Some haematological parameters,copper and selenium level among children of African descent with sickle cell disease in Specialist Hospital Sokoto,Nigeria

Abstract

BACKGROUND:

Sickle cell disease is a genetic disorder of haemoglobin causing myriad of pathology including anaemia.

OBJECTIVES:

The aim of this study was to evaluate some haematological parameters and trace elements of total of forty-five (45) children with Sickle cell disease attending Specialist Hospital Sokoto.

METHOD:

Twenty-five (25) apparently healthy children which were assessed as controls. The haematological parameters were determined using automated method and trace elements (copper and selenium) were determined using colorimetric and atomic absorption spectrophotometry method respectively.

RESULTS:

The Mean WBC and PLT was significantly higher among sickle cell disease subjects when compared to controls individuals ( $p<$ 0.05). The Mean RBC, HCT, HGB, MCV, MCH and MCHC was significantly lower among Sickle cell disease patients when compared to controls ( $p<$ 0.05). The Mean Copper and Selenium value was significantly lower (40.4 $\pm$ 1.44 $\mu$ g/dl and 54.6 $\pm$ 1.60 ng/ml) among Sickle cell disease subjects compared to controls (75.6 $\pm$ 1.30 $\mu$ g/dl and 86.3 $\pm$ 2.30 ng/ml) ( $p<$ 0.05). The WBC, HGB, HGT and Copper values of Sickle cell disease subjects shows a weak positive put non-statistically significant correlation with age ( $p>$ 0.05). The RBC, MCV, MCH, MCHC, PLT, and Selenium values of sickle cell disease patients shows a negative non-statistically significant correlation indicating that the selenium level decreases as the age increases ( $p<$ 0.05).

CONCLUSION:

This study shows that the WBC and platelet count was significantly higher among sickle cell disease subjects compared to controls. The RBC, HCT, HGB, MCV, MCH and MCHC were significantly lower among sickle cell disease patients compared to controls. The serum copper and selenium levels were significantly lower among sickle cell subjects compared to controls. We recommend that trace elements (copper and selenium) and haematological parameters be monitored routinely among sickle cell disease children to optimize the care offered to these individuals.

Keywords

Haematological parameters copper selenium children sickle cell disease in specialist hospital sokoto nigeria

1. Introduction

Sickle Cell Anaemia also known as sickle-cell disease (SCD) is a common genetic condition in Nigeria that occurs due to a haemoglobin disorder (haemoglobinopathies) associated with the inheritance of a mutant haemoglobin genes (HB S) from both parents [1]. SCD was first observed about 1904 by Herrick in the blood of an anaemic West Indian Medical Student [2]. Sickle cell disease (SCD) is the most common life-threatening genetic disorder worldwide [3]. Haemoglobinopathies, mainly thalassemia and sickle-cell anaemia, are widespread globally [4]. Unlike in the developed world where the prevalence of SCD has decreased to $<$ 1% and with a life expectancy of 53–60 years, about 80% of SCD cases occur in sub Saharan Africa, a significant (50–90%) number of children born with SCD do not reach their fifth birthday and SCD continue to account for up to 20% of neonatal mortality [3, 5, 6].

Haemoglobins S differs from haemoglobin A by the substitution of valine for glutamic acid at position 6 ${}^{\rm th}$ in the $\beta$ chain [7]. The substitution of glutamic acid for lysine at position 6 ${}^{\rm th}$ of the $\beta$ globulin chain results in the production of haemoglobin C [8]. Subjects with heterozygous state for Hb S (HbAS) and C (HbAC) are asymptomatic but may present with a mild microcytosis with relative red cell resistance to haemolysis and malaria [9]. Compound heterozygotes HbSC presents with a sickling disorder similar to sickle cell anaemia (SCA), although it is generally milder than in the HbSS form. However, 2% of HbSC patients have more severe disease with frequent vaso-occlusive crisis (VOC) and acute chest syndrome (ACS) [7].

