Key Words

Reference values – cell blood count – reference interval.

Introduction

The concept of reference values was conceived by a group of Scandinavian scientists in the 1970s, and then developed by many works of French and Spanish Societies as well as the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC-LM) And the National Committee for Clinical Laboratory Standards (NCCLS) in the United States during the 1980s. The reference values are the different values that can be obtained from the results of biological tests in healthy individuals. They are in a range format with a lower and upper limit determined according to the international recommendations [1].

Nevertheless, these “normal” values change according to the technical analysis used and according to several parameters, such as age, sex, geographical origin, altitude, pregnancy, alcohol consumption, tobacco or medicines [2]. For this, their determination for each country, or even each region, is important.

This study is motivated by the absence of references values of Moroccan adult population. Its aims were to determine the references values of hematological parameters in a sample of Moroccan population and compare the findings with those available in the literature and similar studies.

Materials and Methods

The selection of the reference sample is the key to success of an accurate determination of the reference values. We have chosen to work with adults, military personnel from all regions of Morocco, who present themselves at the Blood Transfusion Center of the Military Teaching Hospital in Rabat. The choice of donors is based on the possibility that the population is theoretically in good health and that it receives a medical consultation with a pre-donation examination eliminating any suspicion of illness. An information sheet specific to each donor is completed by the doctor who authorizes or prohibits the donation. It includes personal information such as sex and age, a history of allergy, viral, bacterial or parasitic infectious disease, stay abroad, transfusion, surgery, medication or vaccine. Consent was obtained for all participants. Were included in our study, any Moroccan adult male and female, able to donate after the medical consultation. Were excluded, any male under 18 or over 55 years old and a woman under 18 or over 45 years old, pregnancy, suspicion of disease and any positive serology (HIV, HBV, HCV, Syphillis) performed systematically after the blood donation.

Donors who are eligible after the medical consultation are registred. The technician assigns them an identical serial number in tubes, donation pockets and the registry. This number allows the identification of the donor at any time of analysis and links the biological sample to the donor and to the obtained result. Venous blood sampling is performed under vacuum in a 4 mL tube with Ethylen Diamin Tetra Acetic (EDTA). Once the samples are taken, they are sent as soon as possible in racks to the Laboratory of Hematology for analysis in a manner that abides by the personnel’s safety rules and the integrity of the sample. The test tubes are carefully stored. The analysis was realized with BECKMAN COULTER LH 750 HEMATOLOGY ANALYZER. Every morning the technician carries out the controls using normal blood control, low and high pathological blood controls. The parameters studied are: Red Cells, Hemoglobin, Hematocrit, Mean corpuscular volume, Mean corpuscular hemoglobin concentration, Cell distribution index, Platelets, Mean platelet volume, Total and differential white cell count. The results obtained are entered and recorded in the SPSS version 16.0.

Results

The number of donors participating in the study was 1215. After removing the samples with positive serology, abnormalities in biochemical parameters, the number of participants remaining was 996. 874 men (87.7%) and 122 women (12,3%) with a sex ratio (M / F) of 7.1. The age varies between 18 and 55 years with an average of 25.17 years for men and 18 and 45 years with an average of 23.08 years for women.The statistical analysis of our database revealed the presence of some aberrant values which cannot be considered as reference values. To remedy this, these values were eliminated by dividing the values into several percentiles so as to retain only the reference values between the percentile at 2.5 and 97.5 and to abolish all value outside this range. This statistical method was adopted in all the studies carried out for the determination of the reference values of the hemogram [2,3]. Details of results are shown in table 1 and 2.

