Qantification methods of CD4 T

cells in HIV . infection

• By Karthika selwanayagam
• KD/BSCBMS/04/25
 What is HIV
● Human Immunodeficiency Virus.
● Belongs to retroviruses
● Causes AIDS
● Acquired immunodeficiency syndrome .
 Relationship between HIV
infection and CD4 T cells

• HIV primarily targets a type of cell in the immune

system called the CD4 T cells
• HIV bind to the CD4 molecules on the surface of
helper T cells and replicates within them
• There for , the CD4 T cells have two roles during
HIV infection
1. When they are infected, their role is to
reproduce virus particles.
2. When they are not infected, their role is to
activate the humoral and cellular immune responses
 Relationship
• The current method used for staging between
HIV infection in
HIV
settings with infection
limited resources
measurement of CD4+ T cells
is the sole and CD4

T cells
• A gradual decrease in T helper (CD4+) cells is a
characteristic of the normal course of HIV infection
Relationship between HIV
infection and CD4 T cells
• Due to the virus's infection and apoptosis of
CD4+ T cells, the primary mechanism for
programmed cell death, this depletion happens.
• These cells are linked to the
immunopathogenesis of HIV infection and
function as regulators and amplifiers of the
immune response
• CD4+ T cells are considered one of the most
important immunological parameters in HIV-
infected individuals.
Quantification methods of CD4 T
cells in HIV infection.
1. Flow cytometry
2. FACScount
3. Manual CD4 cell count
4. Dynabeads T4-T8 system
5. Capcellia CD4/CD8
6. TRAxCD4 test
7. Microchip Methods etc..
 Quantification methods of CD4 T cells in HIV infection.

• aim of this study was to determine the variation of relative counts

for CD4+ T cells using two different methodologies:
(i) estimating the percentage values using a hematology counter
and a flow cytometer and
(ii) determination of these values only using the flow cytometer.
● Therefore, quantification of CD4+ lymphocytes
(immunophenotyping by flow cytometry) is an essential step in the
evaluation of HIV patients [

● Therefore, flow cytometry using a CD45+ monoclonal antibody

in combination with CD3+, CD4+, and CD8+ antibodies can be
used to determine the total lymphocyte count and the percentage
of lymphocyte subsets .

● Absolute lymphocyte counts can be influenced by biological

factors that affect the total number of white blood cells and
lymphocytes, such as: B. Use of myelosuppressive drugs, acute
 METHODS

• 2. STATISTICAL ANALYSIS
• 1. LABORATORY ANALYSIS

● Here I m.goint to explain with a example.

● There was 81 people selected for the treatment.

 LABORATORY ANALYSIS
• lymphocyte counts obtained by flow cytometry alone and the
combination of the two methods (flow cytometry and blood
counter).
• Biological samples were removed from the vacuum system
"Vacutainer".
• Anticoagulant EDTA-K3 was used and two 5 ml tubes of venous
blood were drawn for analysis.
• All tests were performed within 6 hours after collection.
 Percentage Values of CD4+ T Lymphocytes Estimated by Hematologic
Equipment Associated with Flow Cytometry

• Samples were subjected to cell counting using a Cell-Dyn 3700

hematology counter (Abbott, QC, Canada) and a FACSCalibur
flow cytometer (Becton Dickinson-Biosciences, San Jose, CA,
USA).
• First, absolute absolute lymphocyte counts were obtained using a
hematology instrument.
Percentage Values of CD4+ T Lymphocytes Obtained by
Flow Cytometry

• Immunophenotyping of each sample was performed using the

Multitest/TruCount standard (CD45+/CD3+/CD8+/CD4+
monoclonal antibody) T cell counting protocol and a FACSCalibur
flow cytometer (Becton Dickinson-Biosciences, San Jose, CA). rice
field. Obtain the relative number of CD4+ cells.
 STATISTICAL ANALYSIS
• A Kolmogorov-Smirnov test was performed to check for normality
and the values ​showed a normal distribution.
• Statistical methods used included descriptive analysis (mean and
standard deviation), correlation, and comparisons between the two
methods.
• Data were analyzed using Student`s t-test and means of paired values
​were compared.
• When analyzing different methods, correlations between results were
verified by graphical representation of the Bland-Altman method.
• Data were processed with the statistical program MedCalc .
 Results
• Samples were grouped according to the absolute count of CD4+ T cells,
resulting in the following stratification for the 81 samples analyzed: 18
samples with CD4+ T-cell counts below 200 cells/mL ( cells/mL), 34 samples
between 200 and 500 cells/mL ( cells/mL), and 29 samples with counts above
500 cells/mL ( cells/mL).

