Laboratory guidelines for enumeration CD4 T lymphocytes in the context of HIV AIDS (revised version 2009)
Re
se
vi
d
r
Ve
on
si
20
09
The human immunodeficiency virus selectively invades a
subpopulation of lymphocytes (CD4 T lymphocytes). The progressive
loss of CD4 T lymphocytes eventually results in the loss of an ability to
mount desirable immune response to any pathogen and results in
vulnerability to opportunistic pathogens characteristic of AIDS. The
estimation of peripheral CD4 T lymphocyte counts is used as a tool to
take an informed decision for initiation of antiretroviral treatment,
monitoring disease progression and the effectiveness of antiretroviral
treatment. Several technologies for determining the number of CD4 T
lymphocytes are now available. The technologies are either flow
cytometric or non-flow cytometric. This document describes the
technologies as well as the infrastructure required for these in
developing countries.
World Health House
Indraprastha Estate,
Mahatma Gandhi Marg,
New Delhi-110002, India
Website: www.searo.who.int
SEA-HLM-392
Laboratory guidelines
for enumerating
CD4 T lymphocytes in the
context of HIV/AIDS
SEA-HLM-392 (Revision)
Distribution: General
Laboratory guidelines for
enumerating CD4 T lymphocytes
in the context of HIV/AIDS
© World Health Organization 2009
All rights reserved.
Requests for publications, or for permission to reproduce or translate WHO
publications – whether for sale or for noncommercial distribution – can be
obtained from Publishing and Sales, World Health Organization, Regional
Office for South-East Asia, Indraprastha Estate, Mahatma Gandhi Marg, New
Delhi 110 002, India (fax: +91 11 23370197; e-mail: publications@searo.
who.int).
The designations employed and the presentation of the material in this
publication do not imply the expression of any opinion whatsoever on the part
of the World Health Organization concerning the legal status of any country,
territory, city or area or of its authorities, or concerning the delimitation of its
frontiers or boundaries. Dotted lines on maps represent approximate border
lines for which there may not yet be full agreement.
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.
All reasonable precautions have been taken by the World Health Organization
to verify the information contained in this publication. However, the published
material is being distributed without warranty of any kind, either expressed or
implied. The responsibility for the interpretation and use of the material lies
with the reader. In no event shall the World Health Organization be liable for
damages arising from its use.
This publication does not necessarily represent the decisions or policies of the
World Health Organization.
Printed in India
Contents
Acronyms and abbreviations .......................................................................v
1.
2.
3.
4.
Natural history of HIV infection......................................................... 1
1.1
Magnitude of the problem.................................................................. 1
1.2
Biology of HIV ................................................................................... 3
1.3
Pathogenesis of HIV ........................................................................... 5
1.4
Factors influencing HIV disease progression ........................................ 9
1.5
WHO clinical staging of HIV-related disease in adults and
adolescents aged 15 years or more .................................................. 11
Role of CD4 T lymphocytes in disease progression .......................... 13
2.1
CD4 T lymphocytes and HIV............................................................ 13
2.2
CD4 T lymphocyte counts and antiretroviral therapy ........................ 15
2.3
CD4 T lymphocyte count as indicator of treatment failure ............... 16
2.4
CD4 T lymphocyte count in paediatric population ........................... 18
Principles of flow cytometry ............................................................ 21
3.1
Introduction ..................................................................................... 21
3.2
What is flow cytometry? .................................................................. 21
3.3
Instrument description...................................................................... 22
3.4
Use of fluorochromes in flow cytometry ........................................... 25
3.5
Optimizing the flow cytometer settings ............................................. 26
Methods of CD4 T lymphocyte enumeration .................................. 29
4.1
Introduction ..................................................................................... 29
4.2
Flow cytometric methodologies ........................................................ 29
4.3
Selections of methodology for CD4 T lymphocytes
count estimation .............................................................................. 39
4.4
Importance of reference ranges in interpretation of the CD4 T
lymphocyte counts obtained in the laboratory .................................. 40
Laboratory guidelines for enumerating CD4 T lymphocytes in the context of HIV/AIDS
iii
5.
6.
7.
