Establishment of reference values based on influential characteristics of hematopoietic stem cells and immune cell subsets in the bone marrow

TL;DR

This study establishes reference ranges for hematopoietic stem cells and immune cells in bone marrow, considering factors like age and body weight.

Contribution

The paper provides new reference values for bone marrow cell populations influenced by age and body weight.

Findings

No significant difference in HSC or immune cell counts between males and females.

Younger donors had higher CD34+ and immune cell subset counts.

Healthier body weight correlates with richer bone marrow cellularity.

Abstract

Hematopoietic stem cell transplantation is still a curative treatment for many haematological cancers. Many factors, such as age, sex, ethnic background, smoking status, and body mass index, affect average reference values in different populations. This study aimed to establish a reference range for the absolute numbers and percentages of healthy individuals' hematopoietic stem cells and immune cells in the bone marrow. Seventy-one healthy donors (32 males and 39 females) were enrolled in the study. Following bone marrow harvesting, using flow cytometry, immunophenotyping was performed to determine the absolute number and percentage of CD34+ stem cells and various immune subsets. We found no statistically significant difference in the absolute count of HSCs or immune cell subsets in the bone marrow between males and females. Regarding age, the younger group had more significant CD34+ and immune cell subsets. Donors with healthier body weights tend to have richer bone marrow cellularity. Establishing a reference value for hematopoietic stem cells and immune cells in the bone marrow based on various influential factors is pivotal for defining bone marrow status and donor selection.

Full text

Introduction

1

Hematopoietic stem cells (HSCT) are rare subsets of specialized hematopoietic cells responsible for the lifelong maintenance of bone marrow precursors and progenitor cells. HSCT remains the primary curative treatment for many haematological cancers and benign disorders, such as congenital marrow failure syndrome and thalassemia [1,2]. A successful HSCT with a better clinical outcome depends on the donor site stem cell count, based on the number of CD34^+^ cells or total nucleated bone marrow cells [3].

Among the various factors, age-related changes in the major lymphocyte subsets in bone marrow blood occur from childhood to adulthood [4]. Age-related changes were established as the main factor that helps to assess bone marrow lymphoid cell subsets [5].

Another factor that is important in considering donor selection is sex. In many medical aspects, such as immunocompetence and mortality, sex differences have been well documented in humans [6]. Many studies have shown that sex variations in donors and recipients affect HSCT outcomes [7]. For example, a study showed that HSCT in male recipients resulted in better transplant outcomes in terms of overall survival and disease-free progression survival than did HSCT in female recipients, regardless of donor sex [8,9].

Body mass index (BMI) has gained increased interest in recent decades since obesity has spread worldwide [10]. At the cellular level, an increase or decrease in BMI considerably influences homeostasis [11]. Many research studies have shown that BMI is directly related to blood cells, especially total white blood cells, particularly CD4^+^ and CD8^+^ lymphocytes [12]. Because obesity is more common in women, they tend to be most affected [12]. Therefore, determining the relationship between BMI and the cellular content of the bone marrow is of particular interest.

Immunophenotyping of stem cells and lymphocytes is essential in evaluating the immune system in health and disease [13]. Flow cytometry monitors blood components in patients with leukaemia, immunodeficiency syndrome, or lymphoma and evaluates immune status [14,15]. It is a reliable and accurate tool for rapidly and quantitatively determining cellular components [16]. Many factors, such as environmental factors, infection status, nutritional status, smoking status, sex, age, race, ethical diversity and immunosenescence, can affect the immune system. Therefore, establishing an internal normal range of HSCs and lymphocyte subsets is necessary for each population and worldwide [17,18]. Although similar methods were used to estimate lymphocyte subsets, there was still variation in the percentage of lymphocytes in different populations, as typical reference values may apply to one population but not others [19]. The current study aimed to determine reference values for HSCs and lymphocyte subsets in the bone marrow of healthy donors based on various influential factors, such as age, sex and BMI.

