Analysis of Haematological Parameters in Sickle Cell Trait and Sickle cell Disease patients of Chhattisgarh

 

Sanjana Bhagat

Department of Biotechnology, Govt. Nagarjuna PG College of Science, Raipur 492010, Chhattisgarh, India.

*Corresponding Author E-mail: sanjanabhagat@gmail.com

 

ABSTRACT:

In this study, we examined the hematological parameters between sickle cell disease, sickle cell trait and control individuals. Haematological parameters were compare among SS, AS and AA groups. However, significant difference were observed for HbF ((p<0.000), Hb ((p<0.000) HCT ((p<0.000), MCV ((p<0.000), except for MCH (P=0.757). Haematological parameters were comparable among male and female in study groups. However, no significant difference were observed excluding in SS patient for MCH ((p<0.035), in AS patients for Hb (p< =0.009) and in control (AA) for Hb ((p<0.001) and HCT ((p<0.007). HbF levels were higher in SS (19.39±7.14) patients than AS (1.06±0.74) and control (0.12±0.1). Highly significant difference for HbF level ((p<0.000) was observed among these study groups. Furthermore, diverse ranges of HbF levels were also noted in present study.

 

KEYWORDS: HbF level, Haematological indics, Sickle cell disease, MCH, Haemoglobin.

 

 

INTRODUCTION: 

Sickle-cell disease (SCD) is the most common genetic disorders characterized by autosomal inheritance. Sickle cell disease is caused by point mutation in the sixth position of the β-globin chain, due to single nucleotide substitution, GAG→GTG in codon 6 of the β-globin gene on chromosome 11. In the deoxygenated state, sickle haemoglobin has the propensity to polymerize the erythrocytes and interfere with normal blood flow in micro circulation is the primary indispensable event in the molecular pathogenesis of sickle-cell disease1,2.

 

Several studies indicate that the pathophysiology of sickle cell anemia (SCA) is affected by number of factors like β-globin gene cluster, fetal haemoglobin (HbF) level, chronic inflammation, oxidative stress as well as gender and diverse range of clinical features. Haemoglobin S is resulting from (GTG for GAG) substitution causes polymerization of HbS and red cell sickling on deoxygenated state3-5.

 

A study by Akinbami et al., (2012)6 elucidate that degree of haemolysis is inversely related to haemoglobin concentration and packed cell volume in sickle cell anemia patients. Numerous factors affect haemolysis in SCA patients, percentage of irreversible sickle cell is of greatest significance. The degree of haemoglobin polymer formation, calculated from the mean corpuscular haemoglobin concentration (MCH) and the relative proportion of haemoglobin fraction also correlates closely with the severity of haemolysis7,8. In addition, the packed cell volume (PCV) and mean corpuscular volume (MCH) increased with increasing HbF, but differences were inverse with painfull crises and other complication like acute chest syndrome1,9. The complete clinical studies highlight the phenotypic diversity of SCA10,11.

 

The severity of sickle cell disease has correlated with various clinical phenotypes between different individuals. Among various genetic modulators for clinical severity of SCA the β-globin gene cluster haplotypes, HbF level, haematological parameter and oxidative stress are associated with disease severity level. The hematological parameters are very important for the management of sickle cell disease. In this context, the objective of this study was to determining hematological parameters among sickle cell disease and sickle cell trait patients of Chhattisgarh.

Table 1: Gender and age information of participants.

Parameters

Sickle cell disease (n=100)

 Males                       Females

Sickle cell trait (n=50)

   Males                     Females

Controls  (n= 50)

Males                    Females

Frequency

51(51%)

49(49%)

25(50%)

25(50%)

25(50%)

25(50%)

Mean age ±SD

24.74 ±8.90

22.79 ±7.58

27.92 ±7.39

24.68 ±6.98

26.51±9.89

22.61±6.01

 

Table 2: Haematological indices in both sexes among sickle cell disease, sickle cell trait and control.