Alternate methods for CD4 T lymphocytes enumeration ................. 43
5.1
Introduction ..................................................................................... 43
5.2
Manual method ............................................................................... 43
5.3
Newer methodologies ..................................................................... 44
5.4
Non-CD4 markers for monitoring HIV disease progression and for
initiation and monitoring the success of anti-retroviral therapy.......... 46
Quality management in CD4 T lymphocytes enumeration .............. 47
6.1
Management requirements .............................................................. 47
6.2
Technical requirements .................................................................... 49
Establishing a laboratory for CD4 T lymphocyte enumeration .......... 55
7.1
Suggested requirements at different levels ........................................ 55
7.2
Selection of methodology for CD4 T lymphocyte counts ................. 56
7.3
Establishment of a CD4 T lymphocyte enumeration laboratory ......... 56
Annexes
1.
Collection and transport of specimens ............................................. 59
2.
General biosafety guidelines for immunophenotyping laboratory..... 63
3.
Pipetting techniques ....................................................................... 65
4.
Suggested format for sample transport, receipt and report form ....... 67
5.
Useful references ............................................................................ 71
6.
List of contributors .......................................................................... 75
Acronyms and abbreviations
AIDS
acquired immunodeficiency syndrome
ART
antiretroviral therapy
ARV
antiretroviral
CD
cluster of differentiation
CD4
T-lymphocyte CD4+
CV
Coefficient of variation
EDTA
ethylenediamine tetra-acetate
FBC
full blood count
FSC
forward scatter
FITC
fluorescein Isothiocyanate
FCM
flow cytometer
FACSCount
fluorescent activated cell sorter count
FDA
Food and Drug Administration
HIV
human immunodeficiency virus
IATA
International Air Transport Association
TLC
total lymphocyte count
UN
United Nations
UNAIDS
Joint United Nations Programme on HIV/AIDS
UPS
uninterrupted power supply
VCTC
Voluntary Counseling and Testing Centre
WBC
white blood cells
WHO
World Health Organization
EQAS
External Quality Assessment Scheme
IQAS
Internal Quality Assessment Scheme
GLP
good laboratory practice
nm
nanometer
Laboratory guidelines for enumerating CD4 T lymphocytes in the context of HIV/AIDS
v
vi
μl
microliter
PE
phycoerythrin
PLG
panLeucogating
QA
quality assurance
QC
quality control
OIs
opportunistic infections
SOP
standard operating procedures
SSC
side scatter
SEA
South-East Asia
SEARO
Regional Office for South-East Asia
PEP
post-exposure prophylaxis
US/USA
the United States of America
Laboratory guidelines for enumerating CD4 T lymphocytes in the context of HIV/AIDS
1
Natural history of HIV infection
1.1 Magnitude of the problem
The HIV/AIDS pandemic has severely affected health development and
eroded the cumulative improvements achieved 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 33 million infections and 27 million deaths till date . At
the end of 2007, an estimated more than 20 million people were living with
HIV, with sub-Saharan Africa carrying the highest burden: accounting for 67%
of all people living with HIV.
In 2007, an estimated 2.7 million people became newly infected with
HIV. More than 96% of these new infections are in low- and middle-income
countries. Each day 7400 persons become newly infected with the virus; of
these, half are women and 45% are young people of the age group 15-24
years. Of the estimated 33 million adults living with HIV worldwide, nearly
15.5 million are women.
At the end of 2007, there were an estimated two 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
socioeconomic 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, dropping out of school, lack of love, attention
and affection, psychological distress, stigma, discrimination and isolation, and
malnutrition and illness. Cumulatively, 16 million children worldwide became
orphans after their parents died due to AIDS related illnesses
Laboratory guidelines for enumerating CD4 T lymphocytes in the context of HIV/AIDS
1
HIV burden in the South-East Asia Region
The WHO South-East Asia (SEA) Region comprises the Member countries
of Bangladesh, Bhutan, the Democratic People’s Republic of Korea, India,
Indonesia, Maldives, Myanmar, Nepal, Sri Lanka, Thailand and Timor-Leste.
The overall HIV prevalence in adults in South and South-East Asia is 0.3%,
relatively lower than the 5% prevalence in sub-Saharan Africa. However, due
to the large population of the Member countries, even a low HIV prevalence
effectively means that large numbers of people are living with HIV.
At the end of 2007, there were an estimated 3.6 million people living
with HIV in the SEA Region. This included 0.26 million new infections in 2007.
Approximately 300 000 persons died of AIDS during 2007. Five countries—
India, Thailand, Myanmar, Indonesia and Nepal—account for a majority of the
HIV/AIDS burden in this Region. Long-standing epidemics have resulted in 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 PLHAs in
each Member country of the Region.