Table 1: Results of satistical analysis of our study

Central trend and scattering parameters Min – Max
WOMAN MAN WOMAN MAN
GB (G/L) 7 ,79 ± 1,67 8,70 ± 2,38 [4,40-12,90] [4,70-21,70]
PNN (G/L) 5,14 ± 1,65 5,33 ± 1,98 [0,70-10,60] [0,80-16,60]
LYM (G/L) 2,06 ± 0,87 2,55 ± 0,97 [0,10-5,40] [0,10-7,10]
MON (G/L) 0,34 ± 0,73 0,52 ± 0,53 [0-5,70] [0-7,40]
EOS (G/L) 0,20 ± 0,21 0,23 ± 0,20 [0-0,90] [0-1,50]
BAS (G/L) 0,04 ± 0,16 0,06 ± 0,15 [0-1,20] [0-1,50]
GR (1012/L) 4,66 [4,40-5] 5,79 [5,43-6,17] [3,70-6,86] [2,55-8,52
Hb (g/dL) 13,27 ± 1,58 17,05 ± 1,63 [10-20] [9,10-23,20]
Ht (%) 41,92 ± 5,33 52,64 ± 4,44 [29,50-64,60] [27,30-69]
MCV (fL) 88,89 ± 6,97 90,67 ± 5,69 [64,90-100,40] [62,50-115,90]
MCTH (pg) 28,12 ± 2,92 29,35 ± 2,01 [18,50-32,90] [18,90-35,80]
MCHC (g/dL) 31,59 ± 1,54 32,40 ± 1,28 [27,00-34,10] [27,30-43,90]
CDI (%) 15,06 ± 2,09 13,98 ± 1,22 [11,07-23] [11,40-21,90]
PLT (G/L) 228,06 ± 54,04 200 ± 72,87 [114-373] [45-883]
MPV (fL) 9,67 ± 1,37 9,53 ± 1,30 [6,80-13,10] [1-14,50]

Table 2: Reference values after correction

Central trend and scattering parameters The reference interval after correction
WOMAN MAN WOMAN MAN
GB (G/L) 7 ,79 ± 1,67 8,70 ± 2,38 [5,10-11,58] [4,60-13,81]
PNN (G/L) 5,14 ± 1,65 5,33 ± 1,98 [2,04-9,46] [1,60-9,80]
LYM (G/L) 2,06 ± 0,87 2,55 ± 0,97 [0,42-4,18] [0,58-4,41]
MON (G/L) 0,34 ± 0,73 0,52 ± 0,53 [0-1,18] [0-7,40]
EOS (G/L) 0,20 ± 0,21 0,23 ± 0,20 [0-0,80] [0-0,80]
BAS (G/L) 0,04 ± 0,16 0,06 ± 0,15 [0-0,20] [0-0,50]
GR (1012/L) 4,66 [4,40-5] 5,79 [5,43-6,17] [3,90-5,96] [4,83-7,03]
Hb (g/dL) 13,27 ± 1,58 17,05 ± 1,63 [10,10-16,97] [13,80-20,21]
Ht (%) 41,92 ± 5,33 52,64 ± 4,44 [31,39-51,64] [43,38-61,60]
MCV (fL) 88,89 ± 6,97 90,67 ± 5,69 [69,41-100,80] [77,97-100,56]
MCTH (pg) 28,12 ± 2,92 29,35 ± 2,01 [19,91-31,97] [24,80-32,30]
MCHC (g/dL) 31,59 ± 1,54 32,40 ± 1,28 [27,50-33,90] [29,68-34,20]
CDI (%) 15,06 ± 2,09 13,98 ± 1,22 [12-20,91] [12,18-16,82]
PLT (G/L) 228,06 ± 54,04 200 ± 72,87 [121,12-341] [108-327,25]
MPV (fL) 9,67 ± 1,37 9,53 ± 1,30 [7,41-12,70] [7,30-12,20]

Discussion

The theory of reference intervals was developed to try to answer the question: Is the result of the analysis normal, too high or too low? It aims to describe as accurately as possible the variations of biological parameters used in medicine in subjects considered to be in good health. On the other hand, it is one of the factors contributing to medical decisions, taking into account the specificities of each patient [1], but can also be used in clinical trials as an inclusion / exclusion criterion or for the following of who participate [3]. The different references values of hematological parameters available are the results of studies carried out on essentially in west African or European populations [3,4]. Given the diverse biological and social differences between countries, it is essential to determine references values for each country or validate one of the available values [5]. Details are shown in tables 3 and 4.

