Original Article
Lymphocyte Subsets in Healthy Saudi Donors
Amal Zaghlool AM*, Mariam Mohammed A, Musbuah Hassan A, Ohoud Abdulaziz H, Ohoud Ahmed A, T.A.M.A. Al-bukhari*, H.A. AL-Pakistani*,
AI-Rabia MW*, Tariq H Ashour*
Departments of Hematology and Immunology* and Laboratory Medicine, Faculty of Medicine* and Faculty of Applied Medical Sciences, Umm Al-Qura University, Makkah, KSA E-mail: [email protected]
Received: October 20, 2009 Accepted: December 3, 2009
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ABSTRACT
Background: Lymphocyte and their subsets show variation with race and age.
Aim of the work: The aim of this study was to detect the distribution of lymphocyte subsets in Saudi population (Makkah city) and to compare with different races and with previous Saudi studies.
Subjects and Methods: This study was conducted on 54 healthy subjects; they were 13 males and 41 females with male to female ratio of 1:3. Their ages ranged from 17 to 45 years with a mean age of (22 ± 5.8) years. Lymphocyte and their subsets were analyzed by flow cytometry BD FACS Calibure and their absolute counts were calculated.
Results: The CD3, CD4, CD8, CD16 and CD69 were expressed as percentage and absolute counts. They were compared to other studies (Ethiopia, Dutch, Singapore, Hong Kong and two previous Saudi studies). For all the studied CD markers, all the absolute counts of the different CD markers had higher significant values (P< 0.01) as compared to other studies expect for CD4 of the Dutch study and CD8 of one Saudi study which showed no significant differences from our study (P> 0.05). The CD4/CD8 ratio had higher significant values when compared to other studies (P<
0.01), except for the Dutch study and another Saudi study which showed significant decrease(P<0.05) and no significant difference (P> 0.05) when compared to our results respectively. There were no significant differences when comparing the males to the females groups as regards percentage and absolute counts of the different CD markers studied, except for significant increase of CD3 percentage in the females group (P< 0.05) and higher significant values of CD69 absolute counts in the males group (P< 0.01).
Conclusion: Most of the absolute counts of the different CD markers studied in our work (CD3, CD4, CD8, CD16 and CD69) and the CD4/CD8 ratio are higher when compared to different populations. Our study suggests that there may be true genetic differences in lymphocyte subsets. Every population must establish a normal range of their own; it is recommended that samples from several other areas in Saudi Arabia should therefore be studied to confirm our data.
Keywords: Lymphocytes subsets, flow cytometry, healthy donors.
INTRODUCTION
L
ymphocytes are the primary cells of an immune response. Most lymphocytes are located in lymphoid tissues and only a small portion circulates in the blood. The most important types of lymphocytes are the T cells, B cells, mature B cells (plasma cells) and natural killer cells (NK) 1. Mature T lymphocytes circulate in the blood stream and the lymphatic system, where they search for foreign or abnormal cells and can attack them1.The CD3 antigen is a protein complex that associates with molecules known as T cell receptor (TCR) and the ~ chain to generate an activation signal in T lymphocytes. The TCR, ~ chain and CD3 molecules together comprise the TCR complex2.
CD4+ and CD8+ T cells have different functions and recognize antigens derived from different cellular compartments3,4. CD4+ T cells express a variety of cytokines and cell surface molecules that are important to B cell proliferation, immunoglobulins production, and CD8+ T cell function. Following antigen stimulation, CD4+ T cells differentiate into two major classes of effectors T cells based on their cytokine profiles, which are called T helper type 1 (Th1) and T helper type 2 (Th2l The CD4 molecule is structurally related to immunoglobulins and has an affinity for nonpolymorphic residues on the MHC class II molecule. In this capacity, CD4 presumably increases the efficiency with which CD4+ T cells recognize antigen in the context of class II molecules which are restricted in their expression to B cells, macrophages, dendritic cells, and a few other tissues during inflammation. In addition, the cytoplasmic tail of CD4 promotes signaling by TCR CD8+ T cells are very efficient killers of pathogen-infected cells. Given the ubiquitous expression of class I molecules, mature cytolytic T cells (CTL) can recognize viral infections in a wide array of cells, in distinction to the more- restricted distribution of class II molecules and their recognition by CD4+ T cells.
CTL induce lysis of target cells through the production of perforin, which induces holes in cell membranes, and the expression of Fas ligand (FasL) and TNF-a, which induce apoptosis4.
