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Considering that counting the percentage of CD4 T lymphocytes can add prognostic information regarding patients infected with HIV, the aim of this study was to evaluate the percentage values of CD4+ T lymphocytes from 81 patients determined by flow cytometry and estimated by flow cytometry in conjunction with a hematology counter. Means were compared through the Student's

The natural history of infection with the human immunodeficiency virus (HIV) is characterized by a progressive decline of T helper (CD4+) lymphocytes [

The level of CD4+ T cells is considered to be one of the most important immunological parameters in HIV-infected individuals to evaluate their prognosis and state of immune deficiency, to determine the start of antiretroviral therapy, to monitor the effectiveness of this treatment, to evaluate the need to start or discontinue prophylaxis for opportunist infections [

Thus, quantification of CD4+ lymphocytes (immunophenotyping by flow cytometry) is an indispensable procedure in the evaluation of patients with HIV [

The absolute count of lymphocytes may be influenced by biological factors that affect the total count of leukocytes and lymphocytes, such as the use of drugs that suppress the bone marrow, acute infections (e.g., sepsis, malaria, and tuberculosis), and pregnancy, which can lead to hemodilution [

Several studies have reported that variations in the percentage count of CD4+ T lymphocytes are more stable parameters than variations in the absolute count to assess the progression of the disease [

The main concern regarding the use of counting the percentage of CD4+ T cells is how the variation of results could have an influence on decisions related to the clinical treatment and care of people infected with HIV.

Therefore, the 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.

There were 81 selected individuals with HIV. All participants were informed about the survey, and they freely signed and dated a consent form. The protocol was approved by the Ethics in Research Committee of the State University of Ponta Grossa (no. 0443710-21/2010) and was conducted in accordance with the Helsinki Declaration.

As in immunophenotyping, the determination of CD4+ T cells is the most important immunological parameter in HIV-infected individuals. The percentages of lymphocyte count obtained only by flow cytometry and the combination of the two methods (flow cytometry and hematology counter) were compared.

Biological samples were collected by a vacuum system (Vacutainer) containing the anticoagulant EDTA-K3, and two 5 mL tubes of venous blood were collected for analysis, one by flow cytometry (immunophenotyping) and one for analysis by traditional hematologic equipment (identification by impedance and roughness). All tests were performed within 6 hours of collection.

The samples were subjected to cell count using the Cell-Dyn hematology counter 3700 (Abbott, QC, Canada) and FACSCalibur flow cytometer (Becton Dickinson-Biosciences, San Jose, CA, USA). First, we obtained the total absolute lymphocyte count using hematology equipment. Then, this absolute value of total lymphocyte was combined with the absolute values of CD4+ T lymphocytes obtained by flow cytometry in order to calculate the relative value of CD4+ T lymphocytes.

The immunophenotyping of each sample was carried out using the protocol for T-cell count of the Multitest/TruCount standard (monoclonal antibodies CD45+/CD3+/CD8+/CD4+) by FACSCalibur flow cytometer (Becton Dickinson-Biosciences, San Jose, CA, USA) to obtain the relative count of CD4+ cells.

The Kolmogorov-Smirnov test was conducted to ensure normality, and the values showed normal distribution. The statistical procedures used involved a descriptive analysis (mean and standard deviation), correlation, and comparison between the two methodologies. The data were analyzed using Student’s

In this study, the variability between relative counts for CD4+ T lymphocytes generated by flow cytometry and those estimated by an alternative methodology was analyzed. The estimated method necessitates the combination of results of hematologic equipment (absolute count of total lymphocytes) and cytometry flow (absolute count of CD4+ T lymphocytes).

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 (

The results of the percentage counts of CD4+ T cells obtained directly by flow cytometry were

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

Correlation of percentage values of CD4+,

Correlation of percentage values of CD4+,

Correlation of percentage values of CD4+,

Studying the

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 cells/mL up to 6% for the stratum > 500 cells/mL. A possible explanation for these differences is the form used for the determination of total lymphocytes by the two devices. 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 [

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 (

Similarly, MacLennan et al. [

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 [

In this study, the analysis of agreement between the hematology meter and the flow cytometer showed relatively large limits for the analyzed strata, indicating high variability.

So, although there was a good correlation between the percentage values of CD4+ T lymphocytes estimated by the two methods association, the correlation between individual measurements indicated relatively large limits for all strata of CD4+ cells studied. From a clinical standpoint, the differences given by the limits of agreement of the percentage values of CD4+ T lymphocytes could cause a conflict in decisions regarding treatment and care of people infected with HIV. Therefore, the interpretation of the percentage count of CD4+ T lymphocytes for immune monitoring of patients with human immunodeficiency virus should carefully take into account variations that may occur due to the methodology used.

The authors are grateful to

^{+}T lymphocyte cell death in human immunodeficiency virus infection and AIDS

^{+}T-cell determinations in persons infected with human immunodeficiency virus (HIV)

^{+}-T-cell counting by flow cytometry: CD45 gating for volumetric analysis

^{+}T lymphocyte values in human immunodeficiency virus-negative adults in Botswana

^{+}, CD8

^{+}peripheral blood lymphocytes

^{+}T-cell percentage is an independent predictor of clinical progression in AIDS-free antiretroviral-naive patients with CD4

^{+}T-cell counts >200 cells/mm

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^{+}lymphocytes are independent predictors of disease progression in HIV-infected persons initiating highly active antiretroviral therapy

^{+}T-cell enumeration