring in Southern Africa.

Deborah K Glencross, George Janossy and Wendy S. Stevens   (1 August 2007)

Use of fixed, propidium-iodide -stained cells, produced in house, to further reduce the cost of the Blantyre count for CD4+ cells

Paul E Williams, Graham Read   (8 September 2007)

CD4 counts and HIV therapy: flow cytometry is not the answer 30 July 2007
Michel Erlewyn-Lajeunesse, Locum Consultant Paediatrician Bristol Royal Hospital for Children Send response to journal: Re: CD4 counts and HIV therapy: flow cytometry is not the answer	Dr MacLennan and colleagues are to be commended for seeking an affordable and low-tech’ method for estimating CD4 counts in HIV infected individuals.1 Unfortunately their reliance on flow cytometry, even in its most basic form, will severely restrict the suitability of this technique for general use in resource poor settings. We should not remain fixed on flow cytometry as the only way to estimate severe immuno-suppression in HIV infected individuals. We no longer require an accurate CD4 count, but rather the ability to detect those individuals who are asymptomatic but immuno-suppressed and in need of antiretroviral therapy. Standard laboratory methods are available for this purpose. Flow cytometry is not the only way to estimate CD4 count. Quantative ELISA and Dynabeads can estimate CD4 cell numbers.2 These technologies do not require expensive equipment other than would be standard for most diagnostic laboratories. Whilst the accuracy of these tests raise difficulties of their own, their potential is vast and they should not be dismissed.3 One option could be to use ELISA to calculate a CD4/CD8 ratio. ELISA based CD4/CD8 ratios may just be good enough to predict when to start antiretroviral therapy. The CD4 count by flow cytometry is impractical as a standard method for Africa. We already have the technology for an affordable method, and should reassess quantative techniques in the light of the specific clinical questions posed by our current therapy. References 1. Maclennan CA, Liu MK, White SA, van Oosterhout JJ, Simukonda F, Bwanali J et al. Diagnostic accuracy and clinical utility of a simplified low cost method of counting CD4 cells with flow cytometry in Malawi: diagnostic accuracy study. BMJ 2007;335:190. 2. Idigbe EO, Audu RA, Oparaugo CT, Onwujekwe D, Onubogu CC, Adedoyin J et al. Comparison of Dynabeads and Capcellia methods with FACScount for the estimation of CD4 T-lymphocyte levels in HIV/AIDS patients in Lagos, Nigeria. East Afr.Med.J. 2006;83:105-11. 3. Mwaba P, Cassol S, Nunn A, Pilon R, Chintu C, Janes M et al. Whole blood versus plasma spots for measurement of HIV-1 viral load in HIV -infected African patients. Lancet 2003;362:2067-8. Competing interests: None declared
Consolidation of affordable flow cytometric PanLeucogating testing protocols to ensure sustainable reliable CD4 monitoring in Southern Africa. 1 August 2007
Deborah K Glencross, Principal Pathologist Johannesburg South Africa, George Janossy and Wendy S. Stevens Send response to journal: Re: Consolidation of affordable flow cytometric PanLeucogating testing protocols to ensure sustainable reliable CD4 monitoring in Southern Africa.	To the Editor, BMJ MacLennan et al. (1) are correct in their efforts to raise awareness for affordable CD4 testing in Malawi. They are also to be commended for confronting the wave of misguided patronising calls from well-meaning ‘first-world’ foundations for developing “Holy Grail” low-tech CD4 methods on the basis of a misbelief that African laboratory facilities and professionals are incapable of mastering technologically advanced CD4 testing techniques. The “Blantyre” method (1) as described is however de facto an embodiment of the affordable simplified and highly reliable PanLeucogated (2) CD4 (PLG-CD4) single platform methodology that is currently in use across 52 National Health Laboratory Service (NHLS) laboratories (3) in South Africa (SA). PLG-CD4 (2) is a rational affordable simplification of the international CD4 guidelines (4,5) using the same two antibodies CD45 and CD4 featured in the ‘Blantyre’ method. PLG-CD4 has been widely validated (2,6-13), including in NIH-sponsored US studies (11) where reproducibility of PLG against conventional CD4 guideline methods was improved by 25% and 33%, within- and between- laboratories respectively. PLG-CD4 testing is used to support the Namibian HIV/AIDS programme and in laboratories in Malawi, Mozambique, Angola, Botswana, Swaziland, Zambia (14), the Caribbean (8) and in Thailand (9,11). MacLennan et al (1) wondered whether their method could be “successfully implemented in a resource poor setting”. The answer lies in recognition of a significant SA milestone in the near-decade long fight (15, 16) for the development of affordable CD4 methodologies. The validation (5-12) and subsequent implementation (13) of PLG-CD4 as an affordable and precise, accessible and robust CD4 testing system in the SA National treatment programme in 2004 demonstrates that implementation of simple cheaper flow cytometric CD4 methods is possible. Recommended by the SA National Treasury and Department of Health (17) and through process of tender with well-timed intervention by the Clinton Foundation (18), truly affordable, reliable and precise PLG-CD4 was secured for SA –, with all inclusive sustainable pricing from placement and maintenance of flow cytometers, to supply of all controls including sample preparation and antibody reagents. These are crucial components for a sustainable country- wide network, driven by high volumes of tests within a coordinated national network - currently, the NHLS processes in excess of 250 000 PLG- CD4 tests per month. Further, in answer to the question posed (1) the NHLS has been consistent in supplying accurate CD4% and absolute CD4 counts (13) from laboratories with no initial flow cytometry skills. Certainly, SA provides evidence for the “political will” that the BMJ editorial (19) whole-heartedly welcomes: the necessary investment in quality control and training can, and does, make all the difference (13,14,20,21). PLG-CD4’s sustained success in SA is based on monitored novel simple pipetting quality control (20), regional external quality assessment support (CD4 AF-REQAS/WHO; ref.13,14) and laboratory certification (22), all performed nationally in a coordinated fashion. Training (21) is also considered crucial to consolidate skills (as pointed out by Madhivanan and Krupp, there is no dearth of local basic laboratory skills – ref.19). The large-scale/high-volumes of CD4 testing required to support a consolidated national programme offsets the capital and reagent costs (1,19) . The question still however remains whether the “Blantyre” method will be sustainable or not (1). Without a coordinated national Malawian effort, the fledgling “Blantyre” technology will unfortunately, probably not survive without the driving energy of the Calman MacLennan. Indeed, it is sad to already hear directly from the author of such concerns, even before the methodology made its public debut in the literature (1) (personal correspondence of CM with DKG). The spirit of collaboration between Malawi and SA (CM and DKG) and the bleak outlook for the “Blantyre” initiative should be an example that solutions for reliable and affordable CD4 testing lie in a “collaborating Africa” – the time has come for true cooperation and networking amongst African CD4 laboratory professionals. African leaders have taken the first step by securing the New Partnership for African Development (NEPAD) (23). The stage has therefore been set for the African medical fraternity to embrace this endeavour, take charge and deliver the continent from “crisis management to sustained response” (24). Only such action can make the ideals of the latest WHO initiative “Towards Universal access by 2010” a reality (25). References: 1.MacLennan CA, Liu MK, White SA, van Oosterhout JJ, Simukonda F, Bwanali J, Moore MJ, Zijlstra EE, Drayson MT, Molyneux ME. Diagnostic accuracy and clinical utility of a simplified low cost method of counting CD4 cells with flow cytometry in Malawi: diagnostic accuracy study. BMJ. 2007 Jul 28;335(7612):190. Epub 2007 Jul 17. 