Table 1.1: HIV/AIDS Burden in countries of the South-East Asia Region, 2007
Adult HIV
prevalence (%)
Estimated number of people
living with HIV/AIDS
Bangladesh
se
vi
d
r
Ve
on
si
20
09
The human immunodeficiency virus selectively invades a
subpopulation of lymphocytes (CD4 T lymphocytes). The progressive
loss of CD4 T lymphocytes eventually results in the loss of an ability to
mount desirable immune response to any pathogen and results in
vulnerability to opportunistic pathogens characteristic of AIDS. The
estimation of peripheral CD4 T lymphocyte counts is used as a tool to
take an informed decision for initiation of antiretroviral treatment,
monitoring disease progression and the effectiveness of antiretroviral
treatment. Several technologies for determining the number of CD4 T
lymphocytes are now available. The technologies are either flow
cytometric or non-flow cytometric. This document describes the
technologies as well as the infrastructure required for these in
developing countries.
World Health House
Indraprastha Estate,
Mahatma Gandhi Marg,
New Delhi-110002, India
Website: www.searo.who.int
SEA-HLM-392
Laboratory guidelines
for enumerating
CD4 T lymphocytes in the
context of HIV/AIDS
SEA-HLM-392 (Revision)
Distribution: General
Laboratory guidelines for
enumerating CD4 T lymphocytes
in the context of HIV/AIDS
© World Health Organization 2009
All rights reserved.
Requests for publications, or for permission to reproduce or translate WHO
publications – whether for sale or for noncommercial distribution – can be
obtained from Publishing and Sales, World Health Organization, Regional
Office for South-East Asia, Indraprastha Estate, Mahatma Gandhi Marg, New
Delhi 110 002, India (fax: +91 11 23370197; e-mail: publications@searo.
who.int).
The designations employed and the presentation of the material in this
publication do not imply the expression of any opinion whatsoever on the part
of the World Health Organization concerning the legal status of any country,
territory, city or area or of its authorities, or concerning the delimitation of its
frontiers or boundaries. Dotted lines on maps represent approximate border
lines for which there may not yet be full agreement.
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.
All reasonable precautions have been taken by the World Health Organization
to verify the information contained in this publication. However, the published
material is being distributed without warranty of any kind, either expressed or
implied. The responsibility for the interpretation and use of the material lies
with the reader. In no event shall the World Health Organization be liable for
damages arising from its use.
This publication does not necessarily represent the decisions or policies of the
World Health Organization.
Printed in India
Contents
Acronyms and abbreviations .......................................................................v
1.
2.
3.
4.
Natural history of HIV infection......................................................... 1
1.1
Magnitude of the problem.................................................................. 1
1.2
Biology of HIV ................................................................................... 3
1.3
Pathogenesis of HIV ........................................................................... 5
1.4
Factors influencing HIV disease progression ........................................ 9
1.5
WHO clinical staging of HIV-related disease in adults and
adolescents aged 15 years or more .................................................. 11
Role of CD4 T lymphocytes in disease progression .......................... 13
2.1
CD4 T lymphocytes and HIV............................................................ 13
2.2
CD4 T lymphocyte counts and antiretroviral therapy ........................ 15
2.3
CD4 T lymphocyte count as indicator of treatment failure ............... 16
2.4
CD4 T lymphocyte count in paediatric population ........................... 18
Principles of flow cytometry ............................................................ 21
3.1
Introduction ..................................................................................... 21
3.2
What is flow cytometry? .................................................................. 21
3.3
Instrument description...................................................................... 22
3.4
Use of fluorochromes in flow cytometry ........................................... 25
3.5
Optimizing the flow cytometer settings ............................................. 26
Methods of CD4 T lymphocyte enumeration .................................. 29
4.1
Introduction ..................................................................................... 29
4.2
Flow cytometric methodologies ........................................................ 29
4.3
Selections of methodology for CD4 T lymphocytes
count estimation .............................................................................. 39
4.4
Importance of reference ranges in interpretation of the CD4 T
lymphocyte counts obtained in the laboratory .................................. 40
Laboratory guidelines for enumerating CD4 T lymphocytes in the context of HIV/AIDS
iii
5.
6.
7.
Alternate methods for CD4 T lymphocytes enumeration ................. 43
5.1
Introduction ..................................................................................... 43
5.2
Manual method ............................................................................... 43
5.3
Newer methodologies ..................................................................... 44
5.4
Non-CD4 markers for monitoring HIV disease progression and for
initiation and monitoring the success of anti-retroviral therapy.......... 46
Quality management in CD4 T lymphocytes enumeration .............. 47
6.1
Management requirements .............................................................. 47
6.2
Technical requirements .................................................................... 49
Establishing a laboratory for CD4 T lymphocyte enumeration .......... 55
7.1
Suggested requirements at different levels ........................................ 55
7.2
Selection of methodology for CD4 T lymphocyte counts ................. 56
7.3
Establishment of a CD4 T lymphocyte enumeration laboratory ......... 56
Annexes
1.