 

Haematological Parameters

 

HbF (%)

Hb (g/dl)

HCT (%)

MCV(fl)

MCH (pg)

Sickle Cell Disease (SS)

19.39±7.14

9.29±2.54

29.53 ±10.40

91.29 ±10.88

29.71 ±8.67

Sickle Cell Trait (AS)

1.06±0.74

12.01±2.56

32.08 ±5.55

77.88 ±10.56

28.32 ±5.87

Controls (AA)

0.12±0.13

12.59±2.34

34.86 ±6.54

81.41 ±10.03

28.11 ±4.32

P values

< 0.001*

< 0.001*

< 0.001*

< 0.001*

0.757

 

 

MATERIALS AND METHODS:

The study was performed on hundred sickle cell disease (SS) and fifty sickle cell trait (AS) patients. Fifty control (AA) individuals were also included in the study.

 

Informed consent was obtained from all subjects prior to drawing the blood samples. The 8ml blood was collected in EDTA coated vacutainers. Serum was separated from 3ml blood and stored at -20ºC for analysis. Haematological analysis was carried out using an automated cell counter (BC-3000Plus Auto Hematology Analyzer). The fetal haemoglobin levels were quantified by ion-exchange high-performance liquid chromatography12.

 

 

Statistical analysis was done using SPSS and p value of ≤0.05 was considered statistically significant. Firstly data were analysing for normal distribution and homogeneity of variances assumption according to Shapiro-Wilk test and Levene’s test respectively. Those groups that were not meet the parametric assumptions were further compare by non-parametric test by using Mann-Whitney test and Kruskal-Wallis test.  For the correlation analysis Karl Pearson method was used.

 

RESULT:

Age and gender distribution of study subjects:

A total of 100 sickle cell disease, 50 sickle cell trait and 50 controls were enrolled. Groups consisted of 51(51%) males and 49(49%) females for sickle cell disease patients (SS), while sickle cell trait (AS) were made up of 25(50%) males and 25(50%) females. Controls consisted of 25(50%) males and 25(50%) females (Table 1).

 

The overall mean age of the sickle cell disease patients were 23.84±8.38 years (males 24.74±8.90 and females 22.79±7.58 years) and for sickle cell trait  were 26.3 ±7.37 (males 27.92±7.39 and females 24.68±6.98 years) while in controls 24.88±8.70 (males 26.51±9.89 and females 22.61±6.01years). The minimum and maximum age for participants were (SS= (9-50 years), AS= (14-48 years), Control= (9-53 years) in the present study.

Haematological indices in study groups:

For sickle cell (SS) disease group, the overall mean haemoglobin (Hb) concentration was 9.29±2.54g/dl, mean cell volume (MCV) 91.29±10.88 fl, mean cell haemoglobin (MCH) 29.71±8.67pg,  mean hematocrit (HCT) level 29.53±10.40% and mean fetal haemoglobin (HbF) level 19.39±7.14% and for sickle cell trait (AS) group, the overall mean haemoglobin (Hb) concentration was 12.01±2.56 g/dl, mean cell volume (MCV) 77.88± 10.56 fl, mean cell haemoglobin (MCH) 28.32±5.87pg, and mean hematocrit (HCT) level 32.08±5.55% and mean fetal haemoglobin (HbF) level 1.06±0.74%. While for controls, mean haemoglobin (Hb) concentration was 12.59±2.34g/dl, mean cell volume  81.41±10.03 fl, mean cell haemoglobin 28.11±4.32pg, mean hematocrit 34.86 ±6.54%   and mean fetal haemoglobin concentration 0.12±0.13 % (Table 2).

 

Comparisons were made by Kruskal-Wallis H (One-Way ANOVA), post hoc Mann-Whitney U test

 

In present study we find the statistically highly significant difference (p< 0.001) for all haematological parameters among three groups (SS, AS & AA) except for mean cell haemoglobin concentration (MCH) by using Kruskal-Wallis H (One-Way ANOVA) test (Table 2). In sequence to assess the multiple comparisons by post-hoc we find the same significant differences for all parameter (Table 3) except for haemoglobin concentration (Hb) between AS and control (AA).

