Hematological and biochemical alterations in school-aged children with sickling cell disorders indicate an elevated risk of liver and cardiovascular diseases

INTRODUCTION

Sickling cell disorder is an umbrella term comprising all pathologies that arise due to the presence of sickle hemoglobin (HbS) in at least one beta chain of hemoglobin (Hb) molecule.^[1] It includes all sickle cell trait and sickle cell anemia cases.^[1] It is estimated that approximately 5% of the world’s population carries a gene for sickle cell disease or thalassemia, and 300,000 children are born with Hb disorder every year globally.^[2,3] A previous study from Nepal has also suggested sickling cell disorder as the most common form of hemoglobinopathies, especially in the Western part, among the Tharu population, with an incidence rate of 17.4%.^[4]

Sickling cell disorder-related complications may begin at an early age when the concentration of HbS predominates over fetal hemoglobin. Therefore, early diagnosis and management of such cases seem important.

This study aimed to assess the associated risks of sickling cell disorder in school-going children.

MATERIAL AND METHODS

A cross-sectional study was conducted at the National Public Health Laboratory (NPHL) over 6 months (March–July 2019). Samples were collected from patients below 18 years visiting NPHL with their parents for hemoglobinopathy diagnosis. Informed consent was taken from the guardian of each patient enrolled in the study. Ethical approval was granted by Nepal Health Research Council (Ref. No. 2761) before carrying out this study.

Patients who tested positive for sickling cell disorders during screening were invited to provide a fresh blood sample for this study. Before blood collection, a questionnaire was filled out that consists of detail information of patient’s health status and symptoms related to sickle cell disorders. The study included only patients with sickle cell disorders in a steady state and under 18 years of age. A total of 60 case subjects (patients with sickling cell disorders) and 50 age- and sex-matched control subjects were enrolled in this study. Control subjects were selected from apparently healthy volunteers aged 10–20 years with no personal or family history of Hb-related disorders.

Blood samples were collected in a Tripotassium Ethylenediaminetetraacetic Acid (K3EDTA) vial and a clot activator gel tube. Serum was separated from clot activator gel tube by centrifuging at 1500 rcf for 5 min and used for biochemical analysis while K3EDTA blood was used for hematological investigation. Screening for sickling cell disorder was carried out with the help of capillary electrophoresis (Sebia Minicap Flex-Piercing) by interpreting electrophoretogram in conjunction with peripheral blood smear and complete blood count (CBC) findings. The electrophoretogram generated by the instrument is shown in Figure 1. An ancillary sickling test was also performed using sodium metabisulfite for the confirmation of sickling cell disorder cases.Hematological tests were performed using ABX Pentra XL80 Hematology Analyzer (Horiba Medical Ltd., Kyoto, Japan). K3EDTA blood sample was employed for testing CBC parameters including Hb, red blood cell count (RBC), hematocrit (Hct), mean cell volume (MCV), mean cell hemoglobin (MCH), mean cell hemoglobin concentration (MCHC), red cell distribution width-coefficient of variation (RDW-CV), white blood cell count, granulocytes percentage, and platelet count. The analyzer uses the principle of impedance to calculate blood count and the spectrophotometry method to calculate Hb level. Blood indices (MCV, MCH, and MCHC) were computed from the RBC histogram and Hct by numeric integration of pulses by the instrument.

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Figure 1:

Electrophoretogram generated during screening for sickling cell disorder.

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A biochemical investigation was performed using an AU480 Chemistry Analyzer (Beckman Coulter, Inc., USA) that uses the principle of spectrophotometry and potentiometry for several biochemical assays. The analyzer was validated using standard quality control reference material and commercial kits before performing biochemical tests. The samples were prepared and tested strictly adhering to the manufacturer’s guidelines. The tests performed included lipid profile parameters; triglycerides, total cholesterol (T. Cho.), high-density lipoproteins (HDL) and low-density lipoproteins (LDL), and liver function test parameters; total bilirubin (T. Bil), direct bilirubin, aspartate transaminase (AST), alanine transaminase (ALT), and alkaline phosphatase (ALP).

The obtained data were entered and analyzed using Statistical Package for Social Sciences software version 20.0 (IBM Armonk, NY, USA). Mean and standard deviation was calculated for each parameter separately for case and control subjects. Analysis of variance and Student’s t-test were used to understand the difference between the two groups. A P < 0.05 was considered statistically significant at 95% confidence interval.

RESULTS

A total of 60 sickling cell disorder patients aged 10–20 years were enrolled in the study. The median age of patients was 13 years. The total number of control subjects was 50, with a median age of 18 years. There were 32 male and 28 female among case subjects, and 22 males and 28 females among control subjects. Among the total 60 sickling cell disorder cases, the highest number had heterozygous sickle cell (83.3%), followed by compound heterozygous sickle cell/β-thalassemia (15.0%) and homozygous sickle cell (1.7%), as shown in Table 1.

Hematological finding shows significant differences in Hb, packed cell volume (PCV), MCV, MCHC, RDW-CV, and platelets among case and control subjects. Sickling cell disorder patients had significantly lower values for Hb (10.96 ± 1.96), PCV (33.48 ± 6.04), MCV (68.41 ± 9.49), MCHC (32.76 ± 1.65), and platelets (137.33 ± 73.35) compared to the control subjects – Hb (13.71 ± 2.17), PCV (40.32 ± 6.35), MCV (83.14 ± 10.40), MCHC (34.06 ± 1.42), and platelets (253.04 ± 90.07) (P = 0.000). However, RDW-CV for case subjects was significantly higher (16.61 ± 3.11) than the control subjects (13.87 ± 1.99) (P = 0.000), as shown in Table 2.