Table 3: Female sample results compared to French and Ghanain studies

French study [3] Ghanain study [5] Our study
GB (G/L) 3,91 – 10,88 3,40 – 9,30 5,10 – 11,58
PNN (G/L) 1,74 – 7,10 1,50 – 5,60 2,05 – 9,46
LYM (G/L) 1,26 – 3,64 1,20 – 4,40 0,42 – 4,18
MON (G/L) 0,20 – 0,66 0,20 – 0,90 0 – 1,18
EOS (G/L) 0,04 – 0,52 0,05 – 0,5 0 – 0,80
BAS (G/L) 0 – 0,08 0,01 – 0,05 0 – 0,2
GR (1012/L) 3,96 – 5,12 3,09 – 5,30 3,9 – 5,96
Hb (g/dL) 11,70 – 15 8,8 – 14,40 10,10 – 16,97
Ht (%) 34,70 – 44,40 26,40 – 45,00 31,39 – 51,64
MCV (fL) 78,40 – 95,30 73,00 – 96,00 74,00 – 100,08
MCTH (pg) 26,10 – 32,50 22,30 – 33,60 19,91 – 31,97
MCHC (g/dL) 31,90 – 35,80 30,40 – 36,50 27,50 – 33,90
PLT (G/L) 186 – 440 89 – 403 121,13 – 341
MPV (fL) 7,50 – 10,9 12,6 – 22,9 7,41 – 12,70

Our sampling involved 996 participants. In the French and Ghanain studies, the number is respectively 33,258 and 264 participants [3,4]. Our sample is validated according to IFCCLM and the CLSI (number of individuals ≥ 120) regarding the number, but these recommendations specify that this number is required for each group [1]. Knowing that our sampling is selective, we think it would be wise to select other groups of the Moroccan population to differences and similarities in the different categories of our population.

Table 4: Men sample results compared to French and Ghanain studies

French study [3] Ghanain study [5] Our study
GB (G/L) 4,08 – 10,81 3,50 – 9,20 4,60 – 13,81
PNN (G/L) 1,82 – 6,81 1,50 – 5,90 1,60 – 9,80
LYM (G/L) 1,27 – 3,77 1,20 – 5,20 0,59 – 4,41
MON (G/L) 0,23 – 0,74 0,20 – 1,40 0 – 1,44
EOS (G/L) 0,04 – 0,56 0,05 – 0,5 0 – 0,80
BAS (G/L) 0 – 0,09 0,01 – 0,05 0 – 0,50
GR (1012/L) 4,39 – 5,68 3,79 – 5,96 4,83 – 7,03
Hb (g/dL) 13,40 – 16,70 11,30 – 16,40 13,8 – 20,21
Ht (%) 39,20 – 48,60 33,20 – 50,50 43,38 – 61,60
MCV (fL) 80,20 – 95 70 – 98 77,97 – 100,56
MCTH (pg) 27,20 – 32,80 22,70 – 33,50 24,80 – 32,30
MCHC (g/dL) 32,40 – 36,30 30,60 – 36 29,68 – 34,20
PLT (G/L) 171 – 397 88 – 352 108 – 327,25
MPV (fL) 7,4 – 10,8 12 – 23,4 7,30 – 12,20