2.Glencross D, Scott LE, Jani IV, Barnett D, Janossy G. CD45-assisted PanLeucogating for accurate, cost-effective dual-platform CD4+ T-cell enumeration. Cytometry 2002;50(2):69-77. 3.http://www.nhls.ac.za (accessed 27/07/2007). 4.Mandy FF, Nicholson JK, McDougal JS. Guidelines for performing single-platform absolute CD4+ T-cell determinations with CD45 gating for persons infected with human immunodeficiency virus. Centers for Disease Control and Prevention. MMWR Recomm Rep 2003;52:1-13. 5.Barnett D, Bird G, Hodges E, Linch DC, Matutes E, Newland and Reilly JT. Guidelines for the enumeration of CD4+ lymphocytes in Immunosuppressed Individuals. Clin Lab Haem. 1997; 19:231-41. 6.Janossy G, Jani IV, Bradley NJ, Bikoue A, Pitfield T, Glencross DK. Affordable CD4(+)-T-cell counting by flow cytometry: CD45 gating for volumetric analysis. Clin Diagn Lab Immunol 2002;9(5):1085-94 7.Storie I, Sawle A, Whitby L, Goodfellow K, Granger V, Reilly JT, Barnett D. Flow rate calibration II: a clinical evaluation study using PanLeucogating as a single-platform protocol. Cytometry B Clin Cytom 2003;55(1):8-13 8.Sippy N, Marshall and Abayomi A. Comparison of the PanLeucogating technique with four-colour heterogenous gating for CD4+ T cell enumeration in HIV infected individuals in Barbados. Abstract no. MoPeB3108. 2004 XV Int Conf AIDS, Bangkok. 9.Pattanapanyasat K, Shain H, Noulsri E, Lerdwana S, Thepthai C, Prasertsilpa V, Likanonsakul S, Yothipitak P, Nookhai S, Eksaengsri A. A multi-center evaluation of the PanLeucogating method and the use of generic monoclonal antibody reagents for CD4 enumeration in HIV-infected patients in Thailand. Cytometry B Clin Cytom 2005;65(1):29-36. 10.Ceffa S, Erba F, Assane M, Coelho E, Calgaro M, Brando B. Panleucogating as an accurate and affordable flow cytometric protocol to analyse lymphocyte subsets among HIV-positive patients on HAART treatment in Mozambique. J Biol Regul Homeost Agents 2005;19(3-4):169-75. 11.Pattanapanyasat K, Shain H, Prasertsilpa V, Noulsri E, Lerdwana S, Eksaengsri A. Low cost CD4 enumeration using generic monoclonal antibody reagents and a two-color user-defined MultiSETtrade mark protocol. Cytometry B Clin Cytom. 2006 70:355–60. 12.Denny T, Gelman R, Bergeron M, Forman M, Landay A, Louzao R, Mandy F, Schmitz J, Wilkening C and Glencross DK. A multi-lab study of CD4 counts using flow cytometric PanLeukogating (PLG): a NIAID-DAIDS immunology quality assessment program study. Abstract number 670, 2006 Conf Retrovirus and Opportunistic Inf (CROI). 13.Glencross DK, Aggett HM, Stevens WS, Vercauteren G, Bergeron M, Houle G, and Mandy F. XVI Improved between-laboratory performance of South African NHLS laboratories using PLG CD4 methodology for the National Anti- retroviral treatment programme. Abstract MOPE0105. 2006 XVI Int Conf AIDS, Toronto, Canada. 14.Vercauteren G, Aggett HM, Mandy F and Glencross DK. Impact of the World Health Organisation Regional External Quality Assessment programme (REQAS) on the laboratory performance for CD4 enumeration for monitoring of ARV therapy in Resource limited settings. Abstract No MOPE0138.2006 XVI Int Conf AIDS, Toronto, Canada. 15.Sherman GG, Galpin JS, Patel JM, Mendelow BV, Glencross DK. CD4+ T cell enumeration in HIV infection with limited resources. J Immunol Methods 1999;222(1-2):209-17. 16.www.AFFORDCD4.com (accessed 27/07/2007) 17.http://www.info.gov.za/issues/hiv/documents.htm - p92 (accessed 27/07/2007) Report of the Joint Health and Treasury task team on treatment option to enhance comprehensive treatment for HIV/AIDS in the Public Sector, South Africa. 8th August 2003. 