The effect of T helper (Th)/T cytotoxic (Tc) ratio on disease severity in type A hemophilia patients

Abstract

BACKGROUND AND OBJECTIVES:

The aim of this study was to investigate the T helper (Th) to T cytotoxic (Tc) ratio in children suffering from type A hemophilia disease and to evaluate the correlation of this ratio with disease severity.

MATERIAL AND METHOD:

Two mls of EDTA anti coagulated whole blood was collected. Immunophenotyping of lymphocytes count was carried out by FACS analysis using a double CD4 and CD8 kit. The mean $\pm$ SD of absolute numbers of CD4 and CD8 lymphocytes/ml was calculated and the ratio of CD4/CD8 was evaluated by statistical method.

RESULTS:

Among 80 type A hemophilia patients, 66 (82.5%) were male. The mean age was 15 $\pm$ 3.51 years. 12 (15%) of them were suffering from mild disease and 68 (85%) had sever disease. The CD4 /CD8 ratio was obtained between 0.45 and 1.44 with mean1.79 $\pm$ 0.78. The correlation between this ration and disease severity was 0.019.

CONCLUSION:

The results showed that CD4/CD8 ratio has correlation with disease severity in type A hemophilia patients, however there was no association between this ratio and gender.

Keywords

CD4/CD8 ratio hemophilia disease

Abbreviations

Th	T helper
Tc	T cytotoxic
HBD	Hereditary coagulation bleeding
FXIIID	FXIII deficiency
FX	Factor X
FIX	Factor IX
hFIX	Human coagulation factor 9
FVIII	Factor FVIII
CMI	Cell mediated immunity
CTL	Cytotoxic T cells
Tregs	T regulatory cells
Th	Helper T
cIMT	Carotid intima-media thickness

1. Introduction

Hemophilia A is a congenital bleeding disorder which happens as a result of factor VIII (FVIII) coagulant deficiency or abnormality. FVIII works as a cofactor in factor X (FX) activation in normal clot formation. Lack of FVIII can lead to a defect in clot formation at the injury site. The prevalence of hemophilia in American population has been reported to be 13.2 per 100000 males among white Americans. Hemophilia patients suffer from abnormal bleeding time due to the deficiencies in the amount of factors [1, 2]. Patients with this condition have prolonged bleeding following trauma such as injury, surgery, or having a tooth pulled. On the other hand in severe cases of hemophilia, continuous bleeding happens after minor trauma. This condition may also happen in the absence of injury (spontaneous bleeding). Serious complications can be seen as a cause of bleeding into the all part of body especially in to the joints, muscles, brain, or other internal tissues [3]. The disease is classified as mild, moderate, and severe based on the degree of deficiency of the coagulation factors. A few studies on race have reported ethnicity differences in hemophilic epidemiology. The prevalence of hemophilia in American population has been reported to be 13.2 per 100000 males among white Americans, 11.0 among African-Americans, 11.5 among Hispanic males, and 4.3 among Asian/Pacific Islanders in the United States [4]. In Iran based on various factors such as molecular studies, clinical presentations, type of treatment, development and management of patients with inhibitor, the prevalence of disease is varied. According to the report of Dorgalaleh A, Iran has the ninth largest hemophilia population in the world [5]. In a report of the center of epidemiology of hereditary bleeding (HBD) disorders from Southern Iran, factor VIII deficiency was the most prevalent type (50.4%) among patients [6, 7, 8, 9, 10, 11, 12]. The role of B Cells in breaking and maintaining tolerance to clotting factor VIII in congenital and acquired hemophilia A has been suggested by Amanda M. Actor [11] but the role of cell mediated immunity (CMI) in hemophilia patients remains to be explained. CD4 cells play critical roles in the maintenance of immune system and achieving a regulated effective immune response to pathogens. These cells are activated after interaction with antigen and presented by APCs in association with MHC – class II complex and differentiate into specific subtypes based on the cytokine production which are called Th1, Th2, Th3, Th9 and Th17 each with a characteristic cytokine profile [13, 14, 15]. A study was carried out by Hu et al in 2007 which suggests the role of Th1 cells in initiating the immune response to fVIII and of Th2 cells in the development of strong inhibitor production [16]. Another study that was conducted by XuebinJi in 2014 showed that this disease is an immune mediated disease of adults and children that is characterized by excessive platelet destruction and decreased platelet production by T1 cell (Th1 and Tc1) function [13]. The aim of current study was firstly to investigate the role of cell mediated immunity response by measuring the ratio of CD4/CD8 counts in children suffering from hemophilia A and to investigating the Its association with some variable factors such as sex, age, disease intensity and type of treatment.

