Assessment of cellular immunity by CD4+ and CD8+ lymphocyte counts in 2–12-year-old children with iron-deficiency anemia Khalale P, Patil BU, Ghongade P, Gupta A

ORIGINAL ARTICLE

Year : 2023 | Volume : 12 | Issue : 1 | Page : 55-60

Assessment of cellular immunity by CD4+ and CD8+ lymphocyte counts in 2–12-year-old children with iron-deficiency anemia

Pratiksha Khalale, Bharat Umakant Patil, Pravinkumar Ghongade, Anupama Gupta
Department of Pathology, Mahatma Gandhi Institute of Medical Sciences, Wardha, Maharashtra, India

Date of Submission	06-Nov-2022
Date of Decision	02-Feb-2023
Date of Acceptance	06-Feb-2023
Date of Web Publication	15-Mar-2023

Correspondence Address:
Anupama Gupta
Department of Pathology, Mahatma Gandhi Institute of Medical Sciences, Sevagram, Wardha - 442 102, Maharashtra
India

Source of Support: None, Conflict of Interest: None

Check

DOI: 10.4103/sjhs.sjhs_134_22

Abstract

Background: Iron-deficiency anemia (IDA) in children and infants can have long-term adverse consequences on neurodevelopment and behavior, which can be irrevocable in some cases and detrimental to the immune system. Aim: The aim of this study was to investigate the role of IDA and its effects on immunity in CD8 and CD4 lymphocytes. Settings and Designs: This was a prospective study. Materials and Methods: A total of 40 pediatric cases (2–12 years) were first time diagnosed as IDA based on complete blood count (CBC) parameters, peripheral blood smear, and serum ferritin levels. Flow cytometric immune assays were used to determine the number of CD4+ and CD8+ lymphocytes and the ratio of CD4+/CD8+. Statistical Analysis: Pearson's correlation coefficient and Students' unpaired t-test were used. Results: The difference between the case and control groups in hemoglobin (Hb), red blood cell (RBC) indices, red cell distribution width, RBC count, and serum ferritin is highly significant. A significant linear positive association between CD3+ and CD4+ cell counts and serum ferritin and a significant linear positive correlation between CD4+ cell counts and Hb were observed. However, there was a nonsignificant linear positive association of both parameters, hemoglobin (Hb) and mean corpuscular volume (MCV) with CD3+ cell count, CD4+ cell count, and CD4+/CD8+ cell ratio. Conclusion: Iron deficiency does not affect total white blood cell count or differential leukocyte count on routine CBC, but it can alter cellular immunity (CD3+ cell count, CD4+ cell count, and CD4+/CD8+ cell ratio).

Keywords: Children, immunity, iron-deficiency anemia

How to cite this article:
Khalale P, Patil BU, Ghongade P, Gupta A. Assessment of cellular immunity by CD4+ and CD8+ lymphocyte counts in 2–12-year-old children with iron-deficiency anemia. Saudi J Health Sci 2023;12:55-60

How to cite this URL:
Khalale P, Patil BU, Ghongade P, Gupta A. Assessment of cellular immunity by CD4+ and CD8+ lymphocyte counts in 2–12-year-old children with iron-deficiency anemia. Saudi J Health Sci [serial online] 2023 [cited 2023 Mar 20];12:55-60. Available from: https://www.saudijhealthsci.org/text.asp?2023/12/1/55/371705

Introduction

Deficiencies in various vitamins and minerals can affect the human body through several different mechanisms. Iron deficiency is a prevalent clinical condition observed daily in clinical practice, even though iron is the most abundant metal on earth. It impacts a person's physical, mental, and work performance, especially in premenopausal women and children. In addition to helping red blood cell (RBC) form, iron is essential for our body's defense mechanisms in several ways.^[1] Iron-deficiency anemia (IDA) was labeled as one of the leading causes of global disease burden in 2002.^[2]

Globally anemia is the second leading cause of disability, succumbing to deaths of around ten lakhs every year, with 75% of those fatalities occurring in Africa and Southeast Asia.^[3] It is generally observed that IDA contributes to 50% of anemia cases.^[4] IDA accounts for 2.5 crore disability-adjusted life years (DALYs), or 2.4% of all DALYs occurring globally, in terms of years of healthy life lost.^[5]

