Introduction
Complete blood count (CBC) results are routinely used to evaluate acute or chronic inflammation and infection [1]. This is because leukocytes and platelets exist during a chronic inflammatory environment and release angiogenic factors, proteases, cytokines, and chemokines. Also, CBC is used to assess red blood cells (RBCs), haemoglobin (HGB), and red cell indices in anaemia cases [2]. Leukocytes, also called white blood cells (WBCs), are a major component of the body’s immune system and important players in the intestinal immune system. This system is composed of granulocytes (neutrophils, basophils, and eosinophils) and non-granulocytes (monocytes and lymphocytes) [3]. WBCs arise in the bone marrow and pass into the bloodstream. The WBCs help fight infection by invading viruses, bacteria, and germs that attack the body [4].
Ulcerative colitis (UC) and Crohn’s disease (CD) are known as inflammatory bowel disease (IBD), two major chronic diarrhoeal illnesses, and current research suggests that inflammatory bowel disease is closely related to environmental factors, genetic susceptibility, and the immune system of the intestinal mucosa [5, 6]. The inflammatory process in these illnesses includes many innate immunity cells (neutrophils, macrophages, eosinophils, basophils, and mast cells) that represent the first line of defence against an intruding pathogen that is used by the host immediately or within hours of encountering an antigen. Adaptive immunity T helper (Th) lymphocyte is antigen dependent and antigen specific and, therefore, involves a long time between exposure to the antigen and maximal response [7, 8]. The neutrophils are primary immune cells that respond to infection and are the most frequent type of WBCs, representing 50–70% of the total WBC in blood circulation [9]. Lymphocytes play a main role in innate and adaptive immunity. Lymphocytes comprise 20–50% of total WBCs, comprising T, B, NK, and innate lymphoid cells [9]. After that, different blood composite scores have been proposed as biomarkers of systemic inflammation, including NLR, PLR, LMR, and a new inflammation-related biomarker known as SIRI, which was released in 2016. All the above are highly sensitive markers of inflammation in several inflammatory disorders, for instance, infection, rheumatoid arthritis, cardiovascular disease (CVDs), atherosclerosis, and cancer [10].
IBD patients are at risk of developing anaemia, a status in which the RBCs and the protein haemoglobin in the blood are at lower levels. Anaemia is consistently linked with IBD, and the prevalence ranges from 4% to 67% [11]. This can be caused by problems due to IBD, including blood loss, decreased iron absorption, and decreased absorption of other vitamins and minerals. Pro-inflammatory cytokines are proteins characteristic of IBD attacks, causing bone marrow dysfunction, resulting in decreased haemoglobin production. This is often accompanied by iron deficiency anaemia [12]. The RBC indices in routine blood examination include the following: mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), and mean corpuscular haemoglobin concentration (MCHC), which are widely known as RBC indices. These values are beneficial in explaining the aetiology of anaemia. RBC indices can be determined if the values of RBC count, HGB, and haematocrit (PCV) are known [13]. The size of RBCs indicates MCV, the amount of HGB per RBC indicates MCH, and the amount of HGB per unit volume indicates MCHC, calculated as MCH divided by MCV [14]. Red distribution width (RDW) represents the coefficient of variation of the RBC volume distribution (size) and is expressed as a percentage. RDW as a measure of anisocytosis helps further in the evaluation of anaemia based on morphology [15]. Platelets (PLTs) participate in active disease periods, stimulate mucosal inflammation, and regulate coagulation [16]. In IBD, the PLTs have been changed, including count increases and morphological changes (mean platelet volume (MPV), platelet distribution width (PDW), and plateletcrit (PCT)), which are stimulated by inflammatory agonists [17]. PLTs are acute-phase reactants that are induced by inflammatory cytokines [18]. Rising proof suggests that fibrosis in GIT diseases may also be related to PLT [19]. There are studies dealing with gender differences in the response of white cells, red cells, and platelets in cases of intestinal inflammation in IBD patients.
Aim
This study aims to identify differences in haematological features between genders in patients with IBD, and to investigate whether these differences are related to disease activity and anaemia.
Material and methods
Patients
All charts were reviewed to identify inpatient and outpatient IBD patients seen in the clinic. The study was conducted after approval by the hospital’s Ethics Committee. Furthermore, there was a waiver of informed consent. The diagnosis of inflammatory bowel disease was determined according to standard clinical, endoscopic, histologic, and radiological criteria.
The present study was conducted between October 2023 and March 2024 in the Gastroenterology and Hepatology Specialised Hospital, Najaf, Iraq. The specimens were prospectively collected and scored clinically and endoscopically from IBD patients. In this study, 50 patients (25 male and 25 female) were admitted with symptoms: diarrhoea, constipation, abdominal pain, weight loss, anorexia, gastrointestinal bleeding, or rectal bleeding, and 50 samples were taken for controls (25 male and 25 female) from healthy indicviduals (not suffering from IBD). The study was conducted as an intervention project of the Swiss Inflammatory Bowel Disease Cohort Study with approval from each local Ethics Committee.
