Introduction

Diabetes mellitus (DM) is a group of metabolic disorders characterized by chronic hyperglycemia with abnormalities in the metabolism of carbohydrate, lipid, and protein resulting from defects in insulin secretion, action, or both [1]. The global burden of diabetes among adults has dramatically risen from 108 million in 1980 to 463 million cases of diabetes and 4.2 million deaths in 2019 [2]. More than three-quarters of the global diabetes burden exists in low and middle-income countries and the number of people with diabetes by 2045 is estimated to rise to 700 million worldwide [2]. Sub-Saharan Africa is experiencing a markedly increased prevalence of diabetes. According to the 2019 report of the International Diabetic Federation, 19.4 million (4.9%) adults in the Africa Region and 1.6 million (3.2%) adults in Ethiopia had diabetes [2].

Uncontrolled DM is associated with multiple disorders including metabolic, cellular, and blood disturbances leading to vascular complications [3]. Type 2 diabetes (T2DM) is a part of the metabolic syndrome that comprises dyslipidemia, obesity, hypertension, and changes in hematological parameters [4]. Hematological changes encountered in T2DM patients include changes in the function, structure, and metabolism of red blood cells (RBCs), white blood cells (WBCs), platelet (PLT) and the coagulation systems [5]. These changes may manifest as immunological and coagulation problems, and anemia characterized by a decrease in the RBC count, hemoglobin (Hgb) and hematocrit (Hct) level as compared to non-diabetic individuals [6]. Anemia is a common hematological change in patients with T2DM and often unrecognized, and estimates of its prevalence vary widely [7–9].

Hematological changes in diabetes can be caused by several factors including increased production of reactive oxygen species (ROS) and the formation of advanced glycation end products (AGEs) as a result of the long-term hyperglycemia. Increased production of ROS resulting in oxidative stress, which is implicated in tissue damage and hematological changes such as RBC dysfunction, PLT hyperactivity, and endothelial dysfunction [10–11]. These hematological changes may lead to complications such as anemia, and a state of hypercoagulability, and contribute to cardiovascular disease (CVD) in diabetic patients [12]. Another mechanism is insulin resistance, which is associated with endothelial dysfunction, increased levels of inflammatory markers, and PLT hyperactivity, which accelerates vascular complications in T2DM patients [11].

There has been renewed interest in hematological parameters such as WBC, red blood cell distribution width (RDW), mean platelet volume (MPV), platelet distribution width, and platelet count, as predictors of endothelial dysfunction and inflammation in T2DM [13,14]. An increased WBC count is a classical marker of inflammation and evidence from epidemiological studies suggests an association between WBC count and diabetes risk [15]. Platelets play a vital role in the integrity of normal homeostasis and MPV is the marker for its function [16]. Diabetes is complicated by accelerated atherosclerosis and platelet activation plays a key role in inflammation and the atherothrombosis process contributes to the development of CVD in a patient with T2DM [17]. Mean platelet volume reflects changes either in platelet stimulation or the rate of platelet production and increased MPV has been observed in diabetes patients with coronary heart disease, nephropathy, and retinopathy [18].

Generally, hematological changes have been observed in T2DM patients. Current guidelines on the management of diabetes do not recommend periodic follow-up monitoring of hematological parameters. Although studies regarding the hematological parameters of diabetic patients done in different areas came up with a range of contradictory reports. Some studies showed that there is no statistically significant difference between diabetic patients and healthy controls with respect to RBCs indices [19–21], WBC count, and platelet count [14,19], while the other study showed that RBC, WBC, and PLT indices are significantly higher in diabetic patients than controls [22]. Others reported that RBCs indices except RDW are significantly lower, whereas WBC and PLT indices are significantly higher in the diabetic group than the control group [23,24]. Moreover, there is limited information in Ethiopia, particularly in the study area regarding hematological parameters in T2DM. Parameters obtained from hematologic analyzers can provide insight into changes that occur in hematological indices such as WBC, Hgb, Hct, RBC, PLT, RDW, MPV, and other parameters. Analysis of these parameters could contribute to the following-up of the development of degenerative complications in diabetes [25]. Therefore, this study was aimed to assess hematological parameters of type 2 diabetic adult patients in comparison with screened blood donors at Debre Berhan Referral Hospital, Northeast Ethiopia.