It is generally well understood that neutrophil activities, including phagocytosis and oxidative burst, are impaired by iron deficiency. In contrast, nothing is known about how IDA affects the adaptive immune system. Children with iron deficiency have a higher total lymphocyte count (LC); according to some studies, however, this finding has not been supported by other studies.^[6],[7]

The literature has suggested that it might inhibit humoral immunity (HMI) and cell-mediated immunity (CMI).^[8] Even before anemia develops, IDA has long-term adverse effects on neurodevelopment and behavior in children and infants that can sometimes be permanent.^[9] Among IDA indicators, reduced hemoglobin (Hb) levels appear to be the driving force behind alteration in subsets of lymphocytes instead of reduced levels of iron and ferritin.^[1]

The mechanism through which IDA affects these cells is still unknown. Iron loading and depletion are well controlled by effector molecules such as heme oxygenase, Toll-like receptors, hypoxia factor-1, and nuclear factor-kappa B. This could impact the cell's ability to respond to bacteria attacks.^[10] Because of its growth-differentiating and stimulating behavior, iron is essential in immune cell monitoring, especially for lymphocytes.

This research evaluated the distribution of lymphocyte subsets to investigate the critical role of IDA and its implication on immunity in the pediatric age group. A flow cytometric technique assessed the flow cytometric CD8+ and CD4+ LC as a part of cell-mediated immunity (CMI) in blood smear (PS) (peripheral smear) in IDA.

Materials and Methods

The study was carried out at a tertiary care hospital in central rural India. It is attached to around 1000 bedded tertiary care rural hospitals. The hematology section receives approximately 44,000 specimens each year. This analytical cross-sectional study was carried out in the hematology section of the pathology department from October 01, 2019–September 30, 2021. IDA was diagnosed based on Hb≤11 g%, microcytic hypochromic RBCs on PS, serum ferritin <10 ng/dl,^[11] mean corpuscular volume (MCV <80 FL), and mean corpuscular Hb (MCH <27 pg).

A total of 40 pediatric cases (between age group of 2–12 years), were diagnosed for the first time as IDA, were included in the study. These cases were diagnosed in clinical pathology or hematology section of the hospital. Ten age- and sex-matched seemingly healthy children were recruited from a pediatric clinic as a control group. Their Hb, red cell distribution width (RDW), RBC count, RBC indices, and ferritin levels should be within normal values for their age. The exclusion criteria for cases and control were the same. Written informed consent was obtained from study participants and their caregivers, and all data were kept confidential, de-identified, and used solely for research purposes.

All patients should have normal growth for their age, and nutritional iron deficiency is the only etiology with no other nutritional deficiencies included. Patients with other known or proven causes of immune deficiency, such as protein–energy malnutrition (−2SD and higher), acute and or a chronic systemic inflammatory condition known to influence cellular immune function, or influencing ferritin levels, which is also an acute-phase reactant. Children who have had a previous history of iron therapy, any hematinic, or any multivitamin intake in last 3 months or anemia due to blood loss, any infection, or parasitic infestation at time of study recruitment or any past history of therapy received like immunosuppressive therapy, radiotherapy, and chemotherapy, were excluded from the study.^[6] The cells influencing cell-mediated immunity (CMI), i.e. CD4+, CD8+ lymphocytes, and CD4+/CD8+ cell ratio, were assessed through immunophenotyping by flow cytometry in the study population.

