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Keywords:

  • lymphocyte subsets;
  • flow cytometry;
  • reference ranges;
  • Oman;
  • CD3+CD4+;
  • CD3+CD8+

Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Literature Cited
  7. 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 [4].

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 [9], 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.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Literature Cited
  7. Supporting Information

This study was carried out between April and October 2010 at the Royal Hospital in the Sultanate of Oman. Ethical approval was obtained from the Research and Ethical Review and Approve Committee, Ministry of Health.

Subjects and Sample Collection

A total of 50 healthy adult Omani volunteer donors (25 males and 25 females) were recruited for this study from the Central Blood Bank (CBB) in Muscat, Sultanate of Oman. Selection criteria were based on a questionnaire given to each donor to assess medical, demographic, and lifestyle information. Donors who were suffering from any type of illness, taking any medication or treatment, or those who were cigarette smokers were excluded from the study. All donors were healthy seronegative adults with ages ranging from 18 to 57 years (median 26 years). Informed consent was taken prior to blood donation. Peripheral blood specimens were collected by venipuncture in 2 ml K3EDTA vacuette tubes to be processed within 4 hours of collection.

Flow Cytometry

Appropriately titred directly conjugated MoAbs were added into 5-ml (12 × 75 mm) test tubes containing venous blood. The mixture was incubated as per the manufacturers' recommendations. The samples were then prepared with the TQ-Prep Workstation (Beckman Coulter, Brea, CA) and the ImmunoPrep Reagent System (Beckman Coulter). The cells were subjected to red blood lysis with formic acid (ImmunoPrep A), leukocyte stabilisation with sodium carbonate (ImmunoPrep B), and cell membrane fixation with paraformaldehyde (ImmunoPrep C). The prepared samples were then stored in the dark at 4°C and analyzed within 24 hours.

Antigen expression was analyzed by using 4-color combinations of MoAbs conjugated with fluorescein isothiocyanate (FITC), phycoerythrin (PE), phycoerythrin-Texas red conjugate (ECD), and phycoerythrin-cyanine (PC)-5 (Table 1).

Table 1. Monoclonal antibodies used for Lymphocyte Subset Analysis investigation by 4-color flow cytometry
AntigenConjugateCloneIsotype controlSource
  1. FITC, fluorescein isothiocyanate; PE, phycoerythrin; ECD, phycoerythrin-Texas Red conjugate; PC, phycoerythrin-cyanine.

CD3ECDUCHT1IgG1 MouseBeckman Coulter
CD4PE13B8.2IgG1 MouseBeckman Coulter
CD5FITCBL1aIgG2a MouseBeckman Coulter
CD7PE8H8.1IgG2a MouseBeckman Coulter
CD8FITCB9.11IgG1 MouseBeckman Coulter
CD10PEALB1IgG1 MouseBeckman Coulter
CD16FITC3G8IgG1 MouseBeckman Coulter
CD19ECDJ3-119IgG1 MouseBeckman Coulter
CD20FITCB9E9IgG2a MouseBeckman Coulter
CD45PC-5J.33IgG1 MouseBeckman Coulter
CD56PEN901IgG1 MouseBeckman Coulter

Processed samples were analyzed using Cytomics FC 500 (Beckman Coulter, Miami, FL, USA) flow cytometer equipped with a 488-nm laser air-cooled configuration. Analysis of list mode data was performed using fluorescence gating strategy based on CD45+/SSC log-gating (Fig. 1). For each sample analyzed, a minimum of 20,000 events was acquired. All measurements were performed under the same compensation settings. Analysis was done on peripheral blood lymphocytes: T-cell, B-cell, and NK-cell populations.

image

Figure 1. Flow cytometry gating strategy (density plots): (a) CD45 vs. side scatter (SS) log [FL4/SS log]; gate analysis (dot plots): (b) CD4 vs. CD3 [FL2/FL3 log], (c) CD8 vs. CD3 [FL1/FL3 log].

Download figure to PowerPoint

Quality Control

Internal quality control procedures were used to assess instrument parameters and ensure accurate results. Daily calibration of the flow cytometer was performed using Flow-check and Flow-set fluorospheres (Beckman Coulter) for optical laser alignment and optimal hydrodynamic focussing settings, respectively.

An external quality assurance procedure was also implemented through participation in a performance-monitoring network operated by the Royal College of Pathologists of Australasia Quality Assurance (RCPA) programme.

Statistical Analysis

Analyzed sample data were added into a database, and all statistical analyses were done using IBM SPSS Statistics 19.0. Percentage results were expressed as a median with a 95% confidence range. Reference ranges were determined by interquartile percentile values (from 2.5% to 97.5%). The influence of gender and age in the distribution of peripheral blood lymphocytes were evaluated using the Student's t-test where a P-value of <0.05 was considered significant.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Literature Cited
  7. Supporting Information

Analysis was performed on peripheral blood lymphocytes: T-cells, B-cells, and NK-cell populations.

T-cell Lymphocyte Population

The percentages of total T-cells (CD3), helper T-cells (CD4), and cytotoxic T-cells (CD8) in peripheral blood were determined. In addition, CD5 and CD7 T-cell markers were also investigated.

Reference ranges for CD3, CD5, and CD7 were found to be 57–89 (median 76.5%), 58–85 (median 75%), and 70–89 (median 80%), respectively. Similarly, reference ranges for CD4 and CD8 were found to be 31–58 (median 45%) and 19–43 (median 29.5%), respectively (Table 2).