Materials and methods

2

HSCT center

2.1

This study was conducted at an HSCT centre (established June 2016) of Hiwa Hospital, Suleimanyah/Iraq, with six single-bed, HEPA-filtered, positive-pressure sterile rooms; four double-bed clean rooms; a bone marrow harvest surgical room; an apheresis unit; a manipulation laboratory for cell separation and cryopreservation; and a flow cytometry laboratory [20].

Bone marrow harvesting

2.2

After signing the informed consent and approval from the local ethics committee of the College of Medicine, University of Sulaimani, Sulaymaniyah, Iraq, with approval no. 7/29/701 meeting no. 4), bone marrow (BM) was harvested from healthy donors (n = 71) (Table 1). The BM graft was harvested from the posterior superior iliac crest. First, the harvesting point and surrounding areas were appropriately disinfected with 10 % iodine. Then, a sterile 13G trocar was introduced into the pelvic bone. After that, the bone marrow was harvested using a large syringe prefilled with heparinized normal saline (25,000 IU heparin in 500 ml NS). The graft was then transferred into a sterile bag and filtered in preparation for infusion into the patient. A 2 ml sample was prepared from the filtered BM graft and sent to the flow cytometry laboratory for HSC and immune cell evaluation and enumeration.Table 1. Donor demographic characteristics.Table 1. Gender (n)Male32Female39Age Median (Range)Child7 (2–12)Adolescence15 (13–18)Adult29 (19–41)BMI (kg/m^2^) Median (Range)UnderweightHealthyOverweightObese

Cell enumeration by flow cytometry

2.3

HSC enumeration and viability were assessed using a stem cell enumeration kit (Cat # 664231, Lot # 3234923, BD Biosciences, Brea, CA) and FACS Via flow cytometer (BD Biosciences, USA). The procedure was performed with an accurate count (BD Biosciences, USA) containing known numbers of beads in a freeze-dried palette. For CD34 cell enumeration, 20 μl of stem cell reagent containing anti-CD45 FITC and anti-CD34 PE monoclonal antibodies and 20 μl of 7AAD reagent were added, and for immune cell enumeration, a cocktail of anti-CD3 FITC, anti-CD4 PE, and anti-CD45 Percp Cy5.5 and anti-CD8 APC (Cat # 342417, LOT # 09225 BD Biosciences, Brea, CA), or anti-CD3 FITC, anti-CD16/56 PE, anti-CD45 Percp Cy5.5 and anti-CD19-APC (Cat # 342416, LOT # 21623 BD Biosciences, Brea, CA), were added. Then, 100 μl of freshly harvested bone marrow graft was added by reverse pipetting. The sample was incubated for 20 min at room temperature in the dark. After that, 2 ml of 1x fixative free ammonium chloride RBC lysis buffer was added, and the sample was incubated for an additional 10 min at room temperature in the dark until all the RBCs were lysed. The samples were subsequently analyzed on a FACS VIA flow cytometer, and the data were analyzed with BD Accuri research software. The absolute numbers of CD34^+^ cells (HSCs), CD3^+^ T cells, CD3^+^CD4^+^ T cells, CD3^+^CD8^+^ T cells, CD19^+^ B cells, CD3^−^CD16/56^+^ NK cells and CD3^+^CD16/56^+^ NKT cells were calculated according to the manufacturer's protocol.

Flow cytometry quality control

2.4

The United Kingdom National External Quality Assurance System (UK NEQAS) accredited and continuously monitored the flow cytometry laboratory for CD34^+^ stem cell enumeration, CD34^+^ stem cell viability assessment and immune cell monitoring.

Reference interval verification

2.5

To establish the reference value, the Skewness-Kurtness test was first used to assess the normal distribution of the data. Then, the Box-Cox transformation was used to transform the non-normally distributed data. The healthy donors were grouped based on their age into Children (0–11 years old), Adolescents (12–17 years old) and adults (18 above years old) according to WHO age group classification. Furthermore, for the establishment of reference value based on gender, the donors were grouped into male and female and for the BMI, the donors were grouped into Underweight (below 18.5 kg/m^2^), Healthy (18.5–25 kg/m^2^), Overweight (25–30 kg/m^2^) and Obese (30-above kg/m^2^).

