Indian J Med Res 121, April 2005, pp 539-549
539
Since the first case of a HIV-infected high-risk
male subject was recognized in 19811, HIV infection
has reached pandemic proportions in many countries
worldwide. According to the World Health
Organization (WHO) report, more than five million
people acquired HIV in 2003, bringing the total
number of persons living the HIV to approximately
42 millions2. About 70 per cent of these infected
people live in Sub-Saharan Africa and Asia3. It is
estimated that if not treated, 3 million people will die
every year of HIV/AIDS. Projections for the next
10 yr suggest that the situation will become even more
serious, with a possible 100 million infected people
worldwide2. This situation not only represents a
serious human catastrophe, but also dramatically
impacts on the economic and health fabric in countries
with high HIV prevalence4. Amongst all intervention
strategies applied to curtail the HIV pandemic,
reduction of viral load by efficient antiretroviral
therapy has been considered to be a powerful tool.
In India, an introduction of inexpensive and generic
antiretroviral therapy (ART) has been initiated by
National AIDS Control Organization (NACO). For
the successful inexpensive treatment an affordable
CD4+ T cell count as a tool to monitor HIV progression &
anti-retroviral therapy
Kovit Pattanapanyasat & Madhuri R. Thakar*
Center of Excellence for Flow Cytometry, Office for Research & Development,
Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok, Thailand &
*National AIDS Research Institute, Pune, India
Accepted February 7, 2005
The CD4+ T lymphocytes are the crucial cells in the orchestral events in forming immune
response to the foreign antigen and it is also the primary target cells for human immunodeficiency
virus (HIV). The progressive loss of these cells eventually results in the loss of an ability to
mount desirable immune response to any pathogen and death of the patients in the terminal
stage of HIV infection, i.e., acquired immune deficiency syndrome (AIDS). The longitudinal
monitoring of CD4+ counts is used as a monitoring tool for disease progression and effectiveness
of antiretroviral treatment. Various methods are being used for determination of absolute CD4+
T lymphocytes from the peripheral blood. To date, the flow cytometry is considered as the gold
standard. Different modifications have been tried in the conventional flow cytometry to increase
accuracy and cost-effectivity especially for adapting in resource-poor settings. Principles of the
conventional and modified methodologies are discussed. The non-flow cytometric methodologies
are also there that might be available soon widely. The choice of the methodology should depend
upon the purpose of the assay, the age group of the patients, sample turnover and available
resources.  Importance of maintaining both internal and external quality control systems in
every laboratory performing CD4 count estimation are discussed.
Key words  CD4+ T lymphocytes flow cytometry - quality control
540 INDIAN J MED RES, APRIL  2005
laboratory monitoring is utmost essential. The
enumeration of CD4+ T-lymphocytes in the peripheral
blood is an essential tool for the laboratory monitoring
of HIV infected patients for the progression of
disease and for the assessment of outcome of
antiretroviral treatment.
What are CD4+ T lymphocytes?
The CD4+ T lymphocytes; also known as T helper
cells (CD4+ T-cells), are co-ordinators of the body’s
immune response, e.g., providing help to B cells in
the production of antibody, as well as in augmenting
cellular immune response to antigens. These CD4+
T-cells are the primary target of HIV. The loss of
these cells in HIV infection, both qualitatively and
quantitatively, results in weakening of the immune
response and the ability of the host to respond to
foreign antigens, thus rendering the host susceptible
to infections and leading ultimately to the acquired
immune deficiency syndrome (AIDS).
Hence, the CD4+ T-cell counts are frequently
monitored to assess immune suppression and disease
progression in HIV infected patients. Moreover,
changes in the CD4+ T-cell counts are important
indicator of the response to ART5-7. At present, the
CD4+ T-cell count remains the most important key
surrogate marker for initiation and monitoring ART
and a measure of the effectiveness of the treatment
in clinical trial evaluations.
The National AIDS Control Organization has
published the guideline for initiation of ART8 (Table I).
In adults the absolute numbers of CD4+ T cells
are measured, while in infants and young children the
relative number of these cells (percentage among total
lymphocytes) is more informative. Since the CD4
counts may vary within individuals of different
ethnicity, it is important to establish the reference
ranges of CD4 count for the target population. The
reference range for Indian healthy adult has been
established in a multicentric study carried out at six
different centres from different parts of India9. The
CD4 percentages in Indian adults were found to be
lower as compared to Western countries. The mean
CD4 per cent were found to be 37 per cent (range:
14 to 65%). The CD4:CD8 ratios in southern part of
India were found to be significantly lower as
compared to the values from northern and western
parts of India9. Various studies carried out on Indian
population9-12 have showed the range of absolute CD4
counts as 600 to 1200 cells/µ l.
