Range-reference determination of lymphocyte subsets in Moroccan blood donors

Background: Information on lymphocyte populations (T, B, and Natural killer cells) and subpopulations (CD4 and CD8) in Morocco is scarce if not inexistent. Objective: To establish a reference value of these cells in 242 Moroccan young adult blood donors by flow cytometry. Results: Smokers had significantly higher total leukocyte count (p < 0.001), total lymphocyte count (p < 0.0001) and higher CD3+CD4+ cells (p < 0.0001). The percentage of CD3-CD56+ subsets was affected by smoking (p < 0.01). Our analysis positively correlate with previous observations of an increase of absolute CD4+ T cells, with no changes in other lymphocyte subset cells in smokers. The lymphocyte subpopulation distributions for all antigens were found to be similar to those reported in Saudi and Italian adults, while higher levels were reported for the same gender in other countries, especially Ghana and Kuwait. Conclusion: The international classification standards of the HIV-infected subjects according to their rates of CD4 are applicable to the present study’s population.


Introduction
Human peripheral blood immunologic cell counts may vary among healthy individuals in terms of age, sex, stress, race, smoking habits, diet and physical exercise. 1,2,3,4,5,6 Thes variations may also be due to pathologies that involve the immune system (infections, acquired immunodeficiency syndromes, immuno-heamatological and auto-immune disorders), medication or during immunotherapy. 7ast research has demonstrated country-related differences of CD4 ranges, which are essential biomarkers in the following and the monitoring responses to antiviral therapies in Human Immunodeficiency Virus-infected individuals. 8n this regard, it is of great importance to establish a referential range of lymphocyte subsets (more importantly CD4) for each population; thus, differences between normal and pathologic values can be established, knowing that these pathologic values cannot be reliably quantified if they are not rigorously controlled and compared to the normal ones.These values may be determined through outcome measures such as flow cytometry, being such an easy, straightforward method for immunophenotyping lymphocyte subsets.Therefore, our study was carried out to establish reference value for lymphocyte subsets in a group of healthy Moroccan blood donors by flow cytometry.

Study population
242 healthy military blood donors were the informants in this study.They were all considered free of clinical infection; check-up of blood pressure, pulse rate, and hemoglobin were controlled in the Rabat Military Hospital Blood Transfusion Center.All donors were screened for syphilis, HIV 1,+2, , viral hepatitis B and C infections by routine serological tests respectively Biokit hemaglutination assay, Biorad HIV 1,+2, , Enzygnot AgHBs and Murex HCV and were negative.Blood samples were collected in EDTA-tubes and used within 6 hours of storage at temperature room.Specimens including 222 males and 20 females were analyzed, as well.
The panel used was the same as recommended by the CDC for performing CD4+ T-cell determinations in individuals infected with HIV. 9 Lymphocyte analysis was carried out with a particular attention to the purity of the lymphocyte gate: more than 95% was the maintained purity level.T Lymphocytes were defined with CD3 expression.As to the lymphocyte sub-populations, only cells with double staining were considered positive for T4 and T8 cells respectively CD3+CD4+ and CD3+CD8+ cells.B and NK (Natural Killer) cells were CD19+CD3-and CD56+CD3-respectively.
We resorted to a dual-platform approach to generate absolute lymphocyte subset cell counts using a hematology analyzer (ACT-10).The absolute values for T4 and T8 cells were obtained by multiplying the percentage of lymphocytes which co-express CD3 and CD4, CD3 and CD8 and that the value obtained by the absolute lymphocyte count.CD19 and CD56 absolute values were obtained by multiplying their percentages by the absolute lymphocyte count.The lymphocyte recovery and the consistency of some check sums were rigorously performed as internal quality controls.

Analytical tools
Differences between means of males and females, smokers and non-smokers were compared by Kruskal-Wallis test.The percentages were compared by ANOVA.The data of hematological studies were used for comparison.p-values less than 0,05 were considered to reflect the significance level.

Ethical consideration
All donnors gave informed consent and the donation was voluntary.