 

Table 3: Multiple Comparisons by Mann-Whitney U test

Dependent Variable

Between SS & AS (P values)

Between SS & AA (P values)

Between AS & AA

 (P values)

HbF (%)

< 0.001*

< 0.001*

< 0.001*

Hb (g/dl)

< 0.001*

< 0.001*

0.077

HCT (%)

< 0.022*

< 0.001*

< 0.010*

MCV(fl)

< 0.001*

< 0.001*

< 0.041*

MCH(pg)

0.665

0.742

0.410

Post hoc by Mann-Whitney U test

 

The mean haemoglobin concentration of the males were (9.50±2.57g/dl for SS, 12.78±2.92g/dl for AS, 13.16± 1.99g/dl for AA), mean cell volume (89.69±10.92 fl for SS, 79.29±10.52 fl for AS, 81.53±10.14 fl for AA), mean cell haemoglobin (27.57±4.01 pg for SS, 29.15± 7.57pg for AS, 28.07±4.61 pg for AA), mean hematocrit level (29.65±9.33% for SS, 31.81±5.91% for AS, 36.37 ±5.16% for AA), mean fetal haemoglobin concentration (18.61±6.14% for SS, 1.18±0.73% for AS, 0.11±0.11 % for AA (Table 4).

 

For the females the mean haemoglobin concentration were (9.14±2.51g/dl for SS, 11.25±1.85g/dl for AS, 11.80±2.54g/dl for AA), mean cell volume (91.61± 11.26 fl for SS, 76.48±10.41 fl for AS, 81.23±9.87 fl for AA), mean cell haemoglobin (31.51±10.85 pg for SS, 27.50±3.20 pg for AS, 28.16±3.90pg for AA), mean hematocrit level (29.03±11.13% for SS, 32.34±5.14% for AS, 32.77±7.59% for AA), mean fetal haemoglobin concentration (20.22±7.73 % for SS, 0.94±0.72 % for AS, 0.12±0.15% for AA (Table 4). There was no statistically significant sex wise (male & female) difference observed for all haematological parameters in each groups by using Mann-Whitney U test except  for MCH (p<0.035) in SS patients, Hb (p<0.009) in sickle cell trait and Hb (p<0.001), HCT( p<0.007) in control (Table 4).

 

Table 4: Mean values of haematological indices in both sexes among SS, AS & AA.

Haematological Parameter

Male

Female

p value

Sickle cell disease

HbF(%)

18.61 ±6.14

20.22 ±7.73

0.482

Hb (g/dl)

9.50  ±2.57

9.14 ±2.51

0.41

HCT (%)

29.65 ±9.33

29.03 ±11.13

0.961

MCV(fl)

89.69 ±10.92

91.61 ±11.26

0.214

MCH(pg)

27.57  ±4.01

31.51 ±10.85

< 0.035*

Sickle cell trait

HbF (%)

1.18 ± 0.73

0.94 ±0.72

0.243

Hb (g/dl)

12.78± 2.92

11.25 ±1.85

< 0.009*

HCT (%)

31.81 ±5.91

32.34 ±5.14

0.808

MCV(fl)

79.29 ±10.52

76.48 ±10.41

0.473

MCH(pg)

29.15 ±7.57

27.50 ±3.20

0.432

Control

HbF (%)

0.11 ±0.11

0.12 ±0 .15

0.595

Hb (g/dl)

13.16 ±1.99

11.80±2.54

< 0.001*

HCT (%)

36.37 ±5.16

32.77±7.59

< 0.007*

MCV(fl)

81.53±10.14

81.23 ± 9.87

0.839

MCH(pg)

28.07 ±4.61

28.16± 3.90

0.838

n = number of subjects. Values are mean±SD . Comparisons were made by Mann-Whitney U test

 

Determination of fetal haemoglobin (HbF) level:

In this study the overall mean HbF level in sickle cell patients was (19.39±7.14%) and in sickle cell trait (1.06 ±0.74%) while in controls (0.12±0.13%) were observed with highly statistically (p<0.001) significant difference between them (Table 5). The higher mean HbF level was observed in female (20.22±7.73%) as compare to male (18.61±6.14%) in SS patients and in reverse for sickle cell trait had higher HbF in male (1.18±0.73%) as compare to female (0.94±0.72%). Data presented in Table 6 shows that no correlation was found between fetal haemoglobin levels with haematological parameters among SS patients.

 

Table 5: The HbF percentage in SS, AS and AA groups in present study.

Study groups

HbF (%)

P values

Sickle Cell Disease (SS)

19.39 ±7.14

< 0.001*

Sickle Cell Trait (AS)

1.06 ±0.74

 

Controls (AA)

0.12 ±0.13

 

Comparisons were made by Kruskal-Wallis H (One-Way ANOVA).

 

Table 6: Correlation of Hb, HCT, MCV, MCH with HbF level among sickle cell disease.