Looking at the importance of the CD4 count
estimation in the clinical setting for ART monitoring,
the guidelines addressing the quality control of CD4
testing in persons with HIV infection have also been
developed13-15. In this review, the methodologies of
enumeration of CD4+ T-cells and various available
quality control options are discussed.
Methods for enumerating absolute numbers of
CD4+ T-cells
To date, both automated and manual methods have
been developed for determining the absolute number
of CD4+ T-cells and evaluated in multicentric studies.
These include both flow cytometric and non-flow
cytometric technologies. Most of the technologies
Table I. NACO recommendations for initiating antiretroviral
therapy in adults and adolescents with documented HIV
infection
If CD4 testing available:
WHO stage IV disease irrespective of CD4 cell count
WHO stage III disease with consideration of using CD4
cell counts <350/µ l  to assist decision making
WHO stage I or II disease with CD4 cell count < 200/ µ l
If CD4 testing unavailable:
WHO stage IV disease irrespective of total lymphocyte
count
WHO stage III disease irrespective of total lymphocyte
count
WHO stage II disease with a total lymphocyte count =
1200/µ l
Source: Ref. 8
541
summarized here are commercially available as cell
counting systems and/or kit packages.
Flow cytometric (automated) methodologies: Flow
cytometry is the gold standard method for the
estimation of CD4 counts due to its accuracy,
precision and reproducibility and thus widely used.
This technology is capable of high sample throughput
and great versatility in its applications; CD4 counting
is just one of its numerous uses in the biomedical field.
However, flow cytometry based CD4 counting is
relatively complex, and therefore technically
demanding, costly and needs regular maintenance.
Additionally, it is essential that operators of the flow
cytometer be sufficiently trained in the technical and
biological aspects of CD4 counting. Although, the
system has a high initial and running cost at present,
the introduction of simpler and portable instruments
may change this situation in near future.
Flow cytometry provides options like dual- and
single-platform approach for estimation of absolute
CD4+ T-cell counts.
Dual-platform approach: The dual-platform approach
uses two instruments to generate absolute CD4+ T-
cell counts: a flow cytometer for generating a
percentage CD4+ T-cells among lymphocytes and a
haematological analyzer to enumerate the absolute
lymphocyte counts. An absolute CD4+ T-cell count
is then derived by multiplying percentage of CD4+
T-cells by the absolute lymphocyte count.  Because
the percentage of CD4+ T-cells is obtained from the
reference lymphocyte populations, the purity of the
lymphocytes is therefore needed to be defined with
the greatest precision. Traditionally, in the blood
sample that contains a high proportion of lymphocytes,
a lymphocyte gate for CD4+ T-cell testing is easily
derived from a bivariate histogram or homogeneous
gate that includes forward scatter (FSC, size of the
cell populations) and right angle side scatter (SSC,
granularity of the cell populations) patterns 16.
However, when there is a high proportion of non-
lymphocytes (monocytes, basophils and immature red
blood cells), this traditional FSC/SSC lymphocyte gate
tends to be unreliable as non-lymphocytes have been
shown to contaminate the gate, thus this morphological
gating is now considered as unacceptable17,18. A more
reliable method for assessing lymphocyte gate purity
and lymphoid cell recovery on the basis of differential
CD45 marker density expression has been developed
by Loken et al19. This method also known as CD45
grating, which uses two markers (CD45 and CD14):
CD45 is a pan-leukocyte marker expressed at
different intensities on leukocytes (granulocytes
CD45+; monocyte CD45++; lymphocytes CD45+++
or bright) while the CD14 marker is selectively
expressed by monocytes. This CD45 gating approach
can gate all lymphocytes in the acquired events and
maximize their purity by excluding unwanted non-
lymphocytes. Based on this CD45 gating strategy
together with the advent of the flow cytometry, the
more practical and accurate three-colour (CD3/CD4/
CD45) or four-colour (CD3/CD4/CD8/CD45)
immunophenotyping assays of CD4+ T-cells have
become available13,20-22. In three- or four-colour
immunophenotyping techniques, lymphocytes are
defined as CD45bright with low SSC enabling a very
pure population of lymphocytes (Fig.1A). Once this
lymphocyte gate is established, the percentage of
lymphocytes reactive for CD3+/CD4+ T-cells
(Fig.1B) as in the three-colour assay, and/or CD3+/
CD8+ T-cells as in the four-colour assay can then be
easily determined.