Methods

Venous blood samples were collected from cases and controls under complete aseptic conditions. In Tripotassium ethylene diaminetetraacetic acid (K3-EDTA) anticoagulant tubes, 2 mL of blood was taken for complete blood counts (CBCs), peripheral PSs, and flow cytometric assays. After discussing the study's relevance and methodology with the parents or next guardians, 2 mL of blood in a plain was collected for serum ferritin from individuals whose diagnosis was found to favor a pure iron deficiency. Blood samples were taken from IDA patients just before their scheduled iron therapy. E411 by Cobas (“Elecsys ferritin”) quantitative test kit was used for serum ferritin, an immunoassay for in vitro quantitative determination of ferritin in human serum and plasma. A CBC was performed using an automatic blood counter (Medonic M32 B). Leishman's stain was also used to stain peripheral PSs. The neutrophil/lymphocyte ratio (NLR) and platelet–lymphocyte ratio (PLR) were calculated using the absolute neutrophil, lymphocyte, and platelet counts. Using a Cobas e411 automated chemistry analyzer, serum ferritin was measured using a two-site sandwich direct immunoassay (Roche Diagnostics, Germany). The manufacturer's instructions carried out the assay. T-lymphocyte subsets were counted in whole blood samples using the BD™ multi-check test kit. The test kit had (antibody fluorochrome) CD3/CD8/CD45/CD4 (FITC/PE/PeCP/APC). CD8+ and CD4+ cell counts are determined by immunophenotyping using the BD Canto II flow cytometer. The lymphocyte population was defined utilizing a side and forward scatter histogram. BD FACSCanto™ software was used to calculate the percentage of cell counts in the blood sample.

The institutional ethics committee approved this study (MGIMS/IEC/Path/127/2019 dated October 19, 2019). Informed consent was also taken from every patient in their language regarding their willingness to participate in the study.

Statistical analysis

Pearson's correlation coefficient and the Student's unpaired t-test were used for statistical analysis. The software used in the study was SPSS 27.0 version (IBM software, Chicago, USA), and GraphPad Prism 7.0 (Graphstats Technologies Private Limited, Bangalore, Karnataka-560035, India); P < 0.05 is the significance level.

Results

The study population is divided into two groups: a case group (n = 40) and a control group (n = 10), each of which is further divided into two subgroups: 2–7 years and 8–12 years. Most of our patients in the case group (n = 30, 90%) were between 2 and 7 years old. Only four patients were between the ages of 8 and 12 and were all female.

However, because the number of males and females in the population is almost equal, we considered an equal number of males and females in our control group to counteract the gender bias. No study cases had protein–energy malnutrition, so it excluded the possibility of malnutrition affecting cellular immunity.

The difference between the case and control groups in Hb, RBC indices, RDW, RBC count, and serum ferritin is highly significant. White blood cell (WBC) count, absolute neutrophil count (ANC), absolute LC (ALC), their ratio, PLR, and platelet count are all nonsignificant. When comparing study cases to controls, there is a substantial difference in the percentage of CD4+ cells, CD3+ cells, and CD4+/CD8+ cell ratio. However, the number of CD8+ cells in the case and control groups did not show a remarkable difference [Table 1] and [Figure 1].

Figure 1: Lymphocyte populations of CMI and CD4/CD8 ratio among case and control groups

Click here to view

Table 1: Lymphocyte populations of cell-mediated immunity in case and control groups

Click here to view

The present study revealed a highly substantial positive relationship between the severity of the iron deficiency and CD3+ and CD4+ LCs of CMI in case and control populations. CD8+ cell and other blood cell counts showed no statistically significant correlation. However, the CD4+/CD8+ cell ratio again showed a positive correlation with IDA. A significant linear positive association between CD3+ and CD4+ cell counts and serum ferritin and a significant linear positive correlation between CD4+ cell count and Hb were observed [Table 2]. While Hb has a nonsignificant linear positive correlation with CD3+ cell count, CD4+ cell count, and CD4+/CD8+ cell ratio, mean corpuscular volume (MCV) also has a nonsignificant linear positive correlation with CD3+ cell count, CD4+ cell count, and CD4+/CD8+ cell ratio. Hb: hemoglobin.

Table 2: Correlation between the study population's serum ferritin level and blood cell count

Click here to view

Discussion

Iron deficiency can have deleterious effects on the immune system. Literature shows that it may suppress cell-mediated immunity (CMI) and HMI.^[8] Hassan et al.^[12] evaluated the effect of IDA on humoral, cellular, and nonspecific immunity (phagocytic activity and oxidative burst) and cytokines. At the same time, a few studies^[6],[13] studied the impact of IDA on CD4 T-lymphocytes and CD8 T-lymphocytes and the CD4/CD8 ratio in children. Except for one case, all children in the case and control groups were within the normal weight range according to Indian academy of pediatric (IAP) guidelines.^[14] This shows that no study cases had protein–energy malnutrition, so it excluded the possibility of malnutrition affecting cellular immunity.