Statistical analysis

2.6

The Skewness-Kurtness test was used to assess the data's distribution and determine whether the data was usually or non-normally distributed. The Box-Cox transformation was used to transform the non-normally distributed data. Further analysis of the data was conducted in GraphPad Prism version 9.3.0. The reference values were obtained by identifying minimum and maximum values with the mean ± SEM (standard error of the mean) and the median with standard deviation (SD) of the absolute numbers and percentages of the cells using frequency distribution analysis.

Results

3

Reference values of sex-specific HSCs and immune cell subsets in the bone marrow of healthy donors.

To establish the reference values of sex-related HSCs and immune cell subsets in the bone marrow, 71 healthy donors (male, n = 32; female, n = 39) who donated their marrow grafts to their recipient patients were included in the study. The absolute numbers and percentages of CD34^+^ HSCs were in line with the absolute counts and percentages of lymphocyte subpopulations, including CD3^+^ T cells (total and further assessment of CD3^+^CD4^+^ and CD3^+^CD8^+^ T cells), CD19^+^ B cells, CD3^−^CD16/56^+^ NK cells and CD3^+^CD16/56^+^ NKT cells. The reference values were obtained by identifying minimum and maximum values with the mean±SEM (standard error of the mean) and the median with standard deviation (SD) of the absolute numbers and percentages of the cells of the harvested marrow. Moreover, we explored the variation and influence of sex on the number of bone marrow cells. Based on our investigation, there were no statistically significant differences in the absolute counts of HSCs or immune cell subsets in the bone marrow of male and female individuals, which suggests that sex is not a pivotal influencer of the number of progenitor and immune cells in the bone marrow (Table 2).Table 2. Hematopoietic stem cell and immune cell subset normal values and characteristics based on sex in the bone marrow of healthy donors (absolute numbers and percentages).Table 2. ParametersgroupMeanSEMMedianSDNormal ValuesHSC (abs)All216.910.9211.393.071–552Male220.216.3221.990.781–430Female214.514.8202.395.771–552HSC (%)All0.300.010.300.080.10–0.50Male0.290.010.300.090.16–0.50Female0.300.010.300.080.10–0.50Lymphocyte (cells/μl)All6130381527231691356–17109Male5822493521927891356–13533Female6175527517034181968–17109Lymphocyte (%)All9.680.429.003.425.00–18.00Male9.210.509.002.636.00–15.00Female10.000.629.003.875.00–18.00T cells (cells/μl)All3794189348616301006–8814Male3793287346516261006–8814Female3795255361016531279–7817T cells (%)All64.631.1365.59.2239.00–85.00Male65.741.2466.006.4553.00–78.00Female63.871.7264.0010.7539.00–85.00CD4^+^ T cells (cells/μl)All1773891613767460–3768Male17021231563696460–3442Female18271261696822610–3768CD4^+^ T cells (%)All47.100.9746.57.9034.00–65.00Male46.181.6345.008.4734.00–63.00Female47.741.2048.007.5334.00–65.00CD8^+^ T cells (cells/μl)All1553891494736422–4154Male15451191494676423–2927Female15161221320793518–4154CD8^+^ T cells (%)All40.400.8740.007.1325.00–56.00Male40.481.3739.007.1428.00–56.00Female40.351.1540.007.2225.00–55.00B cells (cells/μl)All13081131114926259–4808Male11811321063751259–2817Female131915910061021298–4804B cells (%)All20.870.8720.007.125.00–44.00Male19.771.0920.005.7010.00–31.00Female21.641.2721.007.945.00–44.00NK cells (cells/μl)All7656952755789–2619Male7829652654689–2286Female72584552549114–2619NK cells (%)All12.210.5112.004.213.00–22.00Male12.960.8513.004.443.00–22.00Female11.690.6411.004.023.00–22.00NKT cells (cells/μl)All3111926516252–730Male3262928516352–624Female2992525216278–730NKT cells (%)All5.400.335.002.741.00–12.00Male5.920.496.002.581.00–11.00Female5.050.455.002.821.00–12.00

Establishment of reference values for HSCs and lymphocytes from the bone marrow according to age