The most common and most severe haemoglobino-pathy is homozygous SS, also called sickle cell anaem-ia [2]. Despite a declining prevalence in the West, the burden on Sub Saharan Africa is expected to increase to 88% of cases by 2050 [10, 11]. No global data regarding the precise numbers of children born with SCD and their haemoglobinopathy profile exist because, in contrast to Western countries, newborn screening for SCD is not available in most low-income countries with the highest predicted burdens. Nigeria is known to bear the highest burden of SCD in the world and therefore the country is in urgent need of policies for prevention and management of SCD [10, 11]. About 5% of the world’s population carries genes responsible for haemoglobinopathies. Nigeria has the largest population of people with sickle cell disorder, with about 150,000 births annually. Sickle haemoglobin (HbS) is present at birth, but most infants don’t show signs until they are six months or shortly before, because the predominant haemoglobin at this time is foetal haemoglobin (HbF) [12]. It has been earlier documented that high level of HbF inhibits sickling in babies with SC [13]. This is because HbF has the ability to decrease the polymerization of deoxygenated HbS, hence preventing red blood cell from forming tactoids which lead to vaso-occlusion [7]. The sickle-cell gene has become common in Africa because the sickle-cell trait confers some resistance to falciparum malaria during a critical period of early childhood, favoring survival of the host and subsequent transmission of the abnormal haemoglobin gene [4]. Although a single abnormal gene may protect against malaria, inheritance of two abnormal genes leads to sickle-cell anaemia and confers no such protection, and malaria remains a major cause of ill-health and death in children with sickle-cell anaemia [4]. It results in an abnormality in the oxygen-carrying protein haemoglobin (haemoglobin S) found in red blood cells [14]. This leads to a rigid, sickle-like shape under certain circumstances. The deformed (sickle) red cells are destroyed prematurely (in as short as 20 days from production) by the cells of the reticulo-endothelial system, leading to a chronic haemolytic anaemia state [9]. A number of health problems may develop, such as attacks of pain, anaemia, swelling in the hands and feet, bacterial infections and stroke [14].

Most SCD patients do not manifest clinically until about age of 6 months when the level of haemoglobin F (HbF) begins to fall [2]. They however may present with pallor, jaundice, hepatosplenomegaly, pain, anae-mia, swelling in the hands and feet, failure to thrive, infections, sickle cell habitus.

Micronutrients are traditionally referred to as trace elements and vitamins of low molecular weight essential to human health. They are important in cellular metabolism, act either as cofactor for metal ion activated enzymes or as specific constituent of metallo-enzymes. They protect the body against oxidative damage, regulate growth activity cognitive development, functioning of the immune and reproductive system [15, 16].

Copper is a trace element vital for the function of many cellular enzymes. It plays a pivotal role in cell physiology as a catalytic cofactor in the redox chemistry of enzymes, mitochondrial respiration, iron absorption, free radical scavenging and elastin cross-linking [17]. Copper plays an important role in our metabolism, largely because it allows many critical enzymes to function properly [18]. Copper is essential for maintaining the strength of the skin, blood vessels, epithelial and connective tissue throughout the body, plays a role in the production of haemoglobin, myelin, melanin and keeps thyroid gland functioning normally [18]. Copper can act as both an antioxidant and a pro-oxidant. Free radicals occur naturally in the body and can damage cell walls, interact with genetic material, and contribute to the development of a number of health problems and diseases. As an antioxidant, Cu scavenges or neutralize free radicals and may reduce or help prevent some of the damage they cause [19]. When copper acts as a pro-oxidant at times, it promotes free radical damage. Maintaining the proper dietary balance of Cu, along with other minerals such as zinc and manganese, is important [20].

Selenium is an essential trace element. Its deficiency causes serious health effects in humans, such as Keshan disease. At least 30 selenoproteins that have been identified in mammals [21]. It has unique physio-chemical properties including antioxidant activity [22].

Full blood count (FBC) is also known as Complete Blood Count (CBC). It is one of the most common laboratory tests performed today. A full blood count (FBC) is a very common laboratory test for most medical investigations. The FBC not only tests for disorders and abnormalities of the blood but, as blood travels throughout the whole body, it can give an indication of disease present in other organs. FBC, as the name suggests, is used to obtain a count of the blood cells in the sample of blood taken. The counts from this small sample are used to estimate the levels of different blood cells within your body’s blood system. Blood is made up from three main types of blood cell: red blood cells, white blood cells and platelets. The number of cells present, the size and proportions of these cells, and the haemoglobin level are measured in FBC. is the oxygen carrying component of red blood cells [23].

Sickle cell anaemia commonly affects growth, leading to low mean weight, low mean height and decreased height velocity. Africa has 70% of the world’s annual figure of 300,000 of new births affected by SCD. In Nigeria the prevalence of sickle cell trait is about 25% while the homozygous state is found in about 3% of the population [2]. Nigeria has the largest population of people with sickle cell disorder, with about 150,000 births annually [7]. In 2006, the WHO declared SCD to be a problem of major public health significance and a burden that must be addressed if recent improvements in overall child survival are to be consolidated [3].