The methodology of CD45 gating has several
advantages: (i) It is easy to distinguish lymphocytes in
the SSC/CD45bright gate even in the presence of a large
amount of debris, which allows the use of lyse/no-wash
staining method; (ii) The isotype controls could be
avoided thus reducing the cost of the reagents; and
(iii) It is possible to determine % lymphocytes among
leukocytes from the SSC/CD45 gate.
Recently, this CD45 gating strategy has been
adopted with a more practical two-colour
PanLeucogating approach22-24. This two-colour
PanLeucogating approach uses total leucocytes as the
common denominator, in which total leucocytes are
identified and gated by their SSC and CD45+
characteristics. CD45 is reactive with fluorochrome-
conjugated antibody. Leucocytes and lymphocytes are
identified and gated by drawing two regions: one
around all leucocytes and the other set on all bright
CD45+ cells with low SSC (Fig. 2A). Lymphocytes
gated in this region are further analyzed for CD4+ T-
PATTANAPANYASAT & THAKAR: CD4+ CELL ESTIMATION
542 INDIAN J MED RES, APRIL  2005
cells. These cells are easily distinguished from non-
CD4+ T-cells and per cent CD4 is then obtained as a
percentage of total lymphocytes (Fig.2B). The same
analysis protocol can also be applied to CD8+
T-cells using CD45/CD8. This CD45-assisted
PanLeucogating technique is now widely accepted,
since it is simple, better, and cost-effective to use in
the resource-poor settings.
The dual platform approach requires the results
of three separate laboratory tests: percentage CD4+
T-cells from the flow cytometer, total white blood
counts and percentage of lymphocytes from the
haematological analyzer. This undoubtedly leads to
wide variability. The basic problems of
haematological analyzer have been described in the
literature25-27. It is now well accepted that the use
of a haematological analyzer as the second platform
for white blood count and the three-or five-part
differential count introduce variable factors into the
calculation of absolute CD4+ T-cells. These are
particularly so in the interlaboratory studies that use
different haematological analyzers28,29. In the
individual patient follow up studies, the absolute
CD4+ T-cell counts obtained from the dual-platform
approach contribute to more variability than per cent
CD4+ T-cell measurement (20-33% within-person
coefficient variation vs. CV of 10-16%). Variation
in the total white blood cell counts (CV of 16-20%)
and lymphocyte differential (CV of 16-26%) over
time in an individual leads to the greater variability
of the absolute CD4+ T-cell counts compared with
per cent CD4+ T-cells. Moreover, the patient
samples contain cell numbers beyond the sensitivity
and linearity range of haematological analyzer ( e.g.,
severely leucopenic patients), the lymphocyte
population defined by flow cytometer may not match
exactly to that of the haematological analyzer.
Further, total white blood cell counts are not
subjected to regular internal quality control and
external quality assurance. In spite of that, this dual-
platform technology is still widely practiced and
recommended in a number of institutional
guidelines13,16,17.
It should be noted that the dual-platform approach
when combined with known density microbeads could
be used as a single-platform flow cytometric method
for determination of absolute number of CD4+ and
CD8+ T-cells.
Fig.1. Representative two-parameter dot plots of a CD3-fluorescein isothicynate (FITC)/CD4- phycoerythrin (PE)/CD45-peridinin
chlorophyll protein complex (PerCP) stained whole blood sample by pan leucogating approach.  (A) The right angle side scatter SSC/
CD45 dot plot shows all leucocyte population with lymphocytes at CD45 bright population (R1). (B) Identification of CD3+/CD4+ T-
cells (upper right quadrant) from R1 cell population.
543
Single-platform approach: The single-platform
approach enables absolute CD4+ T-cell counts to be
derived directly without the need for a haematological
analyzer. This can be assessed either by counting
CD4+ T-cell populations in a precisely determined
blood volume or by using the known numbers of
fluorescent microbeads admixed to a known volume
of CD4- stained blood. Importantly, the precision of
this technology depends on the flow cytometric
measurement and the methodology of pipetting in the
staining process. It is very crucial that the use of
reverse pipetting technique, is the most reliable
dispensing method for absolute CD4+ T-cell count
assay.
To date, single-platform technologies have two
options of microbead-based technologies and the
volumetric technologies. These alternatives are
discussed below:
FACSCountTM microbead-based system: FACSCount
system is the product of Becton Dickinson Biosciences30.