Collection and transport of specimens ............................................. 59
2.
General biosafety guidelines for immunophenotyping laboratory..... 63
3.
Pipetting techniques ....................................................................... 65
4.
Suggested format for sample transport, receipt and report form ....... 67
5.
Useful references ............................................................................ 71
6.
List of contributors .......................................................................... 75
Acronyms and abbreviations
AIDS
acquired immunodeficiency syndrome
ART
antiretroviral therapy
ARV
antiretroviral
CD
cluster of differentiation
CD4
T-lymphocyte CD4+
CV
Coefficient of variation
EDTA
ethylenediamine tetra-acetate
FBC
full blood count
FSC
forward scatter
FITC
fluorescein Isothiocyanate
FCM
flow cytometer
FACSCount
fluorescent activated cell sorter count
FDA
Food and Drug Administration
HIV
human immunodeficiency virus
IATA
International Air Transport Association
TLC
total lymphocyte count
UN
United Nations
UNAIDS
Joint United Nations Programme on HIV/AIDS
UPS
uninterrupted power supply
VCTC
Voluntary Counseling and Testing Centre
WBC
white blood cells
WHO
World Health Organization
EQAS
External Quality Assessment Scheme
IQAS
Internal Quality Assessment Scheme
GLP
good laboratory practice
nm
nanometer
Laboratory guidelines for enumerating CD4 T lymphocytes in the context of HIV/AIDS
v
vi
μl
microliter
PE
phycoerythrin
PLG
panLeucogating
QA
quality assurance
QC
quality control
OIs
opportunistic infections
SOP
standard operating procedures
SSC
side scatter
SEA
South-East Asia
SEARO
Regional Office for South-East Asia
PEP
post-exposure prophylaxis
US/USA
the United States of America
Laboratory guidelines for enumerating CD4 T lymphocytes in the context of HIV/AIDS
1
Natural history of HIV infection
1.1 Magnitude of the problem
The HIV/AIDS pandemic has severely affected health development and
eroded the cumulative improvements achieved 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 33 million infections and 27 million deaths till date . At
the end of 2007, an estimated more than 20 million people were living with
HIV, with sub-Saharan Africa carrying the highest burden: accounting for 67%
of all people living with HIV.
In 2007, an estimated 2.7 million people became newly infected with
HIV. More than 96% of these new infections are in low- and middle-income
countries. Each day 7400 persons become newly infected with the virus; of
these, half are women and 45% are young people of the age group 15-24
years. Of the estimated 33 million adults living with HIV worldwide, nearly
15.5 million are women.
At the end of 2007, there were an estimated two 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
socioeconomic 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, dropping out of school, lack of love, attention
and affection, psychological distress, stigma, discrimination and isolation, and
malnutrition and illness. Cumulatively, 16 million children worldwide became
orphans after their parents died due to AIDS related illnesses
Laboratory guidelines for enumerating CD4 T lymphocytes in the context of HIV/AIDS
1
HIV burden in the South-East Asia Region
The WHO South-East Asia (SEA) Region comprises the Member countries
of Bangladesh, Bhutan, the Democratic People’s Republic of Korea, India,
Indonesia, Maldives, Myanmar, Nepal, Sri Lanka, Thailand and Timor-Leste.
The overall HIV prevalence in adults in South and South-East Asia is 0.3%,
relatively lower than the 5% prevalence in sub-Saharan Africa. However, due
to the large population of the Member countries, even a low HIV prevalence
effectively means that large numbers of people are living with HIV.
At the end of 2007, there were an estimated 3.6 million people living
with HIV in the SEA Region. This included 0.26 million new infections in 2007.
Approximately 300 000 persons died of AIDS during 2007. Five countries—
India, Thailand, Myanmar, Indonesia and Nepal—account for a majority of the
HIV/AIDS burden in this Region. Long-standing epidemics have resulted in 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 PLHAs in
each Member country of the Region.
Table 1.1: HIV/AIDS Burden in countries of the South-East Asia Region, 2007
Adult HIV
prevalence (%)
Estimated number of people
living with HIV/AIDS
Bangladesh