Parameter in SS  patients

Correlation (r2)

P values

Hb and HbF

0.0001

NS

HCT and HbF

0.0061

NS

MCV and HbF

0.0023

NS

MCH and HbF

0.0001

NS

r2 = Pearson correlation coefficient; NS- Not significant

 

This finding suggests that in adult sickle cell patients, their HbF levels remained persistently high excluding the possibility of temporary hematopoietic stress that might affect the expression of the gamma globin gene.

 

DISCUSSION:

The clinical severity and haematological parameters in sickle cell disease patients are influenced by gender, haplotypes, inheritance of α-thalassemia and HbF levels. Several studies shows that difference in hematological parameters may account for clinical complication observed in patients with sickle cell disease.

 

The present study showed that significant difference (p<0.001) observed among three groups (SS, AS, AA) for haematological parameters including HbF, Hb, HCT, MCV except for MCH. Recently Donaldson et al., (2001)9, reported that the HCT or packed cell volume (PCV) and mean corpuscular volume (MCV) were increased with increasing HbF level but differences in the incidence of painful episodes and acute chest syndrome. In this study, we showed that the haematological parameters of sickle cell disease (SS) were compared with sickle cell trait (AA) and controls (AA) individuals. This is in accordance with the previous studies10,13. Our data shows that the haemoglobin (Hb) and HCT value were significantly lower (p<0.001) and reverse MCV value was significantly (p<0.001) higher in SS patients as compare to AS and controls.  In favour of our results same finding were observed by Akinbami et al., (2012)6. These results were expected consideration because SCD patients have higher levels of hemolysis than healthy subjects. Earlier study also reported that the degree of haemolysis is inversely related to haemoglobin concentration and packed cell volume in sickle cell anemia patients14,15,16. Many previous studied reported that WBC and platelets counts were increase in sickle cell disease. Moreover, another study has reported that use of Hydroxyurea for management of sickle cell disease that significantly decreases the WBC counts thus improve the clinical course in SCD patients17.

 

Additionally,  in this study there was no statistically significant sex wise (male & female) difference observed for all haematological parameter in each groups independently except for MCH (p<0.035) in SS patients, Hb (p<0.009) in AS patients, Hb (p<0.001) and HCT (p<0.007) in controls. Hence, the present study showing that Hb and HCT values below the normal reference ranges in female as compare to male in accordance of other studies18.

 

The significant difference in haematological parameters may associate with disease severity level such as pain, inflammation and stroke. Furthermore, study has shown that high leukocyte count, low Hb level and lower HbF level in sickle cell patients have increased risks for sickle cell crises, including stroke19. Interactions between leukocytes, platelets, erythrocytes, plasma proteins, endothelial cells of the blood vessel wall and high leukocyte count are at greater risk of developing vaso-occlusion and severe disease in SCD patients20.

 

This study highlights that significantly (p<0.001) higher level of HbF were observed in SS patients as compared to AS and AA individuals. In agreement to our result, many researchers have reported higher HbF level in sickle cell patient21-23. Recent studies also found association between HbF and disease severity24-26. According to Steinberg and Sebastiani, (2012)5  high HbF is strongly associated with a reduced rate of acute painful episodes, acute chest syndrome and leg ulcers and less conclusive evidence supports an association of HbF with other complication. Furthermore, Steinberg (2005)1suggested that the relationship between reduction of most vaso-occlusive complication of sickle cell anemia with higher HbF levels and the lack of a clear reduction of global disease severity may reflect the absence of a reliable severity index.

 

In conclusion, the results of this study shows that the  mean fetal haemoglobin (HbF) level, mean haemoglobin concentration (Hb), mean hematocrit (HCT) level, mean cell volume (MCV) were significantly higher in SS patients than AS and AA individuals. It was interesting to note that the higher HbF levels were found in SS patients which ameliorate disease severity of SCD patients of Chhattisgarh. Therefore, for management of the SCD complication, hematological parameters are regularly monitored.

 

CONFLICT OF INTEREST:

The author declares no conflicts of interest.

 

ACKNOWLEDGMENTS:

The authors are thankful to all the donors of blood samples for the present study.

 

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Received on 14.03.2023            Modified on 08.07.2023

Accepted on 22.09.2023           © RJPT All right reserved

Research J. Pharm. and Tech 2024; 17(7):3084-3088.

DOI: 10.52711/0974-360X.2024.00483