It is the only available microbead-based single-platform
instrument that designed specifically for enumerating the
absolute CD4+, CD8+ and CD3+ T-cell counts in no-
lyse, no-wash whole blood. The system has been
approved by many international organizations as one of
the predicate method. This system requires ready-to-
use twin-tube reagent tubes. One tube determines the
absolute number of helper/inducer T-cells (CD4+/CD3+)
by using a combination of two-colour monoclonal anti-
human CD3 antibody conjugated to the tandem dye
phycoerythrin + Cy5 (PECy5) and a monoclonal anti-
human CD4 antibody conjugated to phycoerythrin (PE).
The other tube determines the absolute number of
cytotoxic/suppressor T-cells (CD8-+/CD3+). Both tubes
also give the absolute number of total T-cells (CD3) or
CD3+ T-cells, as well as CD4/CD8 ratio. In addition to
the antibody reagents, the reagent tubes also contain a
known number of fluorochrome-labelled reference
beads. These beads function as fluorescence standard
for locating the lymphocytes and also as a quantitation
standard for enumerating the cells.  The control set
consists of fluorescent beads at four different densities:
zero (0 beads/µ l), low (50 beads/µ l), medium
(250 beads/µ l), and high (1000 beads/µ l).
PATTANAPANYASAT & THAKAR: CD4+ CELL ESTIMATION
Fig.2. Representative two-parameter dot plots of a CD45-FITC/CD4-PE stained whole blood sample using PanLeucogating approach.
(A) The right angle side scatter (SSC)/CD45 dot plot shows all leucocyte population (R1) with lymphocyte at CD45  bright population
(R3). (B) Depicts CD4+ T-cells (R2) analyzed from R3 cell population. Calculation of %CD4 T-cells is from the number of events
in R2 divided by the number of events in R3.
544 INDIAN J MED RES, APRIL  2005
When whole blood is added to the reagents,
fluorochrome-labelled antibodies in the reagents bind
specifically to lymphocyte surface antigens. After a
fixative solution is added, the sample is run on the
instrument. The stained cells come in contact with
the green HeNe laser light, which causes the cell to
fluoresce. This fluorescent light provides the
information necessary for the instrument to count the
cells (Fig.3). The calculation of absolute CD3+, CD4+
and CD8+ T-cells is determined automatically by
using the built-in Attractors software program.
Guava® EasyCD4™ volumetric system: The Guava®
EasyCD4™ Reagent kit is the product of Guava
Technologies31. It is based on a micorcapillary cytometry
technology that enables the enormous analytical and
diagnostic power of conventional flow cytometry in a
highly miniaturized single platform. The system contains
two-colour, direct immunofluroscence reagents for
enumeration of mature CD4+ T-cells in human blood.
The kit consists of two monoclonal antibody directly
conjugated to PECy5 and PE for CD3 and CD4 T-cell
antigens. The instrument itself consists of a diode green
laser. Sample acquisition is done by a variable-speed fluid
pump that does not require sheath fluid and the system’s
sampling precision depends on the integrity of fluid
pathway. The CD3+ cells are gated and the CD3+ and
CD4+ cells are identified in the gate (Fig. 4). The system
showed good correlation with conventional flow
cytometry and is easy to operate. It uses smaller blood
volume.  However, there is need for experienced
technical personnel for operating the system.
Partec CyFlow volumetric system : CyFlow is
another desktop single-platform technology made by
Partec GmbH32. It is a volumetric software controlled
absolute count system equipped with either a single
532 nm green solid-state laser used for one
fluorescence parameter or two lasers with a mercury
arc lamp applicable for 2 or 3-colour analyses. Data
acquisition and analysis are performed in real time
with FlowMax software. The system showed good
correlation with the CD4 counts obtained by
conventional flow cytometry. However, experienced
technician is required for accurate measurement.
Non-flow cytometric methods: Although flow
cytometry is the accepted standard method for the
determination of absolute counts of CD4+ and
CD8+ T-cells, the methodology involved the use
of flow cytometer and haematology analyzer, which
is expensive both in term of initial investment and
maintenance as well as requiring highly, trained
personnel. Thus it is unsuitable for routine use in
most laboratories with limited facilities such as
district hospitals. An ideal CD4 testing in a
resource-poor setting would be an assay which is
simple, uses inexpensive instrument and minimum
training period that would reliably identify CD4+
T-cells without sacrificing much accuracy and
precision. There are several alternative non-flow
cytometric technologies available for determination
of absolute CD4+ and CD8+ T-cells. These
Fig. 3. Representative dot plot display of CD4+ and
CD8+t cell counting by FACSCount (Single platform method).