Methods
The standard CBC includes measurement of WBC, WBC differential (neutrophils, lymphocytes, monocytes, eosinophils, and basophils per 1000 cells/µl), RBC, HGB, and haematocrit (HCT) using an automated haematology analyser (CBC Analyzer Swelab alfa, Sweden). Red cell indices (mean corpuscular volume MCV, mean corpuscular haemoglobin MCH, and mean corpuscular haemoglobin concentration MCHC), red cell distribution width (RDW), a platelet count, and plateletcrit (PCT), in addition to the haematological composite scores, including N/L ratio, M/L ratio, P/L ratio, and SIRI, were calculated as follows:
Red cell indices MCH, MCHC, and HCT were calculated as follows, according to Ware [20]:
Haematological composite scores N/L ratio, M/L ratio, P/L ratio, and SIRI were calculated as follows, according to Carrillo-Palau [21]:
Statistical analysis
Results are expressed as mean ± standard error. Data were subjected to analysis using SAS (Statistical Analysis System – version 9.1). Post hoc tests are an integral part of ANOVA. One-way ANOVA and the least significant differences (LSD) post hoc test were performed to assess significant differences among means. P < 0.05 is considered statistically significant.
Results
The results, shown in Table I, show a significant increase (p < 0.05) in WBCs, neutrophil, monocyte, eosinophils, and basophil cells, N/L ratio, P/L ratio, M/L ratio, and SIRI in IBD patients, both males and females, in comparison with healthy individuals. The results also show significant differences between males and females in the studied criteria in healthy and IBD patients.
Table I
Total and differentiated WBC response differences in male (IBD-M) and female (IBD-F) patients with IBD in comparison with apparently healthy subjects
[i] WBCs – white blood cells (× 103/mm3). Lym – lymphocyte (× 103/mm3). Neu – neutrophil (× 103/mm3). Mon – monocyte (× 103/mm3). E&B – eosinophils & basophils (× 103/mm3). N/L ratio – neutrophil-to-lymphocyte ratio. P/L Ratio – platelet-to-lymphocyte ratio. M/L ratio – monocyte-to-lymphocyte ratio. SIRI – Systemic Immune-Inflammatory Index (× 10–3). Lower-case letters denote significant differences between groups (rows).
Furthermore, the results in Table II show a significant decrease (p < 0.05) in RBCs, HGB, MCV, MCH, MCHC, and HCT in male and female IBD patients compared to healthy individuals. The platelet count increased (p < 0.05) significantly in the IBD-M and IBD-F groups compared to the C-M and C-F groups.
Table II
Total RBCs, RBC indices, and platelet responses in male (IBD-M) and fsemale (IBD-F) patients with IBD in comparison with apparently healthy subjects
[i] RBCs – red blood cells (× 106/mm3), HGB – haemoglobin (g/dl), MCV – mean corpuscular volume (fl), MCH – mean corpuscular haemoglobin (pg/cell), MCHC – mean corpuscular haemoglobin concentration (g/dl), HCT – haematocrit (%), RDW – red cell distribution width, PLT – platelets (× 103/mm3), PCT – plateletcrit. Lower-case letters denote significant differences between groups (rows).
Discussion
The current study aimed to detect differences in haematological cell count, red cell indices, platelet count, and haematological composite scores between male and female patients with IBD and healthy individuals. IBD is an idiopathic and inflammatory chronic intestinal disease, described by typical clinical signs like diarrhoea, abdominal pain, and bloody stools [22–24]. As typical markers for inflammation, WBCs and PLTs are considered markers of inflammation because they are typically found in acute and chronic inflammatory settings, where they release chemokines, proteases, cytokines, and angiogenic factors [21]. The present results revealed the inflammatory response of IBD patients as increased total and differentiated WBCs. Recent research talks about the role of WBC counts in IBD pathogenesis [25]. Leukocytes in circulation are key parameters for assessing immune cell homeostasis. Yet, many monitoring studies have associated circulating leukocytes with IBD [25–27]. Neutrophils (NEU) are the master cells of our innate immune system and play a fundamental role in the body’s defence. They rapidly arrive at the site of infection and exhibit a range of influential functions, including phagocytosis, degranulation, and NEU proliferation (NETosis), to eliminate invading pathogens. Neutrophils are now recognised as potent mediators of chronic inflammation. In many chronic inflammatory diseases, their undesirable actions, such as extensive infiltration, hyperactivity, dysfunction of effector cells, and prolonged survival, contribute to disease progression [25, 28].
In an observational cohort study linking monocyte counts to recurrent therapy in IBD patients, 95 patients with IBD were monitored continuously for 16 months. The results showed a significant difference between relapse and improvement in monocyte count, and monocytes and faecal calprotectin were associated with relapse according to multivariate analysis [29]. Another study reported that IBD is related to high levels of eosinophil and basophil, similarly to our current study [30]. Furthermore, lymphocytes were significantly reduced in patients with IBD compared to healthy subjects after adjustment for covariates [21].