Participants in our study were divided into 874 men and 122 women with a sex ratio of 7.1. Women represent only 12.2% of our cohort, and all results must be interpreted with great caution. So we will content ourselves with commenting on the results of all the parameters of our female sample without drawing conclusions before complementing it with other studies with a more representative female population. Female predominance is found in the French study (19,612 men vs 13,646 women) [3] and the Ghanain one involved as many men as women (132) [5]. The mean age for male population is 25.17 years (18-55). 25% of participants are under 20 years old and 75% are under 30 years of age. The youth of our staff is due that the blood transfusion center often receives young recruits and young military personnel for blood donations. For women, the average age is 23.08 years (18-45), 25% are under 20 years old and 75% are under 25 years old. In addition, the age of women was limited to 45 years because during the study, only 5 women between the ages of 45 and 55 could be pre-selected because few military women are still active in this age group. In the Ghanain study, the average age is 36 years (18-59), while in the French study, the average age is 42.5 years (16-69). These averages are higher and can be explained by the participation of people aged over 55 in both studies: 18.83% in the study carried out in France for example [3].

The reference intervals of white cells are identical in women and men [2]. But in our study, as in the French and Ghanaian studies, the values found are outside the published reference intervals for both sexes. The leucopenia of black subjects [6,7] is confirmed in the Ghanain study with limits lower than 3.4G/l and 3.5G/l [5]. For neurophiles, the intervals found in our series are wider with higher values. The lower limit (1.6G/l) is close to the reference value adopted in the laboratory (1.5G/l) and those found in the Ghanain study. The limit found in women is much higher (2.05G/l). The maximum values for women (9.46G/l) and for men (9,8G/l) are higher than the published values as a whole. In the French study, the lower limit for both sexes is also higher but the maximum value is lower than the value adopted in our laboratory. In Ghana, however, the neutrophils reference range is tighter with a lower limit of 1.5G/l and a upper limit of 5.6G/l and 5.9G/l respectively for man and woman. This decline in the number of neutrophils is recognized for black people and is called ethnic neutropenia [2,8,9]. For lymphocytes, we find that the reference intervals found in our study and those of the recent studies are either broad (our study) or tight (French study) compared to published references values. The lowest values are ours: 0.42 and 0.59G/l. These values are recognized as pathological in all of the hemogram references values published in the literature [9,10]. These values require confirmation in our population. The maximum value found in our study and that found in Ghana are higher than that adopted in our laboratory. These values are considered pathological [9,10]. For monocytes in both sexes, there was a large difference between the values obtained in our study and those in the French and Ghanaian studies: our minimum values were lowest (0.01G/l) and the maximum values found for men in our study (1.44G/l) are identical to that of Ghana. These values are higher than those adopted in our laboratory. For eosinophils, the maximal values found in both sexes (0.8G/l) are higher compared to the French study (0.5G/l). It should be noted that in our study more than 95% of the participants had a rate <0.6G/l. It could be inferred from this finding that either the definition of hypereosinophilia should be changed in our context or, before drawing conclusions, a parasitological exam should be systematic to eliminate parasitic infections in subsequent studies. In the Ghanaian study, eosinophils references values were not included. In a study in East and South Africa [11], an increased value (1.53G/l) was found. Its probably due to the prevalence of parasitic infections in the continent. The lower limits in our study are identical to those of the references values adopted in the laboratory, while that of the French study [3] is higher (0.04G/l). For basophils, reference interval for men in our study is wider than in the French study and the references values adopted in our laboratory with a higher upper limit (0.50G/l). While for women, the values found coincide with those adopted in the laboratory. The interval of the French study is narrower and therefore more precise. Very similar results were obtained in the United Kingdom [12].

