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Laboratory Guidelines for Enumerating CD4 T Lymphocytes in the Context of HIV/AIDS SEA-HLM-392 Distribution: Limited

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Laboratory Guidelines for Enumerating CD4 T Lymphocytes in the Context of HIV/AIDS SEA-HLM-392 Distribution: Limited Laboratory Guidelines for enumerating CD4 T Lymphocytes in the context of HIV/AIDS SEA-HLM-392 Distribution: Limited Laboratory Guidelines for enumerating CD4 T Lymphocytes in the context of HIV/AIDS Regional Office for South-East Asia New Delhi, June 2007 WHO Library Cataloguing-in-Publication Data World Health Organization, Regional Office for South-East Asia. Laboratory guidelines for enumerating CD4 T lymphocytes in the context of HIV/AIDS 1. T lymphocytes – methods 2. CD4-positive T lymphocytes – immunology 3. HIV infections – diagnosis 4. Quality assurance, health care 5. Laboratory techniques and procedures – methods 6. Flow cytometry – methods 7. Guidelines. ISBN 978-92-9022-298-9 (NLM classification: WH 200) © World Health Organization 2007 This document is not issued to the general public, and all rights are reserved by the World Health Organization (WHO). The document may not be reviewed, abstracted, quoted, reproduced or translated, in part or in whole, without the prior written permission of WHO. No part of this document may be stored in a retrieval system or transmitted in any form or by any means – electronic, mechanical or other – without the prior written permission of WHO. The mention of specific companies or of certain manufacturers’ products does not imply that they are endorsed or recommended by the World Health Organization in preference to others of a similar nature that are not mentioned. Errors and omissions excepted, the names of proprietary products are distinguished by initial capital letters. The views expressed in documents by named authors are solely the responsibility of those authors. Contents Page 1. Natural history of HIV infection ........................................................................... 1 1.1 Magnitude of problem.............................................................................................. 1 1.2 Biology of HIV.......................................................................................................... 2 1.3 Pathogenesis of HIV ................................................................................................. 5 1.4 Factors influencing HIV disease progression.............................................................. 8 1.5 WHO clinical staging of HIV-related disease in adults and adolescents aged 15 years or more.............................................................................................. 9 2. Role of CD4 T lymphocytes in disease progression .......................................... 11 2.1 CD4 T lymphocytes and HIV.................................................................................. 11 2.2 CD4 T lymphocyte counts and antiretroviral therapy.............................................. 12 2.3 CD4 T lymphocytes counts as indicator of treatment failure ................................... 14 2.4 CD4 T lymphocytes counts in paediatric population............................................... 16 3. Principles of flow cytometry ............................................................................... 18 3.1 Introduction ........................................................................................................... 18 3.2 What is flow cytometry? ......................................................................................... 18 3.3 Instrument description............................................................................................ 19 3.4 Use of fluorochromes in flow cytometry: ................................................................ 22 4. Methods of CD4 T lymphocytes count measurement ...................................... 24 4.1 Introduction ........................................................................................................... 24 4.2 Flow cytometric methodologies .............................................................................. 24 4.3 Selections of Methodology for CD4 T lymphocytes count estimation..................... 32 4.4 Importance of Reference ranges in interpretation of the CD4 T lymphocytes counts obtained by in the laboratory.................................................. 33 5. Alternate methods for CD4 T lymphocytes estimation..................................... 37 5.1 Introduction ........................................................................................................... 37 5.2 Manual methods .................................................................................................... 37 5.3 Methodologies likely to become available in the near future................................... 