In our study, the composite haematological scores are higher in patients with IBD. These results are similar to some other studies [21, 31], which reported that this analysis, performed separately in patients with IBD, was strongly related to the active disease according to disease severity by registration counts of neutrophils, lymphocytes, and monocytes and counting their ratios; N/L ratio, P/L ratio, and L/M ratio, which could be useful as blood-based markers in differentiating active disease in patients with IBD. As demonstrated in this study, the SIRI show significant differences between patients with IBD and healthy controls, and the results were similar to many studies that proved that SIRI was significantly elevated in IBD patients and was closely related to IBD disease activity [21, 32, 33].
The traditional parameters used to diagnose anaemia are RBCs, HGB, and RBC indices; when their levels are lower, it indicates anaemia [14, 34, 35]. In our study, the results of RBC and HGB were significantly lower in the IBD (IBD-M and IBD-F) groups, similarly to [12], who reported that anaemia is one of the most common extraintestinal manifestations of IBD. Patients with IBD are at risk of developing anaemia, a condition in which blood RBCs reduce and HGB protein. In addition to problems caused by IBD, these include decreased iron absorption, decreased mineral and vitamin absorption, and loss of blood. Pro-inflammatory cytokines are proteins characteristic of IBD attacks, causing bone marrow dysfunction, resulting in decreased haemoglobin production. This is often accompanied by iron deficiency anaemia [12].
The results show that the mean rates of RDW were significantly increased in IBD patients. A study [36] was conducted searching for RDW’s capability for disease activity evaluation in CD. The RDW was closely related to WBC, CRP, and PLT levels, but not to ESR. A study [37] evaluated whether RDW can be used as an index of CD activity. Another study [38] performed a systematic review of RDW, assessing it in IBD as a marker of disease activity, to differentiate UC from CD, and as a marker for anaemia assessment in IBD. The studies’ results point out the promising potential of RDW as a surrogate marker of disease activity in IBD [38, 39]. As a result, decreased RBC count, HGB, and MCH levels and increased RDW are related to symptoms of depression [40].
In our study, we determined that PLT and PCT increased significantly. PLT and PCT are biomarkers that are generality suitable for all tested inflammatory biomarkers for evaluation of endoscopic IBD activity [41]; increased levels were the best biomarkers of endoscopic ulcerative colitis activity. PLT-linked indicators, involving PCT and PLT/ALB ratios, had better diagnostic rates for endoscopic CD and UC, consequently, and were much better than current routine laboratory tests. Significantly, these biomarkers may improve intestinal inflammation monitoring and therapeutic efficacy in IBD.
In our study, we investigated the significant differences in the haematological cell count of IBD patients’ in relation to gender, including WBCs, neutrophil, monocyte, eosinophils, and basophils cells, P/L ratio, N/L ratio, M/L ratio, and SIRI increasing in females more than in males, except for the lymphocyte, which decrease in females; these results are similar to [42], which showed that females had greater WBC and PLT counts than males. The sex hormones act as immunomodulators: oestrogen increases immunologic responses, whereas testosterone suppresses the response to infection. Also [43] proved that testosterone induces WBC production. Oestrogens play a multifactorial role in inflammation and autoimmune diseases [44]. In the immune system, oestrogens act on leukocytes and influence their number and function [45]. In the pathophysiology of IBD, studies have shown the complexity of oestrogen signalling, shedding light on the potential mechanisms by which gender- and age-related differences afffect IBD [46]. In other studies, for example [35], there were no significant differences between men and women in both CD and CU.
The results in the present study showed the difference between the C-M and C-F groups, similarly to [43], which proved a sex difference in RBC counts and HGB concentrations in adulthood. The average male adult has a higher RBC and HGB than the average female adult; the sex differences in RBC and HGB numbers have been attributed to the effect of the sex hormone; for example, testosterone stimulates erythropoietin (EPO) production [43].
Our study results showed that females showed greater MCH, MCHC, and RDW than males, similarly to [42], which examined sex and underlying processes behind the well-known sex disparities in adult HGB levels, which are most likely due to the direct effects of sex hormones, including both androgens and oestrogen, on erythropoiesis. There is no proof that females have fewer cellular processes for haeme production, while there is no distinction of how males and females absorb iron [42]. This contradicts some studies [47], which concluded that gender has no significant correlation with red cell indices. In this study, we noticed an increase in PLT count in the IBD-F group than the IBD-C group in males, similarly to [35], which proved that PLTs were higher in females infected with anaemia and IBD than in males [35, 42].
The present results denoted the deleterious in the immunological response of the inflamed intestine. However, these changes can be modulated, as studies have provided robust preliminary evidence that synbiotic administration to IBD patients results in beneficial therapeutic effects [48, 49].
Conclusions
We found an association between haematological features of IBD patients, disease activity, and anaemia in both sexes. Therefore, these features could be proposed as alternative biomarkers of the disease and could also be used in clinical practice. Normal and abnormal blood parameters are essential, as different measures can be used as separate biomarkers to assess the course of patients with different disease conditions. We recommend performing blood tests on patients, as this may help in early detection of the disease and prediction of its course.
