Establishment of the hematology reference intervals in a healthy population of adults in the Northwest of Morocco (Tangier-Tetouan region)

Introduction Among the most useful biological examinations in common medical practice, blood count is the most prescribed. The reference intervals of the hematological parameters of this examination are of major importance for clinical orientations and therapeutic decisions. In Morocco, the reference values used by the laboratories of medical biology and used by doctors are ones collected from Caucasian and European individuals. These values could be different in the Moroccan population. Besides, reference intervals of the blood count specific to the various Moroccan regions are missing. We decided to determine the reference intervals from a population of healthy adults of the Tangier-Tetouan region by following the procedures recommended by the IFCC-CLSI guidelines in 2008 and comparing them to those of the literature. Methods Blood samples were taken from 15840 adult volunteers (8402 men from 18 to 55 years old and 7438 women from 18 to 50 years old) from the regional transfusion center of Tangier and Tetouan during a period between November 2014 and May 2016. The complete blood count was measured by the Sysmex KX21N® analyzer. For each sample a systematic blood smear was done to determine the leukocyte differential. The data analysis was made by the software SPSS 20.0 by using percentiles 2.5th and 97.5th. Results A significant difference between both sexes was noted (p<0,001) for all the hematological parameters (red blood cells, hematocrit, hemoglobin, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, leukocytes, neutrophils, basophils, eosinophils, monocytes, platelets and mean platelet volume) except for the numeration of lymphocytes (p = 0.552). The values of this study were compared with those reported in Arabic, Caucasian and African populations. Said comparisons showed the existence of significant differences. Conclusion This study tries to accentuate the necessity of proceeding with the establishment of reference intervals specific to the blood count of the Moroccan population to avoid errors of diagnosis, allow clinicians to interpret with greater specificity the hematological examinations and to improve the quality of medical care distributed to patients.


Introduction
The exploration of hematopoiesis begins routinely with the establishment of the complete blood count which provides the erythrocytes, leukocytes and platelet values of an individual as well as the morphological characteristics of these blood cells [1]. Indeed, a wide variety of pathologies can result in modifications of the blood count [2]. It is, undoubtedly, one of the most prescribed biological examinations and among the most useful in common medical practice for the evaluation of the state of health in not only sick but also healthy subjects. It is by means of this examination that specific diagnoses can be suggested and that a hematological

Methods
The pre-analytical phases and the analytics of our study were  morphological anomalies of the figurative elements of the blood observed in the blood smear (hypochromia, target red blood corpuscles, Plasmodium, etc). By adopting these criteria, only 14965 reference subjects were finally retained for the study among which 7930 men and 7035 women. It is necessary to note, nevertheless, that our conditions of study were unable to ensure the exclusion of possible patients presenting an iron deficiency and/or affected by thalassemia/hemoglobin diseases. Every subject participating in the study gave their consent freely according to ethical standards. This study was approved by the Regional Health Committee of the Tangier-Tetouan-Al Hoceima region.
Blood sampling: In our study, we followed the standard protocol of taking and preparing of blood samples to minimize the interpersonal variability. To avoid the daily variations of the blood cell parameters and those associated to physical exercise which influences the rate of hemoglobin and that of the leukocytes, we chose a morning schedule for the blood sampling (between 8 and 12 am) after a rest of at least 15 minutes [13,14]. For every VBD, blood samples were withdrawn before the collection of blood from the antecubital vein, in system BD Vacutainer® tubes (13×75 mm) of 5ml containing an anticoagulant the K3-EDTA. Analysis of the samples was performed the same day within 6 hours of collection.
Blood count results according to age: The means, standard deviations, medians and reference intervals of the various parameters of the blood count for men and women according to age groups are presented, respectively, in Table 3 and Table 4. The study showed variations of certain erythrocyte parameters according to age. A significant difference of the values of HGB, HCT, MCV, MCH, MCHC according to age was noted (p < 0.001) for men except for the RBC (p = 0.062). In the women's group, the HGB and the HCT showed a significant difference according to age (p = 0.002 and p<0.001, respectively). For other erythrocyte parameters, we noticed no significant difference, according to age. For leukocyte lineage, the study revealed a slight decrease in the total number of the WBC for the age between 51-55 years for men and the age group 40-50 years for women, but this decrease was not statistically significant for either sexes. The value of the median number of the NEU in each of the age groups was higher for woman than for men without any of significant difference according to age for both sexes. We observed a significant difference according to age of men in the rates of LYM, MON and EOS (p = 0.009, p = 0.002 and p = 0.003, respectively) while there was no significant difference in the number of BAS with age (p = 0.093). On the other hand, in the female genital organ, we noticed a significant difference according to age of women in the number of MON (p = 0.028) and BAS (p < 0.001) while that of the LYM and the EOS were not statistically significant. The values of the median platelet numeration according to age were superior in women with regard to men. According to age, this difference was statistically significant for men (p = 0.001) and for women (p = 0.002). On the other hand, the values of the median MPV according to age were superior for men than for women. This difference according to age was not statistically significant for men (p = 0.699) nor women (p = 0.257).

Blood count results according to the studied provinces:
The means, standard deviations, medians and reference intervals of various blood count parameters for men and women according to the studied provinces are presented in Table 5 and Table 6, respectively. We noted a variation of the erythrocyte parameters to provinces were superior for women than for men. This difference according to provinces was statistically significant for women (p = 0.045) and not significant for men (p = 0.492). The median values of the MPV according to provinces were superior for men than for women. This difference was statistically significant for men (p < 0.001) as well as for women (p = 0.006).

Discussion
The blood count is the laboratory test most frequently prescribed in common medical practice. It offers precious information because it is able to distinguish a normal situation from a pathological one the NEU (0.5×10 9 /L for both sexes) compared to our study ( Table   7). The complete blood count parameters according to sex showed a significant difference in our study between men and women when  (Table 7). Prevalence of carriers of beta thalassemia is of the order of 3% [30]. Also, quality of food, standard of living as well as a higher index of gestation with, consequently, more gestational losses, represents factors that explain the above-mentioned differences. To reiterate, our study should be understood keeping in mind the impossibility of eliminating from our population of study the subjects presenting an iron deficiency or those affected by hemoglobin diseases. The French study of the Regional Institute for the Health [16] and other African populations (8.5-12.7 fL for women versus 8.7-13.1 fL for men) [40].

Conclusion
This study allowed us to establish the reference intervals of the

Competing interests
The authors declare no competing interests.

Authors' contributions
All authors have read and agreed to the final version of this manuscript and have equally contributed to its content and to the management of the case.

Acknowledgments
The authors thank MEGAFLEX Company which contributed to the elaboration of this study by the reagents, the National Center of Blood transfusion of Rabat, the VBD which agreed to participate voluntarily in this study and all the team of the Regional Center of Blood Transfusion of Tangier and Tetouan where the subjects were received and the serological tests were made. Our thanks also go to the regional manager of health of the Tangier Tables   Table 1: Distribution of the studied population according to sex, provinces and age Table 2: Means, standard deviations, medians and reference intervals of the various parameters of the complete blood count of the studied population according to sex        PLT, platelets; MPV, mean platelet volume.
Mann-Whitney U-test for nonnormally distributed parameters was done between males and females: All hematological parameters except lymphocytes showed significant differences between females and males.
p < 0.05 was considered as statistically significant.
a statistically significant.
b statistically non-significant.