NK cells are a subpopulation of lymphocytes involved in cellular immune response that are not restricted by the MHC. Normally NK cells comprise up to 15% of peripheral blood lymphocytes. All NK cells are CD3- and CD56+ 6. NK cells are innate immune effectors that exert damage when activated. The CD16, the low affinity receptor for the Fc fragment of immunoglobulin G (IgG) is a major surface structure on NK cells capable of triggering the antibody dependent cellular cytotoxicity and the expression of genes encoding surface activation molecules and cytokines on engagement by IgG opsonized target 7.
The human CD69 differentiation antigen is one of the earliest cell surface antigen expressed after activation of T and B lymphocytes and other cells of the hematopoietic origin. CD69 is transiently expressed on activated leucocytes including T cells, thymocytes, B cells, NK cells, neutrophils and eosinophils. Once CD69 is expressed on T cells, it acts as a co-stimulatory molecule leading to cell proliferation, secretion and or cytotoxicity 7.
AIM OF THE STUDY
The aim of this study was to detect the distribution of lymphocyte subsets by studying their markers CD3, CD4, CD8, CD16 and CD69 in Saudi population (Makkah city) and to compare them with different races and with previous Saudi studies.
SUBJECTS AND METHODS
This study was carried out on 54 healthy persons who attended the blood bank of King Abdulaziz Hospital and King Faisal Hospital, as well as from medical students of Umm AI-Qura University, Makkah. They were 13 males and 41 females with male to female ratio of 1:3. Their ages ranged from 17 to 45 years with a mean age of 22±
5.8 years.
Sample collection
Three ml of blood were withdrawn from each healthy donor under complete aseptic conditions. They were dispensed into a tubes containing EDT A as anticoagulant substances for performing complete blood count (CBC) and different CD markers analysis by Flow cytometry.
Methods
The studied persons were subjected to the followings:
1. Full clinical history to exclude any disease.
2. Complete hemogram analysis on Sysmex XT2000i and differential blood count.
3. Estimation of percentages of CD3, CD4, CD8, and CD69 on T lymphocytes and CD16 on natural killer cells. The cell surface markers of each CD3, CD4, CD8, CD16 and CD69 were assessed by flow cytometry on BD FACS Calibure by direct immunofluoresence technique. Single and dual analyses were done for all samples. The analysis was done on whole blood, and the lysis of erythrocytes was done by FACS lysing solution (lOX) [BD catalogue No.356457]. The CD3 was labeled with ATC (fluorescein isothiocyanate), and the CD4, CD8, CD16, CD69 were labeled to PE (phycoerythrin).The values were expressed in percentage and their absolute counts were calculated. All CD markers were run along with negative isotypic controls (IgG lIIgG2).
Statistical analysis
The Statistical analysis of this study was done using SPSS program. Quantitative data were described in the form of mean ± SD. The comparison between groups was performed by using independent t test. Correlation coefficients were calculated by Pearson's method. A P> 0.05 was considered statistically not significant while a P<
0.05 and < 0.01 were considered significant and highly significant respectively.
RESULTS
The Clinical data of the studied group:
This study was carried on 54 healthy subjects. They were 13 males, and 41 females with male to female ratio of 1:3. Their ages ranged from 17 - 45 years and their mean age was 22 ± 5.8. The samples were taken from medical students of Umm AL-Qura University and from the blood bank of king Abdulaziz Hospital and King Faisal Hospital (Table 1).
Age/years
Mean± SD Median Range S
=
significantTable1: Clinical data of the studied groups Males group
n = (13) 25.4± 7
27 17-38
Females group
n = (41) 22± 5.2 P<0.05 S
21 17-45 n= number
Total (males &females)
n = (54) 22± 5.8
21 17-45
The comparison of laboratory investigations in male andfemale subjects:
Each of total leucocytic count (TLC), Platelets count and absolute lymphocytes count were not significantly different between males and females (P> 0.05) while lower significant concentrations of hemoglobin were observed in the females group when compared to the males group (P< 0.01) (Table 2, Figure 1).