There is a significant difference in the parameters associated with IDA between the case and control groups, i.e. Hb (P < 0.001), RBC count (P < 0.001), MCV, MCH (P = 0.001), MCH concentration (P = 0.021), RDW (P = 0.026), and serum ferritin (P < 0.001). However, no significant difference is seen in total WBC count (P = 0.60), platelet count (P = 0.24), LC (P = 0.74), neutrophil count (P = 0.37), NLR (P = 0.37), and PLR (P = 0.45) in the case and control groups, and so we can see that iron deficiency does not affect non-RBC parameters of CBC much, to be alerted about immune mediating cell count or function in these children. A few authors^{[8],[15],[16],[17],[18],[19]} reported the same results concerning WBC count, neutrophil count (NC), NLR, and LC. Anani et al.^[13] showed similar results concerning total WBC count.

In the current study, 5 cases had an increased platelet count in the case group. However, this thrombocytosis was statistically insignificant in our study. A few studies^[6],[12] reported similar results regarding platelet count, whereas others suggest that high platelet count is associated with IDA.^{[8],[13],[15],[20]} First-time diagnosed cases of IDA were included in this study, which likely explains the normal platelet count in most of our cases. In a study, Kuku et al.^[21] observed that 520 out of 615 adult IDA patients in the early stages of iron deficiency had normal platelet counts. A significant decrease in the LC influencing CMI in IDA children is represented by a reduction in the percentage of CD4+ cells in the case group compared to the control group. This finding is well correlated with almost all studies in the literature, establishing that the percentage of CD4+ cells is decreased in IDA.^{[6],[8],[13],[15],[16],[19],[20]} At the same time, only two previous studies^[17],[18] reported no significant alteration in the percentage of CD4+ cells in IDA.

The percentage of CD3+ cells in the case group was significantly decreased than in the control group (P = 0.013). Three studies corroborate our finding of a decrease in the percentage of CD3+ cell count in IDA.^{[6],[16],[20]} However, few^[17],[18] again reported no change in the percentage of CD3+ cells to count in IDA. The current study showed a significant decrease in the CD4+/CD8+ cell ratio in the IDA (P = 0.044), which is correlated with other studies.^{[6],[8],[16],[20]} The present study could not identify any significant decrease in the percentage of CD8+ lymphocytes (P = 0.70) in our IDA cases, and many studies correlate with this finding.^{[6],[8],[17],[18],[19]} In contrast, two studies reported a decreased percentage of CD8+ cells.^[15],[20] The main reasons behind the noncorrelation of the percentage of CD4+ cells, CD3+ cells, CD8+ cells, and CD4+/CD8+ cell ratio findings from our study with some other studies can be explained by the difference in the demography. For example, there is a stark difference in study group age. A second possible explanation for noncorrelation may be geographic differences. The third possible explanation, which is very common concerning noncorrelation in any two studies, remains the number of cases taken.

The present study showed that there is a significant positive correlation between serum ferritin level and CD4+% (P = 0.003, r = 0.40) and CD3+% (P = 0.020, r = 0.32) and a nonsignificant positive correlation between

serum ferritin level and ALC (P = 0.74, r = 0.048) and CD4+/ CD8+ ratio (P = 0.23, r = 0.17). In comparison, there was a nonsignificant negative correlation between serum ferritin and ANC (P = 0.68, r = 0.058) and CD8+% (P = 0.99, r = 0.001). Our study showed that there is a significant positive correlation between Hb and CD4+% (P = 0.040, r = 0.29) and a nonsignificant positive correlation between Hb and CD3+% (P = 0.27, r = 0.159) and a CD4+/ CD8+ ratio (P = 0.206, r = 0.182). At the same time, there was a nonsignificant negative correlation between Hb and ALC (P = 0.96, r = 0.007), ANC (P = 0.635, r = 0.069), and CD8+% (P = 0.50, r = 0.096). [Figure 2] and [Table 2]. Anani et al.^[13] also showed similar results to our finding of a nonsignificant negative correlation between Hb% and the percentage of CD8+ cells. The present study showed that there is a nonsignificant positive correlation between MCV and CD4+% (P = 0.36, r = 0.132), CD3+% (P = 0.50, r = 0.096), and CD4+/CD8+ ratio (P = 0.29, r = 0.152). In contrast, there was a nonsignificant negative correlation between MCV and ALC (P = 0.06, r = 0.262), ANC (P = 0.61, r = 0.072), and CD8+% (P = 0.50, r = 0.098). To the best of our knowledge, the present research could not find any studies comparing these data, probably reporting it for the first time. Overall, the current findings largely correlate well with the previous studies, confirming that a few cells of CMI (CD4+ cells and CD3+ cells) are decreased in IDA, and others do not (CD8+ cells).^{[8],[16],[22]}