The samples are stained in a cartridges and acquired in
the equipment.
545
technologies are cost-effective and thus might be
suitable in the local situation in the resource-poor
settings33-35. Currently available manual assays rely
on microscopes (optical or fluorescence).
Additionally, they require the use of other small
equipment such as centrifuges and magnets. These
tests are designed to operate with low sample
throughput in resource-limited laboratories but their
running costs are not always lower when compared
to the automated methods.
Beckman-Coulter cytosphere system: This manual
CD4 count kit36 contains CD4+ cytospheres reagent,
inert latex spheres coated with monoclonal antibody,
used to identify and manually enumerate the absolute
number of CD4+ T-cells by visible light microscopy
in fresh whole blood. In this assay, 10 µ l of cytosphere
monocyte blocking reagent is added to 100 µ l of whole
blood. After incubation, 10 µ l of CD4 Cytosphere
reagent is added to the mixture. Ten µ l of blood-latex
spheres mixture is then pipetted and lysed by lysing
reagent. Finally, the lysed mixture is loaded on the
haemocytometer and read under the light microscope.
Since the diameter of the cytospheres for CD4
reagent is different from the cytosphere monocyte-
blocking reagent, CD4+ T-cells rosetted with
antibody-coated latex beads can be distinguished from
the monocytes rosetted latex beads due to their large
latex sphere size. The Cytosphere system has
advantage for those haematology technicians who are
familiar with the shapes of cells. Here the monocytes
are not removed but appear different under the
microscope, so the bead-covered CD4+ T cells can
be counted.
The Cytosphere system has been found to be
useful alternative to flow cytometry for the estimation
of CD4 T-lymphocyte counts, in resource-poor
settings like Indian laboratories, for monitoring HIV
progression and response to therapy37.
Dynabeads T4-T8 quantitative system: This non-
flow cytometric technology is the product of Dynal38.
The system is based on the use of immunomagnetic
cell isolation method which uses Dynabeads magnetic
particles coated with antibody to CD4 and CD8
antigens to capture and isolate CD4+ and CD8+ T-
cells from the whole blood. In this system, the whole
blood diluted with buffer solution is depleted of
monocyte using monocyte depletion magnetic bead
reagent coated with anti-CD14 antibody. After
incubation on a Dynal rotator, magnetic separation of
monocytes is done using a magnetic particle
concentrator. Aliquot of the supernatant from the
monocyte-depleted blood is diluted and mixed with
magnetic beads coated with anti-CD4 monoclonal
antibody. After incubation, the beads are separated
from the CD4+ T-cells. The isolated CD4+ T-cells
are then stained with gentian violet and trypan blue.
The isolated stained cells are lysed and the nuclei
are identified and counted manually by light
microscopy or in an automated cell counter.
PATTANAPANYASAT & THAKAR: CD4+ CELL ESTIMATION
Fig. 4. Representative dot plot display of CD4 count estimation by Guava equipment. The gate is set up on the CD3+T lymphocytes
and then CD3+ and CD4+ lymphocytes are counted.
546 INDIAN J MED RES, APRIL  2005
The method has been validated in resource poor
settings in Africa39,40. The method showed significant
correlation with that obtained by flow cytometry and
found to be cost effective with US$ 3/test40.
However, these methods are manual and labour
intensive. To scale these up to match-expanding
access to ART may prove challenge. The automation
by addition of image analysis software would increase
the potential of its use in ART monitoring. The system
could be cheaper than other alternatives and will be
useful in small settings if it is backed up with flow
cytometry for quality assurance.
ELISA based counting systems (Capcellia
ELISA and TRAx and Zymmune)
The ELISA systems measure CD4 protein in the
lysed whole blood. The system could be used in 96
well formats, thus could be automated easily and can
be used on large number of samples. The Capcellia
immunocapture assay has been used to estimate CD4
counts in HIV seropositives and compared with the
CD4 counts obtained using flow cytometry and the
plasma viral load. Capcellia for CD4+ and CD8+ T-
cell counts was found to be a cost-effective, user-
friendly assay, which provides counts that correlate
well with HIV-1 load measurements41. Another study
done by the same group42 showed that the test can
be a useful alternative for flow-based method
however, the assay system has certain limitations
inherent to ELISA techniques. Hence, these systems
have not found favour with the investigators.