Table 2: The comparison of laboratory investigations in male andfemale subjects:
Males Females group P and significance
group Mean±SD
Mean±SD
TLCX109/L 6.9± 1.5 7.2±2.4 >0.05 NS
Hemoglobin gl dl 13.3± 1.9 1O.8± 1.6 <0.01 HS
Platelets countX109/L 227.2± 6.1 262.4± 70 >0.05 NS
Absolute lymphocytes 3.5± 0.7 3.1± 0.9 >0.05 NS
countX109/L
TLC= totalleucocytes count HS= Highly significant. NS= Not significant
The Comparison between males andfemales regarding CD markers:
There was significant increase of the CD3 percentage in the female group when compared to the male group (P< 0.05). Meanwhile, the absolute count of CD69 had higher significant values in the male group when compared to the female group (P<
0.01). No other significant differences were detected as regards the percentage of CD4,8, 16,69 and the absolute count of CD3, 4, 8, 16 (P> 0.05 ) (Table 3). As there were no significant differences between male and female groups for most of the
results we gathered them as one group. Table 4 shows the descriptive data of this group.
Figure 1: Picture of a normal lymphocyte
Table3: Comparison between males and females as regards CD markers percentage and absolute count
Males group Females group P & significance
Mean±SD Mean±SD
CD3% 66.4± 11.9 75.9± 10.4 <0.05 S
CD3 absolute 2286.0± 618 2399.0± 814 >0.05 NS count X 109/1
CD4% 28.2± 7.7 32.5± 10.5 >0.05 NS
CD4 absolute 964.6± 326 1008.8± 426.5 >0.05 NS count X109/1
CD8% 29.4± 9.4 28.3± 10.8 >0.05 NS
CD8 absolute 1000.9± 323.3 880.6± 436 >0.05 NS count X109/1
CD4/8 Ratio 0.997± 0.4 1.2± 0.5 >0.05 NS
CDI6% 18.6±6.2 19.0± 8.4 >0.05 NS
CD16 absolute count 635.4± 239 669± 351 >0.05 NS X 109/1
CD69% 19.7 ± 9.0 15.4± 6.8 >0.05 NS
CD69 absolute count 713.8± 347 486.2± 253 <0.01 HS X 109/1
Table4: Descriptive data of CD markers in whole group (male and female) studied.
Mean±SD Median Range
CD3% 73.6± 11.5 74.5 51-90
CD3 absolute 2371.8± 768 2372 650-3600
count X 109/1
CD4% 31.5± 10.0 31 15-62
CD4 absolute 998± 402.0 1010 300-2480
count X 109/1
CD8% 28.6± 10.4 28.5 7-55
CD8 absolute 909± 412 930 245-1925
count X109/1
CD4/CD8 ratio 1.7± 0.5 1 0.5-2.5
CDI6% 18.9± 7.9 17 5-46
CD16 absolute count 609.8± 326 520.6 1200-1840
X 109/1
CD69% 16.5± 7.5 15 4-36
CD69 absolute count 541.0± 292 455 40-1320
X 109/1
The Comparison between our data and other published data (Table 5):
In the present study the CD3 (percentage and absolute count) had higher significant values when compared to the studies done in Singapore and Hong Kong (P< 0.01). On the other hand, there was no significant difference of the CD3 percentage when compared to the previous first Saudi study.
The CD4 percentage had significant decrease values when compared to the Singapore study (P< 0.05) and lower significant values when compared to both the first Saudi and Hong Kong studies (P< 0.01). However, the absolute count of CD4 had significant increase when compared with second Saudi study (P< 0.02), and higher significant values when compared to the first Saudi study, Ethiopia, Singapore and Hong Kong (P< 0.01). In addition, no significant difference was detected when compared to the study done by the Dutch (P> 0.05).
As regards the CD8 percentage there were no significant differences when compared to Singapore, Hong Kong and first Saudi study (P> 0.05). On the other hand, the absolute count of CD8 had higher significant values as compared to the first Saudi study, Dutch, Singapore and Hong Kong (P< 0.01). Also, it showed significant increase when compared to Ethiopian study (P< 0.05). However, there was no significant difference when compared to the second Saudi study (P> 0.05).
The CD4/CD8 ratio had higher significant values when compared to Ethiopia, Singapore and Hong Kong studies (P< 0.01). On the other hand it had lower
significant values (P< 0.01) and no significant difference (p> 0.05) when compared to Dutch study and the first Saudi study respectively.