Figure 2: (a-l) (a): Nonsignificant negative correlation between serum ferritin level and ANC (↑serum ferritin level, ↓ANC), (b) Nonsignificant positive correlation between serum ferritin level and ALC (↑serum ferritin level, ↑ALC), (c) Significant positive correlation between serum ferritin level and percentage of CD3+ lymphocytes (↑serum ferritin level, ↑CD3+ lymphocytes%), (d) Significant positive correlation between serum ferritin level and percentage of CD4+ lymphocytes (↑serum ferritin level, ↑CD4+ lymphocytes%), (e) Nonsignificant negative correlation between the percentage of CD8+ lymphocytes (↑serum ferritin level, slightly ↓CD8+ lymphocytes%), (f) Nonsignificant positive correlation between serum ferritin level and CD4+/CD8+ cell ratio (↑serum ferritin level, ↑CD4+/CD8+ cell ratio), (g) Nonsignificant positive correlation between percentage of CD3+ cell count and Hb (↑Hb, ↑CD3+%), (h) Significant positive correlation between percentage CD4+ cell count and Hb (↑Hb, ↑CD4+%), (i) Nonsignificant positive correlation between CD4+/CD8+ cell ratio and Hb (↑Hb, ↑CD4+/CD8+ cell ratio) (Hb), (j) Nonsignificant positive correlation between the percentage of CD3+ cell count and MCV (↑MCV increases, ↑CD3+ cell count), (k) Nonsignificant positive correlation between the percentage of CD4+ cell count and MCV (↑MCV increases, slight ↑ CD4+ cell count.), (l) Nonsignificant positive correlation between MCV and CD4+/CD8+ cell ratio (↑MCV, slight ↑ CD4+/CD8+ cell ratio) (MCV-mean corpuscular volume). Hb: hemoglobin. ANC: Absolute neutrophil count, ALC: Absolute lymphocyte count, MCV: Mean corpuscular volume

Click here to view

Strengths and limitations of the study

This is the fourth study in India evaluating this critical research area of the increased infectious mortality and morbidity rate in Indian children with IDA. However, the financial constraints could not allow us to take a large study population. However, a decrease in CD3+ and CD4+ cell count and thus a reduction in cellular immunity could be shown significantly in IDA children.

Conclusion

On routine CBC, iron deficiency does not affect total WBC count or differential leukocyte count. Iron deficiency can alter cellular immunity (CD3+ cell count, CD4+ cell count, and CD4+/CD8+ cell ratio.) Further studies taking large case populations are recommended to evaluate the types of infections that are more common in these children with low cellular immunity in IDA and how this could impact the overall health of the children in India.

Acknowledgment

This project was financially aided by the Research Grant Committee of our institute.

Financial support and sponsorship

This study was financially aided by the Research Grant Committee of the institute.

Conflicts of interest

There are no conflicts of interest.