38 5.4 Non-CD4 markers for monitoring HIV disease progression and for initiation and monitoring the success of anti-retroviral therapy ......................... 39 6. Quality Management in CD4 T lymphocytes enumeration ............................ 42 6.1 Management Requirements.................................................................................... 42 6.2 Technical requirements .......................................................................................... 44 Page iii 7. Establishing a laboratory for CD4 T lymphocytes enumeration ....................... 48 7.1 Suggested requirements at different levels .............................................................. 48 7.2 Selection of Methodology for CD4 T lymphocytes counts....................................... 49 7.3 Suggested testing at different levels......................................................................... 49 7.4 Establishment of a CD4 T lymphocytes enumeration laboratory............................. 50 Annexes 1. Collection and transport of specimens............................................................... 52 2. General bio-safety guidelines for immunophenotyping laboratory.................. 55 3. Pipetting techniques............................................................................................ 57 4. Format for sample transport, receipt and report form ..................................... 59 5. Useful references................................................................................................. 61 6. List of contributors............................................................................................... 62 Chapter 1 Natural history of HIV infection 1.1 Magnitude of problem The HIV/AIDS pandemic has severely affected health development and eroded improvements in life expectancy, particularly in countries with the highest prevalence of infection. Since the first cases of acquired immunodeficiency syndrome (AIDS) were reported in 1981, infection with human immunodeficiency virus (HIV) has grown to pandemic proportions, resulting in more than 65 million infections and 25 million deaths. At the end of 2006, an estimated 39.5 million people were living with HIV, with Sub-Saharan Africa carrying the highest burden. In 2006, an estimated 4.3 million people became newly infected with HIV. More than 95% of these new infections are in low and middle income countries. Each day 11,000 persons become newly infected with the virus; of these, half are women and 40% are young people (15-24 years old). Of the estimated 37 million adults living with HIV worldwide, nearly 18 million are women. At the end of 2006, there were an estimated 2.3 million children living with HIV. The increasing number of child deaths due to AIDS threatens to reverse many of the recent gains of child survival programmes. Moreover, the socio-economic impact of HIV/AIDS on children is profound. As their parents fall sick and die of AIDS, children undergo a long trail of painful experiences such as: economic hardship, withdrawal from school; lack of love, attention and affection; psychological distress, stigma, discrimination and isolation, and malnutrition and illness. Cumulatively, there are a total of 16 million children worldwide who have become orphans because their parents died of AIDS. HIV burden in South-East Asia Region The WHO South-East Asia (SEA) Region includes: Bangladesh, Bhutan, the Democratic People’s Republic of Korea, India, Indonesia, Maldives, Myanmar, Nepal, Sri Lanka, Thailand, and Timor-Leste. The overall adult HIV prevalence in south and south-east Asia is 0.7%, relatively much lower Page 1 Laboratory guidelines for enumerating CD4 T lymphocytes in the context of HIV/AIDS than the 5.7% prevalence in sub-Saharan Africa. However, due to the large populations, even low HIV prevalence means that large numbers of people are living with HIV. At the end of 2006, there were an estimated 7.2 million people living with HIV in SEA Region. This included 0.77 million new infections in 2006. Approximately 550,000 persons died of AIDS during 2006. Five countries - India, Thailand, Myanmar, Indonesia and Nepal account for a majority of the burden in this Region. Long-standing epidemics have left a huge burden of people living with HIV/AIDS (PLHA) who need prevention, care and treatment services. Table 1.1 provides the estimated number of PLHA in each country in the Region. Table 1.1: HIV/AIDS Burden in South-East Asian countries, December 2005 Adult HIV Estimated number of people Country Prevalence (%) Living with HIV Bangladesh <0.1 7 500 Bhutan <0.1 < 500 DPR Korea n/a n/a Indonesia 0.2 193 000* India 0.9 5 200 000** Maldives <0.1 <100 Myanmar 1.3 339 000** Nepal 0.5 70 000 Thailand 1.4 541 000 Sri Lanka <0.1 5 000 Timor-Leste <0.1 <100 *data for 2006 ** among persons aged 15-49 years Source: Country reports, National AIDS Programmes, Ministries of Health 1.2 Biology of HIV HIV is a lentivirus
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