Table 5: Comparison between our data and other published data
Present Saudil m Saudil2O) Eth· . wpm 06) Dutch (18) Singapore Hong study (Males) (Males) (Mixed) (Mixed) (10) Kong(17) N(S4) N(209) N(1S0) N(142) N(136S) (Mixed) (Mixed) N(184) N(208)
CD3% 73.6± 72.9± 7.7 67± 7.4 69± 7.7
11.5 P> 0.05 P< 0.01 P< 0.01
NS HS HS
CD3 2371.8± 1547± 453 1370± 400
absolute 768 P< 0.01 P< 0.01
count HS HS
CD4% 31.5± 10 39.4 ± 7.9 35± 6 36± 7.5
P< 0.01 P< 0.03 P< 0.01
HS S HS
CD4 998± 402 869± 310 880± 270 775± 225 993± 139 812± 255 725± 256 absolute P< 0.01 P< 0.02 P< 0.01 P> 0.05 P< 0.01 P< 0.01
count HS S HS NS HS HS
CD8% 28.6± 27.6± 7.5 27± 7 30± 7
10.4 P> 0.05 P> 0.05 P> 0.05
NS NS NS
CD8 906± 412 615± 278 890± 290 747± 333 506± 220 629± 235 589± 205
absolute P< 0.01 P>0.8 P< 0.05 P< 0.01 P< 0.01 P< 0.01
count HS NS S HS HS HS
CD4/CD 1.7± 0.5 1.6± 0.7 1.2± 0.5 2.2± 1.0 1± 0.6 1± 0.4
8 ratio P> 0.05 P< 0.01 P< 0.01 P< 0.01 P< 0.01
NS HS HS HS HS
CDI6% 18.9± 7.9 11.7± 5.9 18± 7 20± 8
P< 0.05 p> 0.05 P>O.4
S NS NS
CD16 608± 326 262± 178 190± 80 250 ± 137 425± 244 394± 194
absolute P< 0.01 P< 0.01 P< 0.01 P< 0.01 P< 0.01
count HS HS HS HS HS
CD69% 16.5± 7.5 CD69 541± 292 absolute
count
The Correlation coefficients between CD markers and other parameters studied:
There was significant positive correlation between the CD4 percentage and CD8 percentage (P< 0.05). As regards the absolute count of CD4 there were significant positive correlation with CD8 percentage, total leucocytes count and absolute lymphocytes count (P< 0.05).
There were significant negative correlation of the CD8 percentage with CD69 percentage and CD4/CD8 ratio (P< 0.05) (Figure 2). On the other hand the absolute count of CD8 showed significant positive correlation with CD4 percentage, total leucocytes count and absolute lymphocytes count (P< 0.05). No other correlation could be detected (P> 0.05) (Table 6).
CD3%
CD4%
CD8%
CD16%
CD69%
TLCX109/1 Lymph. % Lymph.
Absolute countX109/l Hb g/dl Platelets count X
109/1 Agel years
Table 6: Correlation between CD markers and other parameters studied
CD 4% CD4 CD8% CD8 absolute CD41CD8
absolute Count ratio
count
r p r p r p r P r p
0.1 >0.05 0.2 >0.05 0.08 >0.05 0.2 >0.05 -0.3 >0.05
NS NS NS NS NS
0.7 <0.05 0.6 <0.05 0.4 <0.05 0.2 >0.05
S S S NS
0.6 <0.05 0.4 <0.05 0.8 <0.05 -0.7 <0.05
S S S S
-0.01 >0.05 0.1 >0.05 -0.2 >0.05 -0.1 >0.05 0.2 >0.05
NS NS NS NS NS
-0.3 >0.05 0.1 >0.05 -0.4 <0.05 -0.2 >0.05 0.2 >0.05
NS NS S NS NS
0.04 >0.05 0.3 <0.05 0.1 >0.05 0.3 <0.05 0.1 >0.05
NS S NS S NS
0.23 >0.05 0.2 >0.05 -0.3 >0.05 0.2 >0.05 >0.05
NS NS NS NS 0.04 NS
-0.18 >0.05 0.6 <0.05 -0.08 >0.05 0.6 <0.05 0.1 >0.05
NS S NS S NS
-0.1 >0.05 >0.05 -0.04 >0.05 0.1 >0.05 >0.05
NS 0.1 NS NS NS 0.02 NS
0.2 >0.05 >0.05 0.2 >0.05 -0.4 >0.05 -0.1 >0.05
NS 0.1 NS NS NS NS
0.03 >0.05 0.1 >0.05 0.1 >0.05 0.2 >0.05 -0.2 >0.05
NS NS NS NS NS
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CD4/CD8 ratio
Figure 2: The correlation between CD41CD8 ratio and CD8 %age
DISCUSSION
The main aim of our study was to detect the distribution of lymphocyte and their subsets in healthy Saudi males and females. This study was conducted on 54 subjects;
they were 41 females and 13 males with male to female ratio 1:3. Their ages ranged from 17 - 45 years with a mean age of 22 ± 5.8 years.