One study43 compared five alternative methods;
FACSCount (uses flow cytometry), Cytospheres,
Dynabeads, Opti-CIM, and Capcellia for
implementation in developing countries, and concluded
that FACSCount and Dynabeads methods compared
well with standard flow cytometry; of these the
Dynabeads method appears less expensive and
potentially useful for countries with limited economic
resources.
CD4 count estimation from dried blood spots
The technique uses an antibody ‘sandwich’ to
capture and detect CD4 proteins in the sample. One
study in Zambia has shown promise in the
methodology showing good correlation with the
conventional flow cytometry44. However the
technology is not ready for use in the field.
Table II shows details of CD4 count estimation
methods available at present.
Quality assurance in CD4 testing
Since the use of CD4+ T-cell measurement have
critical implications for the effective management of
individuals at risk or infected with HIV or progressing
into AIDS, it is crucial that external quality control is
put in place to ensure that the test results from
individual laboratories are equivalent.
The aim of quality assurance is to ensure that
doctors and patients are getting the same information
from the laboratory tests, no matter which laboratory
they use or the methods used there.
For a good quality control, the diurnal variation in
the CD4 counts should be considered as an important
factor. To avoid such variation it is recommended that
the sample should be collected at a specified time
from an individual. Another point that has to be taken
into account is the storage of sample before
assessment. This period varies with the methodology
used and location of the testing laboratory.
Several sets of guidelines addressing quality control
of flow cytometric CD4+ testing have been developed.
Though there is at present no external quality control
on non-flow cytometric CD4 testing, it is important that
accurate daily within laboratory (internal quality
control) and proficiency test or external quality
assurance programmes (EQA) be employed to ensure
the reliability of CD4 data. Satisfactory performance
in CD4 testing EQA is recommended for HIV research
and clinical trial programmes in many parts of the
world.  For the most parts, internal control samples
from the manufacturer are used to monitor both sample
processing and instrument performance however,
internal quality control practice is not without merit, it
does not provide full-process quality control. There are
very few laboratories, particularly in the developing
world participating in international EQA programmes
such as United Kingdom National External Quality
547
Assessment Schemes or UKNEQAS45,46. Proper
transportation of specimens and reagents is necessary
for quality assessment management and availability of
good quality reagent at remote places. A technology
that is being developed and refined at present is a
fixative that allows blood samples to be stored and
transported over 5 days (or even slightly longer) without
loss of accuracy in test results. The high cost of these
programmes has been a major burden, making their
implementation in resource-poor countries difficult.
However, there are free EQA programmes such as
Quality Assessment and Standardization for
Immunological Measures Relevant to HIV/AIDS
(QASI)47, in spite of their irregular schedules; they are
useful and cost saving.
In summary, the various technologies currently
available for enumerating CD4+ T-cells are based
on distinct principles and have different operational
characteristics. New manual technologies based on
micro-arrays and dipsticks are being developed and
probably will become available in a few years time.
It is necessary to consider the purpose of the assay
(whether it is being used for monitoring or for
research), the age group of the patients, sample
turnover and available resources (financial, human
and infrastructure) before making the choice for the
test. It is also required to participate in the quality
control programme to impart reliability to the results.
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Table II. Available CD4 enumeration technologies
Type of assay Principle Remarks
Flow  cytometry PanLeucogating/ Cost effective without
based assays A combination of CD4 and CD45 compromising quality
antibodies22,24
Volumetric method (Partec)32 Cheap (2 US$ per test)
Could be useful in field
Requires trained personnel
Guava Technology33 Cheap (2 to 4 US$ per test)
works on CD3+ lymphocyte gating simple to operate
Requires trained personnel
Microscope based Uses Dynabeads to identify CD4 Might be useful at peripheral
assays  lymphocytes34,35,38-40 centres
Cost effective,
Requires fluorescence
microscope
Uses cytosphere for detecting CD4 Cost-effective
cells, works on haematology principle Good correlation with standard
using shapes of the cells for flow cytometry
interpretation36,37
ELISA based assays Measurement of CD4 protein Detects CD4 protein in whole
(Capcellia ELISA)41,42 blood
Measurement of CD4 protein from Detects CD4 protein in whole
dried blood spot44 blood
Superscript numerals represent reference numbers
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Reprint requests: Dr Kovit Pattanapanyasat, Center of Excellence for Flow Cytometry, Office for Research and Development,
Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand
e-mail: grkpy@mahidol.ac.th
PATTANAPANYASAT & THAKAR: CD4+ CELL ESTIMATION