Determination of T-Lymphocyte Subsets in a North African Population (Tunisia): Establishment of Normal Ranges and Results in HIV-Infected Individuals

Human Immunodeficiency Virus (HIV) infection is characterized by a depletion of the CD4+ T-lymphocytes. Absolute counts of CD3+CD4+ cells have been used in monitoring the progression of this disease and in assessing clinical trials of drugs developed to treat HIV infection and related complications [6,10]. In 1993, the definition of AIDS was revised to include HIV-infected persons whose CD4+ Tlymphocytes were ≤ 200 per μl, even in the absence of opportunistic infections or neoplasm. Both the CDC (Center for Disease Control) and WHO (World Health Organization) have emphasized the need to study lymphocyte subsets in normal populations [3]. It is thus necessary for a testing laboratory to establish normal ranges for these subsets in its local population. A number of studies have been done on lymphocyte subsets in healthy individuals. There is, however, a paucity of published data about Tunisians and North Africans in general. Therefore, our first objective in the present study was the establishment of normal ranges for CD3+CD4+ and CD3+CD8+ cells in a healthy Tunisian population. After that, the second step was the assessment of lymphocyte subsets in HIV infected individuals.


INTRODUCTION
Human Immunodeficiency Virus (HIV) infection is characterized by a depletion of the CD4+ T-lymphocytes.
Absolute counts of CD3+CD4+ cells have been used in monitoring the progression of this disease and in assessing clinical trials of drugs developed to treat HIV infection and related complications [6,10]. In 1993, the definition of AIDS was revised to include HIV-infected persons whose CD4+ Tlymphocytes were ≤ 200 per µl, even in the absence of opportunistic infections or neoplasm.
Both the CDC (Center for Disease Control) and WHO (World Health Organization) have emphasized the need to study lymphocyte subsets in normal populations [3]. It is thus necessary for a testing laboratory to establish normal ranges for these subsets in its local population. A number of studies have been done on lymphocyte subsets in healthy individuals. There is, however, a paucity of published data about Tunisians and North Africans in general. Therefore, our first objective in the present study was the establishment of normal ranges for CD3+CD4+ and CD3+CD8+ cells in a healthy Tunisian population. After that, the second step was the assessment of lymphocyte subsets in HIV infected individuals.

Subjects
The population studied comprised 52 healthy blood donors of both sexes, aged from 19 to 56, and 53 HIV-1 seropositive patients, also of both sexes and aged from 23 to 57.

Flow cytometric analysis
Aliquots of blood samples were stained with a panel of TriTEST reagents, using TruCount Absolute Count tubes (Becton Dickinson). Data were collected on the cytometer (FacsCalibur, Becton Dickinson) using MultiSet software.  Table 3.

RESULTS AND DISCUSSION
This report provides data on CD3+, CD3+CD4+ and CD3+CD8+ lymphocyte counts, and CD4:CD8 ratios, in an HIV seronegative North African (Tunisian) population (Tables 1  and 2). There is little published data of this type.
When we compare these Tunisian normal values to results obtained in other countries, we note that: Both the percentage and the absolute count of CD3+ and CD3+CD4+ cells are very similar to those published in French [2], Belgian [7] and Australian studies [3]. Both the percentage and the absolute count of CD3+CD8+ cells are slightly decreased compared to those obtained in the study of Blanc et al. [2] using the same cytometer mark, the same reagents and the same analysis method, but the differences are not significative.
Finally, when we consider the CD4:CD8 ratio calculated from the percentages of CD3+CD4+ and CD3+CD8+ cells, we note that it is much higher than the value reported by Hannet et al. [7] (average = 1.2; min = 1; max = 1.5) and could be explained by the high CD8+ value in this Belgian study.   Comparing HIV seronegative patients with HIV seropositive subjects, the latter group has significantly lower percentages and numbers of CD3+CD4+ cells (p < 10 −3 ), higher numbers of CD3+CD8+ cells (p < 10 −3 ) and a lower CD4:CD8 ratio (p < 10 −3 ). There is no difference in the percentage and the number of CD3+ cells between these two groups.
These results are in accord with those obtained all over the world. The depletion of CD3+CD4+ cells is not clearly explained at present; however, an increase of the CD3+CD8+ cells is explained by expansion of the cytotoxic T-lymphocyte population during HIV infection.
We used, in our laboratory, the CD38 marker in a two-stain combination with CD8, in HIV seropositive samples, and we found that nearly all of the CD3+CD8+ cells were CD38 positive, concluding that these cytotoxic T-lymphocytes were activated, most probably by the virus.
Considering the distribution of HIV seropositive patients according to their clinicobiological stage (CDC classification; 1993), we note that the majority of patients are in the second (43.6%) and the third (51.3%) clinical categories (B and C), and most of them are in the third biological category (20.5% B3; 43.6% C3). The paucity of subjects of the first clinical category (A) in our data can be explained by the fact that the HIV seropositive patients are diagnosed late, and when they come to our laboratory for a T-lymphocytes subsets count, they are already at the B or C category. The lack of antiretroviral medicines and the natural progression of the disease also explain the bigger number of patients in the third biological categories with an average number of 75 CD3+CD4+ cell/mm 3 for the 20.5% patients (B3 category) and 57 CD3+CD4+ cell/mm 3 for the 43.6% patients (C3 category).

CONCLUSION
In Tunisia, HIV infection is relatively not extensive: among the 10 million inhabitants, the total number of declared seropositive individuals since the beginning of the epidemic until now, is one thousand (0.01%); two hundred of them have AIDS and every year, there is a mean of 46 new declared cases. HIV infection is not a major problem for Public Health in Tunisia, ahead of other pathologies like hepatitis or cancers. However, the extent of the epidemic in the world causes us to be worried about the future. Therefore, we started in our laboratory by establishing normal ranges for T-lymphocytes subsets counts that could be a reference for all North Africa. Following this, we can monitor the efficacy of treatment available to our patients.