In this work, the comparison between males and females showed no significant difference except for CD3 percentage (higher in females than in males) and CD69 absolute count (higher in males than in females) as shown in Table 3. The significant increase in CD3 percentage is in acccordanance with Reichert et aI., (1991l The CD69 is a T cell activation marker. Quantification of both CD3 and CD69 does not play a significant role in clinical application. In contrast to our study, previous workers reported that lymphocyte subsets differ significantly between males and females9. In addition, another group of investigators showed a significantly lower natural killer cell percentage and count in women than in menlO. In addition, We did not find any gender differences in CD4 absolute count which is in agreement with previous authors 10, but in contrast to other studies that have reported gender differences in CD4 absolute counts11,12,13.
Differences in immune cell numbers between genders may be secondary to the differential influences of sex hormones shown in murine studies 14. The mechanisms involved may include the modulation of thymic involution by sex hormones or the
effect of binding to cell receptors for the sex steroid present on T ce1l15.
In our work, as there were no significant differences between males and females regarding most of CD markers, we gathered them as one group and compared to previous published results. The absolute CD4 T cell counts had higher significant values when compared to different studies from Africa16 and Asia10,17 (p< 0.01) (Table 5). This observation supports the hypothesis that specific genetic factors influence peripheral blood CD4 T cell count18,19. Moreover, lymphocyte subsets may be affected by race, geographical location, gender, circadian changes and physical
. 16
exercIse .
Based on reported differences in normal values for lymphocyte subsets in different populations, there are questions whether CD4lymphocytes count threshold in Europe, America and Africa are appropriate for Saudi Arabia. Few studies have examined the normal values for lymphocytes subset in Saudi Arabia20,21,22. Therefore it is important to determine the CD4 cut off value in Saudi Arabia. As there is no significant difference in CD4 value according to gender a single reference range could be suitable for healthy Saudi adults.
The CD8 absolute count showed highly significant increase when compared to other studies (p< 0.01) 10,16,17,18,21. Apart from racial differences these discrepancies could be attributed to other factors such as smoking, diet, climate and hygienic conditions.
The mean of CD41 CD8 ratio in our results was 1.7 with a range from 0.5 to 2.5; this reflected the wide ranges obtained for both CD4 and CD8 absolute count. The mean
ratio of our result showed higher significant values when compared to other studies (Ethiopia 16, Singapore 10 and Hong Kong 17) (P<O.01). On the other hand, there was no significant difference detected when compared to previous Saudi study (p> 0.05) 21. It showed significant decrease when compared to the Dutch study18 (P< 0.05).
Moreover, the CD4/CD8 ratio showed significant negative correlation with CD8 percentage (P< 0.05).
The CD16 is a marker associated with Natural killer lymphocytes even though it is not a specific marker as are CD3, CD4 and CD8 for T cells. In our study, the CD16
b I ' 'f' I h' h h d h d' 101617 182021
a so ute count was slgm 1cant y 19 er w en compare to ot er stu 1es ' , , , , as shown in Table 5. It has been shown that CD16 increase in subjects who assessed their general health and nutritional status as perfect23
.
The age in our study did not show any significant correlation with CD4, CD8 and CD4/CD8 ratio, (P>0.05) as shown in Table 6. The impact of age on lymphocyte subsets is not well established with different studies reporting different results. A group of workers demonstrated that the number of CD4 increases while the number of CD8 decreases with age9. On the other hand another study showed that the number of both CD4 and CD8 increases with age24. The inconsistency arises because most studies looked at patients of different age ranges. Most of the contributors of our study were between 17 -45 of age, therefore it would be difficult to show any definite conclusion about the effect of age on lymphocyte subsets.
CONCLUSION
In conclusion, most of the absolute counts of the different CD markers studied in our work (CD3, CD4, CD8, CD16 and CD69) and the CD4/CD8 ratio are higher when compared to different populations. Our study suggests that there may be a true genetic differences in lymphocyte subsets. Every population must establish a normal range of their own. It is recommended that samples from several other areas in Saudi Arabia should therefore be studied to confirm and correlate with our data.
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