3.1

Furthermore, to identify the reference values of HSCs and lymphocyte subpopulations in the bone marrow of healthy individuals based on age, 38 children, 15 adolescents and 18 adults were recruited for the study. The absolute numbers and percentages of CD34^+^ cells, CD3^+^ T cells, CD3^+^CD4^+^ cells, CD3^+^CD8^+^ T cells, CD19^+^ B cells, CD3^−^CD16/56^+^ NK cells and CD3^+^CD16/56^+^ NKT cells were calculated. The reference values are the minimum and maximum ± SEM and the median ± SD. Several parameters differed according to the age group. Compared with adults, children had significantly greater numbers of CD34^+^ and CD3^+^ T cells; CD3^+^CD4^+^ and CD3^+^CD8^+^ T cells; and CD19^+^ B cells and CD3^−^CD16/56+ NK cells. The differences were statistically significant compared to those in the adult group, except for the CD3^+^CD16/56+ NKT cells. However, compared with adolescents, children were significantly different only in the number of CD34^+^ cells, total CD3^+^ T cells, total CD19^+^ B cells, and total CD3^−^CD16/56+ NK cells. HSCs and lymphocyte subpopulations in the bone marrow tended to be higher in the adolescent group than in the adult group (Table 3).Table 3. Hematopoietic stem cell and immune cell subset normal values and characteristics based on age in the bone marrow of healthy donors (absolute numbers and percentages).Table 3. ParametersGroupsMeanSEMMedianSDNormal ValuesHSC (cells/μl)Child255.913.9245.185.7117.9–552.4Adolescence176.712.2173.448.997.8–254.4Adult175.924.9143.9105.871.4–430.2HSC (%)Child0.320.010.300.080.18–0.50Adolescence0.260.010.240.070.16–0.40Adult0.300.020.280.080.18–0.43Lymphocyte (cells/μl)Child7779520719532062659–17109Adolescence5016413433116533529–9072Adult3560331311214431356–6165Lymphocyte (%)Child11.020.5711.003.545.00–18.00Adolescence7.880.537.002.015.00–11.00Adult8.040.758.002.516.00–15.00T cells (cells/μl)Child4568251476715521764–8814Adolescence3494350294114001406–6557Adult2612243246210611006–4616T cells (%)Child60.211.0960.006.7539.00–72.00Adolescence70.422.0271.507.5754.00–85.00Adult73.091.6075.005.3364.99–83.00CD4^+^ T cells (cells/μl)Child20491152016713605–3557Adolescence16031711415663610–2980Adult13021261333552460–2712CD4^+^ T cells (%)Child44.811.2843.007.9434.00–65.00Adolescence49.502.1451.008.0034.00–63.00Adult52.271.6652.005.5343.00–59.00CD8^+^ T cells (cells/μl)Child18421251763776604–4154Adolescence13701481167592566–2808Adult10781051002457422–1832CD8^+^ T cells (%)Child41.071.2240.007.5225.00–55.00Adolescence39.712.3040.008.6130.00–56.00Adult38.451.2137.004.0334.00–45.00B cells (cells/μl)Child17891561583950345–4808Adolescence968102976410313–1634Adult51866430102259–1319B cells (%)Child23.731.0024.006.1812.00–44.00Adolescence17.921.7617.006.595.00–30.00Adult14.721.0915.003.639.00–22.00NK cells (cells/μl)Child104993962579211–2619Adolescence50574445296121–1383Adult3914836321089–973NK cells (%)Child13.230.7013.004.333.00–22.00Adolescence9.920.8710.003.293.00–15.00Adult11.901.1912.003.968.00–20.00NKT cells (cells/μl)Child3372733316952–730Adolescence1693226613082–522Adult273821816999–618NKT cells (%)Child4.680.424.002.621.00–12.00Adolescence5.850.616.502.312.00–9.00Adult7.540.737.002.425.00–12.00

The influence of BMI on the cellularity of bone marrow in terms of HSCs and immune cell subpopulations