Materials and methods

Results

Demographic, anthropometric, and clinical characteristics of study participants

A total of 268 (134 T2DM patients and 134 controls) study participants were included in this study. The T2DM patients and control groups did not differ in terms of age and sex (p>0.05). Majority of the study participants 85 (63.40%) were males for both T2DM patients and controls. The mean age (mean ±SD) was 43.08±9.30 for T2DM patients and 42.71±8.60 years for controls. Out of the total study participants, around 63 (47.00%) and 76 (56.70%) have a higher educational level for T2DM and controls, respectively. About 113 (84.30%) and 107 (79.90%) were from urban residences for T2DM and controls, respectively. Regarding the anthropometric and clinical characteristics of the study participants, statistically significantly higher values of WHR (p<0.001), WC (p<0.001), BMI (p<0.001), systolic BP (p<0.001), and diastolic BP (p = 0.002) were observed in T2DM patients compared to controls. The median of FBG levels was 159.20 (136.00–185.00) and the median duration of DM since diagnosis was 7.0 (4.0–9.0) years in T2DM patients (Table 1).

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Table 1. Socio-demographic, anthropometric, and clinical characteristics of type 2 diabetic adult patients and control group, at Debre Berhan Referral Hospital, Northeast Ethiopia, 2020 (n = 268).

https://doi.org/10.1371/journal.pone.0253286.t001

Comparison of hematological parameters of the study participants

There is statistically significant difference between T2DM patients and control group with respect to WBC (P = 0.004), absolute neutrophil (P = 0.02), absolute lymphocyte (P<0.001), absolute eosinophil (P<0.001), and absolute basophil counts (P<0.001). Regarding the RBC indices, the mean Hgb in patients with T2DM patients (15.70±1.20) was significantly lower than the control (16.2±1.30) group (P = 0.007). The median (IQR) of RDW was significantly increased in T2DM groups than the control group (P<0.001). Additionally, there were significantly higher mean platelet count (P = 0.013) and mean MPV (P = 0.010) in the T2DM group than the control group (Table 2).

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Table 2. Comparison of hematological parameters of type 2 diabetic adult patients and control group, at Debre Berhan Referral Hospital, Northeast Ethiopia, 2020 (n = 268).

https://doi.org/10.1371/journal.pone.0253286.t002

Factors associated with anemia among type 2 diabetic adult patients

Categorical variables were created from hemoglobin after the hemoglobin was adjusted for an average altitude of 3000 meters with a factor of 1.9 based on the World Health Organization (WHO) recommendation [30]. Associations of anemia with the predictor variables were evaluated using univariable and multivariable logistic regression analysis in diabetic individuals. In the bivariable logistic regression analysis gender, duration of DM, anti-DM drug, and milk consumption were associated with anemia with a p-value of less than 0.2. Variables with a p-value of less than 0.2 were included in the multivariable logistic regression model; duration of diabetes, and milk consumption were significantly associated with anemia. Patients who had been diagnosed with diabetes for 7 years or above were 3 times more likely to have anemia compared to those who have had it for less than 7 years (AOR = 3.05, 95% CI = 1.12–8.34). Type 2 diabetic patients with milk consumption were 4.6 times more likely to have anemia compared to those who do not consume milk (AOR = 4.60, 95%CI = 1.50–14.00) (Table 3).

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Table 3. Factors associated with anemia among type 2 diabetic adult patients at Debre Berhan Referral Hospital, Northeast Ethiopia, 2020 (n = 134).

https://doi.org/10.1371/journal.pone.0253286.t003

Discussion

It has been documented that hematological change is a common complication of DM and represents a significant and under-recognized burden in these patients [31]. In the present study, there was a statistically significant difference in total WBC count, absolute count of neutrophil, lymphocyte, eosinophil, basophil, platelet count, Hgb, RDW and MPV) of T2DM patients compared to control. The mean RBC count was lower in T2DM patients as compared to the control group, but the difference was not statistically significant. This finding is in coherence with the report of studies conducted in India [32], Libya [33], Sudan [21], and Addis Ababa, Ethiopia [34]. The possible explanation for decreased RBCs count might be that persistent hyperglycemia causes increased production of ROS and nonenzymatic glycosylation of Hgb and RBC membrane proteins leading to reduced deformability, increased aggregation, and accelerated aging of RBCs [10,35,36]. These changes in RBCs are also shown to markedly increase blood viscosity that adversely affects the microcirculation in diabetes, leading to microangiopathy [35]. In contrary to our findings, studies carried out in Pakistan [22] and Gondar, northwest Ethiopia [23] reported higher RBC count and Hgb concentration in T2DM patients than controls. This might be explained by the effect of insulin resistance, which is associated with the stimulation of erythroid progenitors increasing RBC count, and increased levels of Hgb and HCT [37].