Some lipid profile test parameters showed significant differences among case and control subjects. Triglyceride level was significantly higher in the case subjects (95.70 ± 38.15) as compared to that of the control groups (67.30 ± 29.47) (P = 0.000). On the contrary, HDL cholesterol level was significantly lower in case subjects (37.93 ± 9.35) than that of control subjects (42.64 ± 10.01) (P = 0.012), as shown in Table 3.

Most of the liver function test parameters were relatively elevated in sickling cell disorder cases compared to healthy controls. T. Bil, ALT, AST, and ALP were significantly increased in case subjects. The mean value for T.Bil, ALT, AST, and ALP of case subjects was 1.01 ± 1.01, 28.33 ± 14.83, 34.53 ± 11.60, and 320.96 ± 158.76, respectively, whereas that for control subjects was 0.52 ± 0.33, 11.72 ± 7.12, 17.26 ± 12.39, and 201.00 ± 82.15, respectively. The P-value for T. Bil was 0.002, and that for ALT, AST, and ALP were 0.000, as shown in Table 3.

DISCUSSION

Sickling cell disorder has been shown to alter several biological phenomena inside our body, characterized by vaso-occlusive crisis, hemolytic anemia, and organ damage.^[5] Due to the genetic cause of the disease, its effect begins at an early age and continue throughout life. Sickling cell disorder cases are distributed heterogeneously worldwide, with a significant number found in sub-Saharan Africa, parts of the Mediterranean, the Middle East, and India.^[3,6] In Nepal, a higher prevalence is observed in the western region.^[4] In this study, we assessed the risks associated with sickling cell disorder in Nepal through hematological and biochemical analysis and found that this population faces an increased risk for liver and cardiovascular diseases.

This study found significant differences in most hematological parameters between sickling cell disorder patients and control subjects. Hb, PCV, MCV, MCHC, and platelet levels were notably lower in the case group compared to the control group. A study carried out in India by Nagose and Rathod also showed decreased values for hematological parameters among sickle cell anemia subjects in accordance with our study.^[7] Similarly, a survey by Akinbami et al. observed decreased levels of Hb, PCV, and MCV in homozygous sickle cell disease patients, aligning with our results.^[8] However, the same survey reported increased levels of MCHC and platelets in sickle cell disease patients, which contrasts with our findings.^[8] In addition, a survey by Antwi-Boasiako et al. in Ghana, noted decreased Hb and PCV levels but increased MCV, MCHC, and platelet levels in sickle cell disease patients compared to controls.^[9] These differences may stem from variations in the selection criteria for case subjects between our study and that of Akinbami et al.^[8] Our finding is also comparable to the result of Clarence et al. who reported a decreased value of PCV in patients with sickle cell anemia.^[10] Overall, hematological values appear to differ significantly between individuals with sickling cell disorders and healthy controls. These variations may be attributed to factors such as the sickling phenomenon, the presence of other Hb variants, hypoxia, dehydration, and acidosis in affected patients.

This study also noted significant changes in liver function test parameters among case and control subjects. T. Bil, ALT, AST, and ALP were significantly increased in sickling cell disorder patients compared to those of controls. Increased value for T. Bil was also reported among sickle cell disease children by a study carried out in India by Biswal et al., similar to our study.^[11] The increased value for T. Bil, ALT, and AST in our study is further supported by Clarence et al., who found similar results in sickle cell anemia patients.^[10] Likewise, a similar finding was also observed in a study carried out in Nigeria by Yahaya among sickle cell anemia patients.^[12] In ddition, a study by Kotila et al. also suggests an increased value of ALT, AST, and ALP in some portions of the study population.^[13] The evidence gained from our study population indicates that sickling cell disorder patients have significantly elevated levels of liver function test parameters. Abnormally elevated levels of such parameters might be an indication of liver dysfunction that puts sickling cell disorder patients at an elevated risk for the development of liver disease.

Sickling cell disorder patients in this study were also found to have significantly different levels of lipid profile parameters. The study noted a considerably lower value for HDL cholesterol but a substantially higher value for triglycerides in sickling cell disorder patients compared to control subjects. They also had decreased levels of T. Cho. and LDL cholesterol compared to the control group. These findings are comparable to the study of Yalcinkaya et al. and El-Hazmi et al. who also demonstrated lower values for total, HDL and LDL cholesterol in sickle cell disease patients.^[14,15] A case–control study conducted in the Tema metropolitan area similarly reported significantly lower HDL cholesterol levels, consistent with our findings. Other findings from that study are also comparable to ours.^[16] Minor variations in certain parameters may be attributed to differences in the age groups of study participants or the selection of case subjects based on disease severity. Overall, this study accounted for significantly increased triglyceride and decreased HDL cholesterol value, which is a significant predictor of cardiovascular disease. This suggests that sickling cell disorder patients aged 10–20 years in steady-state are at higher risk for cardiovascular disease.

CONCLUSION

This study concludes that patients with sickling cell disorders exhibit significantly altered hematological, lipid profile, and liver function test parameters, putting the patients at a higher risk of developing liver and cardiovascular complications. Therefore, it is recommended that patients with sickling cell disorders undergo regular monitoring, with a focus on preventive measures to address liver and cardiovascular complications.