3.2

The BMI has attracted increased interest in medical research in recent decades. Therefore, the BMI of the healthy donors was calculated as the weight in kilograms (kg) divided by the square of the height in meters (m^2^), and the donors were subsequently categorized into underweight, healthy, overweight and obese based on their BMI. The reference values are the minimum and maximum ± SEM and the median and SD of HSCs and bone marrow lymphocyte numbers and percentages, respectively. We found that the body mass indices of all three age groups affect the number of bone marrow stem cells and immune cell subsets in the bone marrow of healthy donors. Our investigation shows donors with healthier body weights and even underweight individuals have more significant absolute numbers of HSCs and mature immune cells in the bone marrow than overweight and obese donors (Table 4).Table 4. Hematopoietic stem cell and immune cell subset normal values and characteristics based on BMI in the bone marrow of healthy donors (absolute numbers and percentages).Table 4. ParametersGroupsMeanSEMMedianSDNormal ValuesHSC (cells/μl)Underweight25114.9224188.29113–552Healthy19117.8118075.5681–370Overweight18727.7518287.7676–321Obese11722.118749.4571–177HSC (%)Underweight0.340.020.320.060.28–0.47Healthy0.280.010.260.080.16–0.50Overweight0.270.020.270.090.16–0.43Obese0.320.020.320.080.20–0.45Lymphocyte (cells/μl)Underweight7837535.773673169.32659–17109Healthy4815518.640222260.52562–11294Overweight4604582.445581931.72230–9072Obese3154834.720991866.51356–5272Lymphocyte (%)Underweight8.680.678.751.905.00–11.00Healthy9.240.549.002.735.00–15.00Overweight8.070.518.001.846.00–12.00Obese7.870.667.501.886.00–10.00T cells (cells/μl)Underweight4706255.148351509.41764–8814Healthy3135283.827421237.31788–6253Overweight3244392.833171302.81681–6077Obese2256627.115521402.11006–4248T cells (%)Underweight60.251.8859.005.3353.00–68.00Healthy66.721.6369.008.1853.00–85.00Overweight67.232.4667.008.8752.00–83.00Obese68.502.2368.006.3260.00–80.00CD4^+^ T cells (cells/μl)Underweight2117131.42024777.4605–3768Healthy1572133.81436583.6831–2849Overweight1560201.31464667.9769–2792Obese1111296.2792662.3460–1857CD4^+^ T cells (%)Underweight43.371.8143.505.1235.00–50.00Healthy47.641.6148.008.0735.00–63.00Overweight49.232.0550.007.4034.00–59.00Obese47.251.6746.504.7440.00–56.00CD8^+^ T cells (cells/μl)Underweight1908123.11908728.5797–4154Healthy1201125.31059546.1604–2757Overweight1335184.11396610.3727–2808Obese944294.7628659.1422–2015CD8^+^ T cells (%)Underweight41.122.1239.506.0133.00–52.00Healthy40.321.3540.006.7730.00–55.00Overweight39.841.7238.006.2033.00–56.00Obese41.121.7942.005.0832.00–47.00B cells (cells/μl)Underweight1747173.315951010.4313–4804Healthy985136.5797595.3313–2766Overweight781138.1557458.2259–1518Obese591165.2358369.4277–1012B cells (%)Underweight23.251.2723.503.6118.00–29.00Healthy19.041.2120.006.075.00–29.00Overweight18.001.6218.005.849.00–28.00Obese18.621.7917.005.0614.00–30.00NK cells (cells/μl)Underweight100990.7876536.9211–2619Healthy619122.4418533.8238–2153Overweight545106.8475354.4121–1383Obese252111.1152248.289–688NK cells (%)Underweight15.001.7015.004.818.00–22.00Healthy12.520.7312.003.686.00–22.00Overweight13.461.3213.004.788.00–20.00Obese10.001.3010.003.703.00–15.00NKT cells (cells/μl)Underweight35426.2362153.152–730Healthy25231.3220136.578–624Overweight35455.9322185.4119–618Obese1148.410618.899–145NKT cells (%)Underweight5.120.765.502.162.00–8.00Healthy5.800.495.002.462.00–12.00Overweight6.610.856.003.092.00–12.00Obese6.250.816.502.312.00–9.00

Discussion

4

Lymphocyte immunophenotyping is crucial for assessing the immune system because ethnic variety and environmental influences impact the immune system, and each population must have an internal normal range for its lymphocyte subsets [21]. Numerous cell subpopulations' concentrations and proportions vary according to age, sex, geography, and ethnicity. Understanding the typical levels of these cells and how they differ in healthy populations will help advance clinical practice for more accurate diagnosis and prognosis and scientific study [22]. Numerous studies have examined racial variations in the distributions of distinct lymphocyte subpopulations [23].