Regarding RDW, the present study revealed that RDW values were significantly higher in T2DM patients than control groups. This finding is in harmony with the findings of previous studies in Pakistan [22], Saudi Arabia [24], Addis Ababa, Ethiopia [34], and Gondar, northwest Ethiopia [23]. Higher RDW indicates the presence of heterogeneity among the circulating RBCs, which is related to the impairment of erythropoiesis and degradation of RBCs [38]. Chronic inflammation and increased level of oxidative stress are common in diabetes and they are known to reduce RBCs’ survival that results in variation in RBCs size and decreased RBCs count [39].

In the current study, we observed that the Hgb as an index of anemia was significantly lower among T2DM patients as compared to controls. This finding is supported by previous studies carried out in Bangladesh [40], India [32], Libya [33], and Nigeria [41] that have been reported significantly lower Hgb in T2DM patients than the control group. The prevalence of anemia was 17.9% (95% CI: 11.5–24.5), which is comparable with studies conducted in Saudi Arabia 22% [6], Australia 17.8% [7], and Sudan 18.3% [21]. In contrast, our prevalence estimate was lower than those reported by studies conducted in India 71.4% [32], Nigeria 45.2% [41], and Dessie, northeast Ethiopia 26% [42]. This discrepancy might be due to a difference in the characteristics of the study population and sample size variation. In our study the participants were adults and most of them were males.

According to the WHO classification of anemia public health significance in populations [30], the current study showed a mild (5.0–19.9%) prevalence of anemia and a public health problem among T2DM. The etiology of anemia in T2DM is multifactorial and includes chronic hyperglycemia, inflammation, oxidative stress, AGEs, nutritional deficiencies, drugs, and hormonal changes in addition to kidney disease [36,43]. Chronic inflammation in diabetes is characterized by increased inflammatory cytokines such as interleukin 6 (IL-6), and interleukin (IL-1). These pro-inflammatory cytokines are thought to changes the sensitivity of erythroid progenitors to erythropoietin and promote apoptosis of immature RBCs and decrease the number of circulating RBCs resulting anemia of inflammation [43]. Whatever the cause, the consequences of anemia complicating diabetes appear adverse, including evidence of increased all-cause and cardiovascular mortality [7,43].

On logistic regression analysis, the duration of DM was significantly associated with anemia similar to the studies from Nigeria [41]. Patients who had DM for more than 7 years were 3 times more likely to have anemia compared to those who had DM for 7 years or less. Evidence suggested that the longer the duration of the disease the higher the inflammatory process, resulting in increased IL-6 with anti-erythropoietic effect, causing a decrease in the number of circulating RBCs and consequently causing a reduction of circulating hemoglobin [43]. Milk consumption (AOR = 4.60, 95% CI = 1.50–14.0) was significantly associated with anemia. Milk consumption might bring this effect by altering the absorption of iron due to its high calcium and casein contents as well as the low content of iron and folate in milk.

Regarding WBC indices, the present study demonstrated that total WBC count, absolute neutrophil count, and absolute lymphocyte count were significantly higher in the T2DM group compared to the control group. This is in corroboration with the report of previous studies conducted in Turkey [20], Bangladesh [40], Libya [33], and Gondar, northwest Ethiopia [23]. Absolute eosinophil and basophil count also showed a significant increase in the diabetic group than the control group in consonance with the study conducted in Saudi Arabia [24] and Bangladesh [40]. The pattern of WBC disturbances in T2DM patients are not widely available in the literature; in the current study 3.7% had leukocytosis, 1.5% had neutrophilia, 4.5%) had eosinophilia, and 5.2% had basophilia.

The mechanism underlying this increase of the total and differential WBC counts in T2DM patients might be explained by the effect of hyperglycemia and the pathogenesis of T2DM. The available biological data have strongly suggested that T2DM is an inflammatory disease [44]. Increased WBC count is a classical marker of inflammation and evidence from epidemiological studies suggests an association between WBC count and diabetes risk [15]. Although defects in insulin action on the peripheral tissues lead to a chronic low-grade inflammatory state and induce the secretion of proinflammatory cytokines, which promote differentiation and maturation of leukocytes [15,45]. Additionally, in hyperglycemic state leukocytes are activated by AGEs, oxidative stress, and cytokines that increase the state of inflammations and the development of vascular complications in diabetes [12,36]. Neutrophils and monocytes are also suggested to be a marker of inflammation, which is associated with the progression of complications [20,36].