18.Clinton Foundation: http://www.clintonfoundation.org/cf-pgm-hs-ai- work3.htm (accessed 27/07/2007. 19.Madhivanan P and Krupp K. Technological challenges in diagnosis and management of HOV infection in resource limited settings. BMJ. 2007 Jul 28;335(7612):165-66. 20.http://www/PLGCD4.net (accessed 27/07/2007). 21.Scott LE, Glencross DK. Monitoring reproducibility of single analysis, single platform CD4 cell counts in a high volume, low resource laboratory setting using sequential bead count rates. Cytometry B Clin Cytom 2005;67(1):31-2. 22.http://www.sanas.co.za (accessed 27/07/2007. 23.NEPAD http://www.nepad.org/2005/files/home.php (accessed 27/07/2007). 24.Piot P. AIDS: from crisis management to sustained strategic response. Lancet 2006;368:526-531. 25.http://www.who.int/hiv/pub/advocacy/universalaccess/en/index.html (accessed 27/07/2007. Competing interests: The NHLS of SA (employer of DKG and WSS) is the sole owner of patent rights for PLG CD4. Licensing of PLG-CD4 by the NHLS to Beckman Coulter was undertaken with the proviso of affordable distribution and technical support in resource-poor countries. PLG CD4 was “CE” marked in 2004 and US FDA 510(k) approved in 2005.
Use of fixed, propidium-iodide -stained cells, produced in house, to further reduce the cost of the Blantyre count for CD4+ cells 8 September 2007
Paul E Williams, Consultant Clinical Immunologist University Hospital of Wales, CF14 4XW, Graham Read Send response to journal: Re: Use of fixed, propidium-iodide -stained cells, produced in house, to further reduce the cost of the Blantyre count for CD4+ cells	We read with great interest the recent paper from Maclennan et al. (1) that described a flow cytometry procedure (Blantyre count) for enumerating CD4+ cells that uses minimised volumes of blood and reagents to reduce the cost and make the test affordable for patients in Africa with HIV. In the full version of the text published on bmj.com on 17th July (2) Table 1 gives the reagent costs in US$ for both the absolute count and percentage CD4 counts: CD4-PE antibody CD45_FITC antibody Red cell lysing solution Fluorescent beads Total 0.056 0.042 0.014 0.329 0.44 The fluorescent beads (CytoCount fluorescent microbeads; DAKO, Denmark) comprise almost three quarters (74.8%) of the reagent costs. We have previously described (3) the development of beads that may be prepared in-house by fixing and staining human leukocytes. Such beads have been shown to perform in a highly satisfactory manner in the routine enumeration of CD3, CD4 and CD8 cells at a reagent cost of less than $0.02 per assay. This material has been patented for use in Europe and North America but we did not seek patent protection in Africa, Asia or any Third World country to enable its use in such countries. We estimate that the cost of the Blantyre count could be reduced by 70% (from $0.44 to $0.132) if they were to produce their own counting beads using the method we described. We would be pleased to provide material to the Blantyre group for them to evaluate under local conditions if requested. References: 1. MacLennan CA, Liu MK, White SA, van Oosterhout JJ, Simukonda F, Bwanali J, Moore MJ, Zjilstra EE, Drayson MT, Molyneux ME. Diagnostic accuracy and clinical utility of a simplified low cost method of counting CD4 cells with flow cytometry in Malawi: diagnostic accuracy study. BMJ. 2007 ; 335: 190-4. 2. ibid. BMJ.2007 July 28; 335(7612):190. Epub 2007 Jul 17. 3. Harrison GM, Bennett AJ, Moody M, Read GF, Williams PE. Use of formalin-fixed, propidium-iodide-stained human leukocytes as a standard for enumerating CD4+ T lymphocytes in a single-platform assay. Clinical & Diagnostic Laboratory Immunology 2001; 8(2): 397-401. Competing Interests: PEW and the Cardiff and Vale NHS Trust own the patent rights of the above material (‘CellBeads’) in Europe and North America. Yours faithfully Dr PE Williams DM FRCP FRCPath Consultant Clinical Immunologist Dr G F Read Principal Clinical Scientist Competing interests: None declared