Data obtained from different nations not only demonstrate heterogeneity in the number of lymphocytes and their subsets but also, despite the use of comparable methodologies, reference ranges from studies conducted in one population do not apply to others [24].

Our study is the only study in the region in which lymphocyte subsets were evaluated based on BM samples, and the majority of the other studies are based on PB lymphocyte subsets; moreover, no distinct studies on BM-based samples could be detected; for this reason, we compare our results with comparable results in PB samples from other countries.

In our study, there was no noticeable difference in lymphocyte count or subset of lymphocytes between males and females, apart from mild deviation in the total lymphocyte count, absolute CD4^+^ T cell and CD19^+^ B cell count and percentage count, which were slightly greater in females than in males but were not significant. Based on age, all the lymphocyte subsets showed a dramatic reduction in count with increasing age, being highest in the childhood period and then in Adolescence and lowest in adulthood, which agrees with the findings of studies performed in Saudi Arabia in all age groups [25]. The T cell population (CD3^+^) was threefold more significant than the B cell population (CD19^+^) in all age groups, both males and females. Still, this ratio increased with age until it reached fivefold greater in adulthood. This result also matches that in Saudi Arabia and Iran [21,25] but does not match those obtained from previous studies in which the B cells were equivalent to the T cells in average adult BM [26]. The CD4:CD8 ratio is approximately 1:1 in males and females and all age groups; this ratio is slightly lower than that reported in previous studies [21,27,28] but by that of Saudi Arabia [25]. NK cells (CD56^+^) were nearly in the same range in all age groups and in both males and females, which is compatible with the results of other studies [21,27,28].

Compared to our neighbouring country (Iran), our result is more significant for all the subsets, especially for CD8^+^ T cells and CD19^+^ B cells, in both adult males and females [21]. However, compared to the results for Saudi Arabia, our results are more significant regarding the absolute counts of all the subsets. At the same time, they are nearly consistent in terms of their percentages. The percentage and absolute count of NK cells (CD56^+^) were lower in our adult group than in their study [25]. Additionally, our lymphocyte subset range is greater than that of several European (Germany), Asian (Hong Kong, Korea), Latin American (Peru, Cuba, Brazil) and African (Tanzania) countries, apart from CD19^+^ cells, which are the same as those of adult Cuban individuals [27]. These findings support that notional lymphocyte subset values are significantly influenced by age, sex, race, and environmental factors [28]. Since more significant donor-site stem cell counts are generally believed to lead to better clinical outcomes, these stem cell counts are essential [29].

There is constant trafficking of hematopoietic stem cells between the PB and extravascular marrow spaces. Consequently, it is not believed that the stem cell qualities of the BM and PB stem cell pools differ [30]. The variation in the cellularity component of the bone marrow with age relies on the ontological changes that occur in the bone marrow niche composition [31]. For instance, the developmental stages from foetal life to neonatal to adult life led to changes in extracellular matrix markers, proliferation capability and other developmental potentials, affecting the differences in average values [32,33]. The cellularity, proliferation capacity, trophic factor generation and differentiation potential of HSCs and lymphocytes in the bone marrow have yet to be well studied. One study showed that age, but not sex, impacts bone marrow cellularity; males and females have similar proportions of HSCs and mature lymphocytes in the bone marrow [34].