The number of red cells differs visibly by sex. This difference is well proven [2]. For men, the reference interval has a width more or less equal to that of the Ghanaian study, contrary to the French study and the literature that are narrower. Our minimum values (4.83 × 1012 / L) and maximum values (7.03 x 1012 / L) are higher. For women, the minimum value (3.90 x 1012 / L) is almost the same for our study and those in the literature. In the Ghanain one, the minimum value is lower (3.09 x 1012 / L). For the maximum value it is higher in our study (5.96 x 1012 / L). Other data in the literature admit as a minimum value for women 3.90 x 1012 / L [10]. The rate of hemoglobin differs according to age, physiological state and sex [2]. For men, the minimum (13,80 g / dL) and the maximum value (20,21 g / dl) are higher than the published ones. In Ghana, these two values are lower. In France, the minimum value is discretely higher and the maximum value lower. For women, the minimum value (10.10 g / dL) is lower than those found in the literature. This value defines anemia. This low value was found in the Ghanaian study with a Hb level at 8.80 g / dL. In France, values are also discreetly low. This decrease in Hb is not explained by the incidence of hemoglobinopaties in the black population and its etiology remains debated [2,9,13]. This rate may also be explained by nutritional behavior of populations. In our study, low Hb may be due to our selection criteria that exclude post menopausal women of the study, so menstruation may explain the low level compared with men, especially since a difference in the rate of Hb is established by the Regional Institute for Health [14]. But no conclusion can be selected because our female sample is very small and other studies with more representative samples are needed to confirm this conclusion. Regarding hematocrit, the width of the reference interval of our study for men is similar to that of the Ghanaian study. But the minimum value (43.38%) and the maximum value (61.6%) are both higher than those published. For women, our minimum value (31.39%) is between Ghana and France, while the highest value (51.64%) is the highest of all studies. For the MGV, CCMH, and TCMH, literature allows the same results for both sexes, but we will analyze the results of these parameters separately for men and women because differences were raised. Concerning the MVG, for men, the minimum value (77.97 fL) is lower than that found in France and that published in the literature but higher than the Ghanaian study. The maximum value (100.56 fL) is higher. For women, the baseline is almost identical to the Ghanaian study with a minimum value (74.00 fL) between Ghana and France and a higher value (100.08 fL). Concerning the TCMH, for men and women the minimum values found in our study and those in Ghana are lower than those of France. The lowest value is ours (19.91pg). For the upper limits found, they are identical to the published values Those of Ghana and France are discreetly higher. Concerning CCMH, for men: the reference range for our study is tighter, with minimal values (29.68 g / dL) and maximum (34.20 g / dL) lower than published studies. For women, the same distribution of values as in humans is observed, with a minimum value (27.50 g / dL) and a maximum value (33.90 g / dL) lower than the other studies. It is noted that the lowest values for Hb, TCMH and CCMH are found in the two African countries: Morocco and Ghana. These three erythrocytic parameters are calculated from the level of HB, HTE and the number of GR. Changes to the latter are therefore the cause of changes affecting the VGM, the TCMH and the CCMH. For platelets, the reference interval is tighter, with lower inferior values than those of the literature and those found in France for both men and women. Lower values are noted in Ghana (88 x 109 / L). Our values are considered pathological values defining thrombocytopenia. However, a platelet count on citrate tube should be performed to confirm these results. The lower values of the French study (186 x 109 / L, 171 x 109 / L) are above published values. The upper limits in our population (341 x 109 / L, 327.25 x 109 / L) are lower than the values of recent studies [3,4]. It should be noted that, despite similarities in the variations in female and male values, the reference range for women is significantly higher than for men. This observation has been well established in previous studies [10, 14, 15]. For MPV, both men and women, the minimum values are close to those of the French study, while the Ghanaian study is much higher and higher.

Conclusion

The hemogram is undeniably the biological examination most prescribed by physicians because it allows to orient towards other explorations or to institute a treatment when a disease is diagnosed. References values are changed according to several parameters such as age, sex, smoking but also ethnic origin, pregnancy and the consumption of drugs or alcohol. The frequent combination of these factors leads to the achievement of reference values for each population and even for each analysis laboratory for a better interpretation of the results. Our study revealed differences from the commonly used reference values. These disparities will subsequently have to be studied more thoroughly and extended to a more quantitatively and qualitatively better distributed population on the national territory in order to make them more reliable so that they can be used.