Results
The age of the scrutinized population ranged between 19 and 49 years with a means value of 27,8 years.The distribution of individuals according to their sex and smoking habits is shown in table I: The predominant sex in the studied group was male.Indeed, the sex ration M/F is 11,1: Table 1 shows that 71% of individuals are non-smokers.Table 2 shows the complete blood count for the study group.The absolute counts of human leukocyte and total lymphocyte in the study population are respectively 6,822x103±1,687x103 and 1876±500 cells/μl.No statistically significant differences were found between males and females.The relative and absolute numbers of lymphocyte subpopulations with their corresponding standard deviations are given in table 3.For normal values of various lymphocyte components, we found that the mean values of 1876 cells/μl (28,44%) for total lymphocytes, 871 cells/μl (53%) for CD3+CD4+ cells, 637,54 cells/μl (33%) for CD3+CD8+ cells, 211 cells/μl (11,73%) for CD3-CD56+ and 159 cells/μl (5,26%) for CD19.The p values were determined by the Kruskall-Wallis test.
In contrast, absolute leukocyte counts according to smoking status showed several significant differences in lymphocyte subpopulations between smokers and non-smokers.In addition to an increased number of peripheral white blood cells, smokers also had a higher total count of blood lymphocytes.This increase is mostly due to an increase of T lymphocytes (CD3+), especially of CD4+ lymphocytes.Statistically, no significant increase could be detected for CD8+ cells, B and NK lymphocytes.

Discussion
The present study was undertaken to determine relative and absolute numbers in human peripheral blood lymphocyte subsets among 242 Moroccan blood donors to establish a reference range.Our population was relatively young, with 90% of male sex and 70% non-smokers.This is not surprising for Moroccan military population, because the majority of the Moroccan military people are males, retiring at 50 years old.All individuals had a normal complete blood count.To the best of our knowledge, this is the first study reported for the Moroccan population concerning relative and absolute baseline lymphocyte/μl subsets.
The number of the circulating white blood cells was shown to be influenced by smoking habits, which confirmed the previous data concerning the impacts of cigarette smoke on blood cells.Indeed, leukocytosis in smokers has been well established based on cross-sectionaland longitudinal studies in the general population. 10,11Moreover, the absolute CD4 lymphocyte counts were higher in smokers than in non smokers.In contrast, the other lymphocyte subpopulations investigated were not affected by smoking.The smoking effect has been reported to increase the number of T cells and mainly CD4 peripheral blood lymphocytes. 11,12hen we compared lymphocyte subset counts (CD4 and CD8) found in the present study with other published series in other countries such as Saudi Arabia and Italy similar ranges were found. 13,14In contrast to other countries, especially Ghana and Kuwait, higher level of CD4 cells was reported for the same gender. 15,16In those studies, the smoking habits of healthy control groups were not reported.Despite using the same panel of monoclonal antibodies as recommended by the CDC 9 observed differences might be due to differences in the methodological variables which were not subject to any restrictive criteria due to a lack of a wide consensus such as lysing solutions, time and temperature of sample incubation, fixative employment and staining technique. 14,17,18We have used a dual platform to determine the lymphocyte subset values; but did not use triple or quadruple staining, which might reduce inter-laboratory variations as reported in many studies 19,20 In addition, these differences of lymphocyte numbers can be associated with sex and age.Population parameters, however, were not well defined.

Conclusion
This study cannot be representative of Moroccan population.However, one can consider the values that we found as nor mal values in a young representative sample and provide the background level of immunity in the general population aged 19-49 years.CD4 counts offer a measure of a person's immune function and can also be used to monitor trends in infection in the young population over time.The smoking habits of healthy control groups, however need to be taken into account especially when comparing the numbers of T cells and CD4 lymphocyte subpopulations from patients with different diseases to normal controls.Moreover, the value added of the present study resides in its finding vis-à-vis the age group of people most affected with HIV.
Through this study, we concluded that the international standards of classification of the HIVinfected subjects according to their rates of CD4 are applicable to the young Moroccans population.