Many reports disagree about the influence of age on the CD34^+^ cell count, and some studies have reported that the CD34^+^ cell count decreases with increasing age [35]. Other works have shown that the functionality of HSCs decreases with increasing age, but the number of CD34^+^ cells is not strongly affected by increasing age [36]. Several others have reported that the CD34^+^ population in the bone marrow is expanding in individuals over 70 years [35]. Several reports have shown that the fraction of CD34^+^ cells in the bone marrow decreases during childhood, possibly due to the increased percentage of lymphocytes in the BM during age [37]. This variation in CD34^+^ cell quantification performed in different centres makes comparing different reports difficult [37]. It has been reported in some studies that the CD34^+^ cell count is inversely associated with female sex and positively associated with body mass index [38].

BMI has a significant role in determining the CD34^+^ cell count and mobilization, and the majority of reports indicate that the greater the BMI and obesity are, the more critical the CD34^+^ cell count [35]. BMI and obesity are associated with various adverse health issues, ranging from cardiovascular disease to diabetes type II, dyslipidemia, arthritis, hypertension, asthma and overall poor health conditions [39]. Our study's results showed no significant difference in the percentage or absolute count of CD34^+^ cells between males and females. Still, the absolute count was more important in children than adults, which agrees with the results of previous studies [29]. However, in our study, the percentage of CD34^+^ cells was the same in all age groups, but the absolute count was more significant in children than adults. These findings prove that BM cellularity is more important in the pediatric age group than in the adult age group [40]. Our results for CD34^+^ cell counts are lower than those recorded in previous studies, in which the median percentage of CD34^+^ cells was 1.1 %, while that in our study was 0.3 % [29,37].

Additionally, the median percentage of CD34^+^ cells in other studies was significantly more significant in children and adolescents (1.38 %) than in adults (0.62 %). In contrast, in our study, the median percentage in all age groups was 0.3 % [29]. Our study showed no difference in the range of CD34^+^ cell counts between the sexes, and these results are compatible with those of other studies [37].

Conclusion

5

Establishing a reference value for hematopoietic stem cells and immune cells in the bone marrow based on various influential factors is pivotal for donor selection and defining the bone marrow status.

Availability of data

The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.

Informed consent

The donors agreed on the bone marrow harvest procedure by signing an informed consent form.

Ethical approval

The procedure performed in this study was approved by the College of Medicine Research Committee, University of Sulaimani, Kurdistan Regional Government, Kurdistan, Iraq (approval no. 7/29/701 meeting no. 4).

Statement of human and animal rights

All procedures performed in this study involving human participants were ethically approved by the College of Medicine Research Committee, University of Sulaimani, Kurdistan Regional Government, Kurdistan, Iraq.

CRediT authorship contribution statement

Rebar N. Mohammed: Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Software, Methodology, Formal analysis, Data curation, Conceptualization. Najmaddin S.H. Khoshnaw: Writing – review & editing, Writing – original draft, Formal analysis, Conceptualization. Vian Faeq Mohammed: Writing – review & editing, Visualization, Validation, Supervision, Data curation, Conceptualization. Dastan O. Hassan: Writing – review & editing, Validation, Formal analysis, Data curation. Chra Nawfal Abdullah: Writing – review & editing, Writing – original draft, Validation, Formal analysis, Data curation. Tavan Ismael Mahmood: Writing – review & editing, Project administration, Investigation, Formal analysis, Data curation. Huda A. Abbass: Writing – review & editing, Writing – original draft, Software, Conceptualization. Dereen Ahmed: Writing – review & editing, Methodology, Formal analysis, Data curation. Kani D. Noori: Writing – review & editing, Writing – original draft, Visualization, Investigation, Conceptualization. Lanja I. Saeed: Methodology, Formal analysis, Conceptualization. Salah Mohammed Salih: Writing – review & editing, Validation, Methodology, Data curation. Hiwa S. Sidiq: Methodology, Formal analysis, Data curation. Dlnya Omer Ali: Visualization, Formal analysis, Data curation. Alan Shwan: Writing – review & editing, Writing – original draft, Software, Resources, Data curation, Conceptualization. Ignazio Majolino: Writing – review & editing, Visualization, Validation, Supervision, Formal analysis, Data curation, Conceptualization. Francesco Ipsevich: Writing – review & editing, Validation, Supervision, Investigation, Formal analysis, Data curation, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.