2. The method of study

This study was carried out on 80 children suffering from hemophilia A disease. After collecting the basic personal information, informed verbal consent was obtained. Having a history of viral infections, gastrointestinal infections, and having any chronic infection (autoimmune diseases) were considered as exclusion criteria. These patients were investigated based on some variables such as gender, age and severity of disease. Two milliliters of EDTA and coagulated whole blood was collected. Immunophenotyping of lymphocyte count was carried out by FACS (Fluorescent Antibody Cell Sorter, Becton Dickinson, Singapore, BD) using a double CD4 and CD8 kit (Model: FR868).Based on the instruction of kit, 100 $\mu$ l from each samples were taken and added to clot tube and 10 $\mu$ l of antibody was added. Again 100 $\mu$ l from the same sample was added to the clot tube separately and added 10 $\mu$ l of control sample. Then these two samples were incubated for 30 minutes. Then, 100 $\mu$ l of Reagent A (UNIQUE-LYSE Code A100) was added, vortexed well and incubated in room temperature for 10 minutes in a dark room. After that, one ml reagent B was added to the tube and was incubated for 20 minutes in a dark room. After these stages, the cytometry and cell count were carried out within two minutes and the results were read later on. The sample cells supernatant were analyzed by flow cytometry instrument (PARTEC PAS II). All samples were stained on the same day and tested within two hours. 488 nm blue laser that read the color range between 520–570 and 670. The values of mean and standard deviation of each lymphocyte subpopulation were estimated. The mean $\pm$ SD of absolute numbers of CD4 and CD8 lymphocytes/ml were obtained. Based on T-independent test, the mean CD4/CD8 ratio in patients with different variables were calculated and evaluation of significance was done by calculating the standard error of difference between two means. The significant correlation between CD4/CD8 ratio and some variables were evaluated by statistical test and SPSS software. $P$ value of less than 0.05 was considered significant.

3. Results

From 80 hemophilia patients, 66 (82.5%) were male. The patients’ age was between 1 and 45 years old with mean and standard deviation of 15 $\pm$ 3.51. 12 (15%) of patients were suffering from mild disease and 68 (85%) had sever disease. CD4 count was between 100 and 1610 with the mean and standard deviation of 568.22 $\pm$ 289.18 and CD8 count was between 115 and 1430 with the mean and standard deviation of 349.61 $\pm$ 217.16. The CD4/CD8 ratio varied between 0.54 to 4.44 with the mean and standard deviation of 1.79 $\pm$ 0.78 (Table 1).

Table 1
Description of some variables among patients

Variables	Related variables	Number	Percent
Gender	Male	66	82.5
	Female	14	17.5
Disease severity	Mild	12	15
	Severe	68	85

Based on T-independent test, the mean CD4/CD8 ratio in patients with severe disease was higher than the patients with mild disease ( $P$ Value $=$ 0.019). There was no significant difference between male and female patients in regarding CD4/CD8 ratios ( $P$ value $=$ 0.095).

