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- Materials and Methods
- Literature Cited
- Supporting Information
Country-specific reference ranges for adult peripheral blood lymphocyte subsets have been established in a few countries around the world; however, there have been no specific comprehensive studies in the Gulf Cooperation Council (GCC) and Middle East, which investigated age and gender-specific reference ranges. Demographic and environmental factors may contribute to variations in these subsets around the world, and thus there is a great necessity for each country to establish its own reference ranges. Hence, the aim of this study is to establish lymphocyte subsets reference ranges for Omani healthy adults. Total, age, and gender-specific reference ranges were established using four-color flow cytometry analysis with an extensive panel of monoclonal antibodies in 50 healthy adult males and females aged between 18 and 57. Reference values were expressed as median and 95% confidence intervals for T cells—CD3+: 76.5 (57–89), CD4+: 45 (31–58), CD5+: 75 (58–85), CD7+: 80 (70–89), CD8+: 29.5 (19–43); B cells—CD10+: 1 (1–3), CD19+: 14 (6–23), CD20+: 14 (6–23), and NK cells—CD16+: 9 (3–22), CD56+: 13 (5–24), CD3−/(CD16+/CD56+): 7 (3–20). In comparison with other published studies, the lymphocyte subsets reference ranges in healthy Omani adults were similar to those reported in the rest of the world. These observations have important clinical implications in lymphocyte subset analysis in Oman, especially in the management of immunological disorders. The reference ranges established by this study can be adopted as a reference for clinical practice decisions. © 2013 International Society for Advancement of Cytometry
Analysis of peripheral blood lymphocyte subsets has become an essential tool in the evaluation of immunological and pathological disorders. Establishment of a normal reference range is fundamental for precise elucidation of immunophenotyping data based on age, gender, ethnicity, genetic differences, and environmental factors.
Flow cytometry is today the most precise and reliable tool for the assessment of immunological status [1-3]. Although various methodologies and gating strategies have been established, the CD45 gating strategy is the most appropriate approach for better identifying the lymphocyte population accurately and reliably .
Although very few studies were done for reference ranges for lymphocyte subsets in the Middle East and the GCC (Gulf Cooperation Council) [5-8], these studies have many limitations. To mention a few, the flow cytometry approach of the gating strategy used was the FSC (forward scatter) versus SSC (side scatter) approach, and a limited panel of monoclonal antibodies (MoAbs) was used. Some of the studies did not take into account gender and/or age differences [7-9]. To the best of our knowledge, there is only one published Omani study , which was performed only on males, with a limited panel of MoAbs and an FSC versus SSC approach for identification of the lymphocytes, and the accuracy and reliability of the data remained limited.
Hence, the objective of our study is to identify the reference ranges for healthy Omani donors for T-cell, B-cell, and NK-cell lymphocyte subsets with a comprehensive and informative panel of MoAbs with the CD45+/SSC log-gating strategy, in both males and females in a broad age spectrum.
- Top of page
- Materials and Methods
- Literature Cited
- Supporting Information
Clinical diagnosis can be more accurate when a reliable reference range is made available with regard to the local population. This, in most cases, is enhanced with the rapid technological advancements in the field of flow cytometry [2, 10-12]. Variation between laboratories can now be minimised on a global level with similar documented procedures being followed throughout, such as sample preparation techniques, wash steps, and gating strategies with lymphocyte subsets . In addition, variations in population genetics and environmental factors need to be addressed individually by each region or country, as it may play a major role in the definition of the so-called normal reference ranges. Thus, in our study, we established the reference ranges of extended sets of lymphocyte subsets in both males and females, in a broad age spectrum with an extensive and comprehensive panel of MoAbs using CD45+/SSC log-gating strategy to identify the lymphocytes.
T-cell Lymphocyte Population
The T-cell subset is mainly recognised by mature T-cells (CD3), T-helper cells (CD4), and T-cytotoxic cells (CD8) [14, 15]. Ability of T-cells to recognise foreign antigens in association with Major histocompatibility complex (MHC) class antigens I and II are facilitated by accessory molecules CD4 and CD8, respectively .