References

1.	AlRajeh L, Zaher A, Alghamdi A, Alsheikh R, AlSultan O. Effects of iron deficiency and its indicators on lymphocyte subsets: A study at King Fahd Hospital of the university, Saudi Arabia. J Blood Med 2022;13:61-7.
2.	World Health Organization. The World Health Organization Report 2002: Reducing Risks, Promoting Healthy Life. Avenue Appia 20, 1211 Geneva, Switzerland: World Health Organization Library Cataloguing Publication Data; 2002. p. 232.
3.	World Bank. Public Health at a Glance.World bank: 818 H Street, NW Washington, DC 20433 USA; 2004.
4.	World Health Organization. Iron Deficiency Anemia: Assessment, Prevention, and Control. A Guide for Programme Managers. Geneva: World Health Organization; 2001.
5.	Ministry of Health & Family Welfare. Guidelines for control of iron deficiency anaemia. National Iron Plus Initiative. New Delhi: MoHFW, Government of India; 2013.
6.	Aly SS, Fayed HM, Ismail AM, Abdel Hakeem GL. Assessment of peripheral blood lymphocyte subsets in children with iron deficiency anemia. BMC Pediatr 2018;18:49.
7.	Sadeghian MH, Keramati MR, Ayatollahi H, Manavifar L, Enaiati H, Mahmoudi M. Serum immunoglobulins in patients with iron deficiency anemia. Indian J Hematol Blood Transfus 2010;26:45-8.
8.	Das I, Saha K, Mukhopadhyay D, Roy S, Raychaudhuri G, Chatterjee M, et al. Impact of iron deficiency anemia on cell-mediated and humoral immunity in children: A case control study. J Nat Sci Biol Med 2014;5:158-63.
9.	Baker RD, Greer FR, Committee on Nutrition American Academy of Pediatrics. Diagnosis and prevention of iron deficiency and iron-deficiency anemia in infants and young children (0-3 years of age). Pediatrics 2010;126:1040-50.
10.	Beard JL. Iron biology in immune function, muscle metabolism and neuronal functioning. J Nutr 2001;131:568S-79S.
11.	World Health Organisation. WHO Guideline on Use of Ferritin Concentrations to assess Iron Status in Individuals and Populations. Geneva: World Health Organisation; 2020.
12.	Hassan TH, Badr MA, Karam NA, Zkaria M, El Saadany HF, Abdel Rahman DM, et al. Impact of iron deficiency anemia on the function of the immune system in children. Medicine (Baltimore) 2016;95:e5395.
13.	Anani MM, Omar HH, El-Kelani A, Hashem AA. Impact of iron deficiency anemia on CD4 and CD8-T lymphocytes among preschool-school children. Comp Clin Path 2017;26:1063-8.
14.	Indian Academics of Pediatric. World Health Organisation. India: Indian Academics of Pediatric; 2006.
15.	Attia MA, Essa SA, Nosair NA, Amin AM, El-Agamy OA. Effect of iron deficiency anemia and its treatment on cell mediated immunity. Indian J Hematol Blood Transfus 2009;25:70-7.
16.	Mullick S, Rusia U, Sikka M, Faridi MA. Impact of iron deficiency anaemia on T lymphocytes and their subsets in children. Indian J Med Res 2006;124:647-54. [PUBMED] [Full text]
17.	Thibault H, Galan P, Selz F, Preziosi P, Olivier C, Badoual J, et al. The immune response in iron-deficient young children: Effect of iron supplementation on cell-mediated immunity. Eur J Pediatr 1993;152:120-4.
18.	Ekiz C, Agaoglu L, Karakas Z, Gurel N, Yalcin I. The effect of iron deficiency anemia on the function of the immune system. Hematol J 2005;5:579-83.
19.	Berger J, Schneider D, Dyck J-L, Joseph A, Aplogan A, Galan P, et al. Iron deficiency, cell-mediated immunity and infection among 6-36 month old children living in rural Togo. Nutr Res 1992;12:39-49.
20.	Reza Keramati M, Sadeghian MH, Ayatollahi H, Mahmoudi M, Khajedaluea M, Tavasolian H, et al. Peripheral blood lymphocyte subset counts in pre-menopausal women with iron-deficiency anaemia. Malays J Med Sci 2011;18:38-44.
21.	Kuku I, Kaya E, Yologlu S, Gokdeniz R, Baydin A. Platelet counts in adults with iron deficiency anemia. Platelets 2009;20:401-5.
22.	Khanna KL. Lymphocytic count in premenopausal women with iron deficiency anemia. Int J Contemp Med Res IJCMR 2019;6:10-2.