Table 2

The comparison of CD4/CD8 ratio based on different variables

Variables	Related variables	Mean $\pm$ SD	$P$ value
Gender	Male	1.703 $\pm$ 0.699	0.095
	Female	2.118 $\pm$ 1.098
Disease severity	Mild	2.289 $\pm$ 1.093	0.019
	Severe	1.712 $\pm$ 0.698

4. Discussion

Hemophilia disease is a genetic bleeding disorder that causes recurrent bleeding mainly internal bleeding of joints, muscles, and brain. The disease is related to the function of immune system and the role of cell mediated immunity (CMI) especially the role of CD4 and CD8 lymphocytes in this disease are not well understood therefore we designed this study to evaluate the T helper (Th) to T cytotoxic (Tc) ratio in children suffering from hemophilia disease. The results of this study were not found in any literature therefore this study provides the first estimates of CD4 and CD8 T lymphocyte counts and CD4/CD8 ratio among the type A hemophilia patients. A wide variation in mean CD4 and CD8 count have been reported in studies conducted by Krishna Ray and S.M. Gupta et al. in normal healthy individuals, HIVpositive individuals and AIDS patients [17]. Wei Lu et al. in their study on virologically suppressed HIV-positive patients reported that the CD4:CD8 ratio can contribute to the immunological evaluation and this may has a role for monitoring both immune dysfunction and viral reservoir size in immune-based clinical trials [18]. Another study has shown that there is an association between markers of age and CD4/CD8 ratio in virally suppressed HIV-infected patients with immunological recovery [19]. Some other studies indicated that there is an association between the CD4/CD8 ratio and carotid intima-media thickness (cIMT) progression in treated HIV-infected patients which can be used as a marker of coronary heart disease and a predictor of cardiovascular events [20]. So far, there are no large-scale studies with sufficient statistical power to clearly assess the association between the CD4/CD8 ratio with other pathological conditions. None of these studies examined the role of CD4 and CD8 individually and CD4/CD8 ratio. On the other hand, as the CD4/CD8 ratio is considered as a marker for both immune senescence and immune activation in pathogenicity of disease, so we conducted a study focused on factors contributing to CD4/CD8 T cell ratio and clinical outcome in type A hemophilia.CD4/CD8 ratio of less than 1.0 is considered as a surrogate marker of immunosenescence and represents an independent predictor for disease or healthy statuses. Hemophilia disease like other clot abnormalities such as ITP could be a T1 cell (Th and Tc) predominant disease although the precise mechanisms await further functional assay. Our results indicated that the CD4/CD8 ratio was between 0.45 and 1.44 with a mean of 1.79 $\pm$ 0.78 and there was a significant correlation between disease severity and this ratio ( $P$ value $=$ 0.019). However, its correlation with the gender was not significant. Similar to these finding can be seen in a previous study that suggested CD4/CD8 ratio to be an independent factor for both non-AIDS morbidity and mortality in long-term treated HIV-positive patients which was independent of CD4 T cell count [19]. Finally, our study indicated that CD4 and CD8 T cell count and their ratio can be a predictive value for disease severity and may provide a surrogate marker of these cells cytokine balance. And it might provide a potential immunotherapy program for hemophilia disease by shifting the cytokine patterns. However, a larger group should be studied to thoroughly assess the variations in lymphocyte subpopulations in this patients.

5. Conclusion

The results showed that CD4/CD8 ratio has correlation with disease severity in type A hemophilia patients, However there was no association between this ratio and gender.

Author contribution

HK, MN, GM and AA participated in Conception and design of the study, library searches and assembling relevant literature, critical review of the paper, supervising writing of the paper, database management. The remaining authors participated in data collection, library searches and assembling relevant literature, writing the paper, and critical review of the paper. All the authors have read the final version of the manuscript and approved it.

We confirm that we have read the Journal’s position on issues involved in ethical publication and affirm that this report is consistent with those guidelines.

Footnotes

Acknowledgments

The authors especially respect and thank all staff from Islamic Azad University, Zahedan branch, Zahedan-Iran for providing us an opportunity to do the project work and giving us all support and guidance which made us complete the project duly. We are extremely thankful to Dean and the Vice dean in research affairs of Islamic Azad University, Zahedan branch, Zahedan-Iran for providing such a nice support and guidance. Second Affiliated hospital for making contributions to this difficult work over the long term and the staff who provided the sincere and warm help. The authors also thank the all individuals who participated in this research project. The authors also declare that there are no conflicts of interest that could be perceived as prejudicing the impartiality of the research reported.

Conflict of interest

Neither of the authors has any conflict of interest to disclose.

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