In our study, we have further investigated the T-cell subset by CD5 and CD7. CD5, which is present on the surface of T-cells, acts as a receptor in T-cell proliferation. It is also involved in the tyrosine kinase-linked T-cell receptor (TCR). CD7 is a glycoprotein and is also implicated in the development and activation of T-cells .
The reference ranges for CD3, CD4, and CD8 were similar to other studies [16-20], except for CD8 [16, 19, 21].
Gender-related study has shown significant differences in reference ranges between males and females for CD3 (P = 0.001), CD4 (P = 0.0049), CD5 (P = 0.008), and CD7 (P = 0.003) similar to Caucasian [17, 18], Brazilian , and Indian  studies. Variations in levels of T-cells between males and females could be attributed to differences in cytokine regulation  and acceleration of thymocyte apoptosis by male androgens, which have an effect on the peripheral T-cell subset .
Our study has shown that females have a higher T-cell subset count for CD3, CD4, CD5, and CD7 compared to males, except in CD8 T-cytotoxic cells.
No age-related differences have been identified between the two age groups in T-cell lymphocyte populations (≤26 years and >26 years), similar to previous studies [5, 6, 9, 16-18, 20, 21, 25] with the exception of CD7. We found that in our study, the younger age group expresses a higher percentage of CD7 compared with the older age group (P = 0.038), which could be attributed to CD7 being expressed early in the T-cell ontogeny .
B-cell Lymphocyte Population
The B-cell subset is usually recognized by the expression of two tyrosine-kinase linked receptor markers, namely CD19 and CD20. CD19 and CD20 are linked with maturity and differentiation of B-cells .
In our study, another B-cell marker CD10 was evaluated. CD10 is a 100-kDa transmembrane type II molecule that is associated with mature and immature B-lymphocytes .
CD19 and CD20 have shown the same reference ranges in our study similar to a Caucasian study, which also studied both the markers . Other studies only evaluated CD19 as B-cell subset marker and we are in line with them, i.e. Omani, Caucasian, Iranian, and African studies [6, 9, 17, 21]. This shows that there is little or no variation among B-cell subsets around the globe.
With regard to gender-related differences, we have found significant differences for both CD19 and CD20 between males and females (P-value ≤0.001) in contrast to all other studies [5, 6, 16-19, 21, 28]. Consequently, the variation in our study could be attributed to being specific to the demographic and environmental factors in the region. Nevertheless, our study found no significant difference for CD10 between males and females.
Age-related study has shown no significant differences between the two age groups. This is in line with all other age-related studies performed on other populations [9, 16, 17, 19, 21]. Some studies have not evaluated age-related differences because of the broad range of ages obtained [6, 8]. A specific study should be performed with healthy donors in each age group to ascertain any difference.
NK-cell Lymphocyte Population
Levels of peripheral blood NK-cell lymphocytes are assessed by the subset of lymphocytes expressing both CD16 and CD56 and lacking CD3 expression [15, 16].
CD16 is a receptor molecule also expressed by a large number of NK cells and mediates antibody-dependent cellular cytotoxic (ADCC) function . CD56 is a neural-cell adhesion molecule expressed on all lymphocytes involved in non-MHC related cytotoxicity [15, 16].
Our NK-cell reference range has shown higher reference range limits in comparison with some published studies [9, 16, 18, 19] and lower reference range limits in comparison with Caucasian and African studies [17, 21]. However, our reference range was similar to an Iranian study , which could be attributed to environmental and ethnic factors.
No significant gender-related difference has been found, keeping in line with an Iranian study  but not with Caucasian [16-19], Asian , or African [21, 29] studies, which could partly be attributed to the factors mentioned above.
With regard to age, no significant differences have been found in our study. Our observations were similar to all other age-related studies [7, 9, 16-18, 21].
In conclusion, we have identified reference ranges for a wide range of lymphocyte subsets, which can now be used as a reference when evaluating patient samples in Oman. This is the first study that investigated reference ranges for both genders with an extensive panel of MoAbs and the CD45+/SSC log-gating strategy. The main limitation of our study is the small sample size; the other limitation being no proper age-group reference values evaluated due to limited donors and the broad range of donors.