• The results of the percentage counts of CD4+ T cells obtained directly by

flow cytometry were for stratum CD4+ < 200 cells/mL, for stratum 200–
500 cells/mL, and for stratum > 500 cells/mL. However, the estimated values
obtained by the hematological counter were , , and , respectively, for each of
these strata. There were identified significant differences between values for
the relative counts from these two methodologies for every studied stratum
• The correlation between the percentages of CD4+ T lymphocytes
obtained by the two methodologies for the three strata of CD4 cells
studied is shown in Figures 1(a), 2(a), and 3(a), as well as the
agreement represented by the Bland-Altman analysis shown in
Figures 1(b), 2(b), and 3(b).

• Figure 1
• Correlation of percentage values of CD4+, (a) and limits of
agreement between the values estimated by Bland-Altman analysis
(b) obtained by the hematology counter and the flow cytometer in the
stratum of CD4 count <200 cells/mL.
Figure 1 a and b
Figure 2 Correlation of percentage values of CD4+, (a) and limits of
agreement between the values estimated by Bland-Altman analysis
(b) obtained by the hematology counter and the flow cytometer in
the stratum of CD4 count between 200 and 500 cells/mL.
Figure 3 a & b: Correlation of percentage values of CD4+, (a) and
limits of agreement between the values estimated by Bland-
Altman analysis (b) obtained by the hematology counter and the
flow cytometer in the stratum of CD4 count >500 cells/mL.
• 0% for the stratum of CD4+ < 200 cells/mL, and the limits of
agreement were from −2.
• In the CD4 strata between 200 and 500 cells/mL, lymphocyte
counts above 500 cells/mL were observed as well as broader
concordance limits between 2.
• 6%), respectively, compared to the extract of CD4+ < 200
cells/mL.
• It was noted that the estimate of the count of CD4+ T cells from the
hematology counter was higher in relative values for the three strata
studied, ranging from about 1% for the stratum CD4+ < 200> 500
cells/mL.
• The additional variability of the count is due to a greater inaccuracy in
the way in which the hematologic equipment classifies total
lymphocytes
• The results corroborate information reported earlier showing that the
lymphocyte count obtained from hematologic analyzers is prone to
errors and, on the other hand, the use of a gate on CD45+ cells labeled
with an associated dispersion parameter light using the flow cytometer
provides better precision and accuracy in the quantification of
lymphocytes in relation to the parameters of cell volume and
conductance of hematologic counters
• Comparing the percentage of CD4+ T cells in the stratum of CD4+
< 200 cells/mL by the hematology counter and flow cytometry
showed that the measures have a strong correlation ().
• To the stratum of CD4+ between 200 and 500 cells/mL, it is noted
that the measures are moderately correlated ().
• Despite the correlation, Figure 2(b) (Bland-Altman) demonstrates
broader limits of agreement of approximately %.
• This is similar to the lymphocyte count above 500 cells/mL with
limits of agreement approximately %, as shown in Figure 3(b)
(Bland-Altman), although this stratum showed a strong
correlation .
• assessing the use of flow cytometry to provide only the absolute count of CD4+ T
cells (associated with total lymphocyte count in hematology analyzers to obtain the
percentage of CD4+ T lymphocytes), obtained bias of 0.
• 92% and limits of agreement between 5.
• 66% through FacsCount and Multitest/Tubs Trucount method and in FACSCalibur
flow cytometer, for the absolute count of CD4+ T cells below 200 cells/mL.
• The main importance of using percentage values of CD4+ T lymphocytes is in the
absolute count changes in response to stimuli that are independent of HIV
infection, and the percentages are less subject to this variability [13].
• Considering the percentage values of CD4+ T lymphocytes for evaluation of HIV-
infected individuals, the stratum CD4+ < 200> 500 cells/mL, the count could be
underestimated by up to 15.
 ADVANTAGE and
DISADVANTAGES
• Reference standard used in • High initiated cost of
many resource-rich equipment
environment • High cost of reagents
• Provides CD4 cells percentage • Operators required high level
as well as absolute count of skill and technical expertise
• Largely automated • Difficulties of getting
• Possible to do more equipment serviced
economically with primary • Whole blood lysis required
gating
 Conclusion
●Thus, although there was a good correlation between the percentages
of CD4+ T lymphocytes estimated by the association of the two
methods, the correlation between individual measurements was not
comparable for all layers of CD4+ cells examined. marked a significant
boundary.
●From a clinical perspective, differences in CD4+ T lymphocyte
percentages presented by agreed limits may conflict with decisions
regarding treatment and care of HIV-infected persons.
●Therefore, when interpreting CD4+ T lymphocyte percentages for
immunosurveillance in patients with human immunodeficiency virus,
variability caused by the methodology used should be carefully
considered.
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