Table 2. Peripheral blood lymphocyte phenotype reference values
PhenotypeDescriptionNPercentagea
  1. a

    Median (95% confidence range; 2.5–97.5% percentiles).

CD3+T-cell5076.5 (57–89)
CD4+T-helper (Th) cell5045 (31–58)
CD5+T-cell5075 (58–85)
CD7+T-cell5080 (70–89)
CD8+T-cytotoxic (Tc) cell5029.5 (19–43)
CD10+B-cell501 (1–3)
CD19+B-cell5014 (6–23)
CD20+B-cell5014 (6–23)
CD16+NK-cell509 (3–22)
CD56+NK-cell5013 (5–24)
CD3/(CD16+/CD56+)NK-cell507 (3–20)

The distribution of these T-cell subsets' reference ranges based on gender is shown in Table 3. Apart from cytotoxic T-cells CD8, expression of CD3, CD4, CD5, and CD7 between the two genders were significant (P = 0.001, P = 0.049, P = 0.008, P = 0.003, respectively).

Table 3. Gender-related differences in the peripheral blood lymphocyte phenotype reference values
 NPercentagea
PhenotypeMaleFemaleMaleFemaleP-value
  1. a

    Median (95% confidence range; 2.5–97.5% percentiles); NS, not significant.

CD3+252571 (57–84)79 (58–90)0.001
CD4+252542 (29–57)49 (31–63)0.049
CD5+252571 (56–83)77 (60–90)0.008
CD7+252578 (68–73)82 (72–91)0.003
CD8+252530 (17–43)29 (19–45)NS
CD10+25252 (1–3)1 (1–3)NS
CD19+252515 (11–26)12 (6–21)0.001
CD20+252515 (11–25)12 (6–21)<0.001
CD16+25259 (3–22)7 (2–31)0.043
CD56+252513 (5–24)12 (4–30)NS
CD3/(CD16+/CD56+)25257 (3–20)6 (1–28)NS

The distribution of these T-cell subsets' reference ranges based on age is shown in Table 4. The population was divided into two groups: ≤26 years and >26 years in the analysis of age-related differences based on the median. No significant age-related differences were observed in the distribution of these T-cell subsets, apart from CD7.

Table 4. Age-related differences in the peripheral blood lymphocyte phenotype reference values
 NPercentagea
PhenotypeAge (≤26)Age (>26)Age (≤26)Age (>26)P-value
  1. a

    Median (95% confidence range; 2.5–97.5% percentiles); 26 years was the median age of the study cohort; NS, not significant.

CD3+262476 (57–90)76.5 (57–89)NS
CD4+262445 (29–58)46 (38–63)NS
CD5+262475 (56–90)75 (58–85)NS
CD7+262482 (70–91)78 (68–88)0.038
CD8+262428.5 (21–45)31 (17–43)NS
CD10+26241 (1–3)1 (1–3)NS
CD19+262414 (6–23)14 (6–26)NS
CD20+262413.5 (6–23)14 (6–25)NS
CD16+26249 (2–31)9 (3–20)NS
CD56+262414 (4–30)12 (5–24)NS
CD3/(CD16+/CD56+)26248 (1–28)6 (3–19)NS

CD7 was found to be significantly higher in the younger population group when compared with the older population group (P = 0.038).

B-cell Lymphocyte Population

The percentages of B-cells were evaluated with the expression of CD10, CD19, and CD20. Reference ranges for CD10, CD19, and CD20 were found to be 1–3 (median 1%), 6–23 (median 14%), and 6–23 (median 14%), respectively (Table 2).

The distribution of these B-cell subsets' reference ranges based on gender is shown in Table 3. CD10 showed no significant difference between the two genders, whilst CD19 and CD20 indicated significant gender-related differences.

In any of the B-cell lymphocyte reference ranges, no significant differences were found in the ≤26 years and >26 years age groups (Table 4).

NK-cell Lymphocyte Population

The percentages of NK-cells were investigated using CD3, CD16, and CD56 MoAbs. The NK cells were defined by CD3/(CD16+/CD56+) phenotype in our cohort study. The reference range for CD3/(CD16+/CD56+) was 3–20 (median 7%) (Table 2).

With regard to NK-cells, analysis revealed no significant difference between the two genders (Table 3).

Similar to the B-cell subset, the NK-cell subset also showed no significant age-related difference in the study cohort (Table 4).

Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Literature Cited
  7. 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 [13]. 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 [15].

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 [15].

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 [22], and Indian [20] studies. Variations in levels of T-cells between males and females could be attributed to differences in cytokine regulation [23] and acceleration of thymocyte apoptosis by male androgens, which have an effect on the peripheral T-cell subset [24].

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 [26].

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 [16].

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 [27].

CD19 and CD20 have shown the same reference ranges in our study similar to a Caucasian study, which also studied both the markers [16]. 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 [16]. 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 [6], which could be attributed to environmental and ethnic factors.

No significant gender-related difference has been found, keeping in line with an Iranian study [6] but not with Caucasian [16-19], Asian [28], 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.

Literature Cited

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Literature Cited
  7. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Literature Cited
  7. Supporting Information

Additional Supporting Information (MIFlowCyt checklist) may be found in the online version of this article.

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