In the present study, the differential white cell counts showed that majority of 127 (94.8%) of T2DM patients had a normal neutrophil count and 5 (3.7%) had neutropenia. Diabetic neutrophils have been associated with impaired deformability, chemotaxis, phagocytosis, bactericidal activity, and they also die sooner than normal [46], which might also explain the neutropenia in the present study. Regarding absolute monocyte count, the present study showed that there was no significant difference between the diabetic and control group. This is in agreement with the study conducted in Ethiopia [23,34] and contrary to a study conducted in Turkey [20] and Bangladesh [40]. Additionally, 5 (3.7%) of T2DM patients had monocytopenia. The reason for this might be that several stimuli, including pro-inflammatory as well as metabolic stimuli, increase the recruitment of monocytes to peripheral tissues, where they differentiate to macrophages and dendritic cells. The destination of monocytes is therefore not the bloodstream and hence peripheral enumeration is not representative of monocyte tissue presence [44].

In this study, analysis of the platelet indices demonstrated that MPV and platelet counts were significantly higher in T2DM compared to the controls. Similar to this study, studies conducted in Nigeria [47] and Ethiopia [34] found that platelet counts were significantly higher in the diabetes group as compared to the controls. The increased MPV among T2DM patients in this study is also in agreement with several studies [22–24,34]. The reason might be that platelet counts and MPV are indicators of thrombotic potential and risk of vascular complications in diabetes. There might be a release of S100A8/A9 by neutrophils which triggers IL-6 production and thrombopoietin synthesis from hepatocytes, leading to bone marrow stimulation to recruit greater numbers of reticulated platelets, which are associated with both atheroprogression and atherothrombosis [48].

The other reason might be due to differences in platelet function between diabetic and control individuals. Evidence suggests that platelets from patients with T2DM have increased reactivity and baseline activation compared to healthy controls [49]. The function of platelet and its size are said to be related and large circulating platelets are reflected by higher MPV which is the marker of the average size, and platelet activity [50]. Diabetic platelets are larger with denser granules and they are enzymatically and functionally hyperactive to produce more prothrombotic factors like thromboxane A2, platelet factor 4, serotonin, and P-selectin than smaller platelets and hence cause an increased tendency to thrombotic events [51]. Platelet hyperactivity in diabetics patients is also attributed to a multitude of factors including insulin resistance, oxidative stress, endothelial dysfunction, inflammation, and hyperglycemia [11,49,52].

Additionally, the present study assessed the correlation of hematological parameters with several cardio-metabolic risk factors. In our study, RBC and RDW were correlated with the duration of diabetes. This relationship might be explained by the effect of chronic hyperglycemia in which lower RBC count and elevated RDW are frequently observed in diabetics with a long duration of disease and vascular complication [53]. Lymphocyte count showed significant and weak positive correlation with SBP and, similar to the study conducted in Turkey [20] and Gondar, Ethiopia [23]. Lymphocyte, and basophil count, and MCH significantly correlated with DBP.

The association between blood pressure and hematological parameters might due to the development of endothelial dysfunction and hypertension in diabetes mellitus. Evidence suggested that hyperglycemia triggers damage to the vascular bed by several cellular mechanisms including accumulation of ROS and AGEs; creating an imbalance between vasodilators and vasoconstrictors [11]. This condition can lead to increased vasoconstriction and vascular remodeling ultimately affecting the blood cells. In respect to the correlation of hematological parameters with body adiposity, platelet count was statistically correlated with BMI and WHR. Moreover, MPV achieved a statistically significant correlation with BMI. A similar study on adult Nigerian people with T2DM found that platelet count and MPV were statistically correlated with BMI. The observed relationship might be due to the fact that obesity is associated with systemic inflammation that could play a role in platelet activation and the production of larger platelets [54].

According to our extensive literature, there has been no comprehensive study assessing the hematological parameters in type 2 diabetes patients; which is addressed by the current study. Despite its strength, our study has some limitations. One limitation of this study is that we cannot determine a cause-effect relationship due to the cross-sectional nature of our study design. Also, morphological and coagulation profile study were not assessed. Another limitation is that it was a single Hospital-based study, thus the observed prevalence of anemia may not reflect its actual burden among patients with type 2 diabetes in the general community.

Supporting information

Acknowledgments

We would like to thank the data collectors and the study participants. Additionally, we would like to thank individuals who have in one way or another contributed to the completion of this research.