The oxidative damage in sickle RBCs causes a decrease in trace element specifically copper, zinc and selenium [24]. Trace elements include; Zinc, Copper, Selenium, Manganese, Chromium, Magnesium, Fluoride, Cobalt, Iron, and Iodine [25]. Copper plays an important role in our metabolism, largely because it allows many critical enzymes to function properly. Cu plays a role in the production of haemoglobin, myelin, melanin and it also keeps thyroid gland functioning normally. Copper can act as both an antioxidant and a pro-oxidant. As an antioxidant, Cu scavenges or neutralize free radicals and may reduce or help prevent some of the damage they cause. When copper acts as a pro-oxidant at times, it promotes free radical damage. The levels of trace elements and magnesium have earlier been reported to be lower in SCD patients compared with HbAA control subjects and proteinuria impacted on the levels of these elements [26].

There is paucity of information in literature concerning the micronutrients status of sickle cell disease patients in this locality. Therefore, we sought to evaluate some micronutrients status in sickle cell patients. In addition, there is a limited data on the full blood count parameters among children with sickle cell disease in Sokoto, North Western Nigeria it is hoped that the information obtained, at the end of this study would be helpful to health care practitioners and health policy makers in this area, in curbing this disorder particularly among children in this vulnerable age group. The aim of this study is to determine the full blood count parameters, copper and selenium level among sickle cell disease children in Specialist Hospital Sokoto in Sokoto North Western Nigeria.

2. Materials and method

2.1 Study area

This study was carried out in the Paediatric Clinic Specialist Hospital, Sokoto, North–Western Nigeria. Specialist Hospital Sokoto is a tertiary institution located within the Sokoto metropolis. Sokoto is the capital city of Sokoto State. Sokoto State occupies 25,973 square kilometers and is situated along latitude 13 ${}^{\circ}$ 3’39 N and longitude 5 ${}^{\circ}$ 14’2 ${}^{\circ}$ E. The State is located in the extreme Northwest of Nigeria, near to the confluence of the Sokoto River and the Rima River. The State is in the dry Sahel, surrounded by sandy savannah and isolated hills, with an annual average temperature of 28.3 ${}^{\circ}$ C (82.9 ${}^{\circ}$ F). Sokoto is, on the whole, a very hot area. It shares borders with Niger Republic to the North, Zamfara State to the East, Kebbi State to the South-East and Benin Republic to the West. However, maximum daytime temperatures are for most of the year generally under 40 ${}^{\circ}$ C (104.0 ${}^{\circ}$ F) and the dryness makes the heat unbearable. The warmest months are February to April when day time temperature can exceed 45 ${}^{\circ}$ C (113.0 ${}^{\circ}$ F). The rainy season is from

June to October during which shower are a daily occurrence. Sokoto city is a major commerce center in leather crafts and agricultural products. As at 2006, the state has a population of 3.6 million [27]. However, based on the population annual growth of 3%, the calculated projected population for Sokoto State now stands at around 4.9 million.

2.2 Study population

The subjects for this study included forty-five (45) children with SCD aged between 1–15 years. Twenty-five (25) age – matched healthy children without any history of SCD were monitored as controls. Both subjects and controls participants were recruited from among the children visiting the Paediatric Department of Specialist Hospital Sokoto, Sokoto North-Western Nigeria.

3. Study subjects/selection

3.1 Inclusion criteria

Children who met the following inclusion criteria were consecutively recruited as subject for this study.

ii. i.
Children aged 1–15 years with confirmed sickle cell disease and who are in stable state visiting Specialist Hospital Sokoto, Nigeria.
ii.
Children whose parents and guardian gave a written informed consent for their ward to participate in this study.

3.2 Exclusion criteria

The following were excluded from participating as subjects in the study.

ii. i.
Sickle cell disease children $<$ 1 or $>$ 15 years and children whose parents/guardian decline to give a written informed consent for their ward to participate in this study were excluded from participation in the study.
ii.
Non-sickle disease children.

3.3 Study design

This research was a case-control study to assess the effect of Sickle cell disease on the full blood count, copper and selenium levels of children with sickle cell disease. Complete blood count parameters, copper and selenium was determined among 45 sickle cell disease children (subjects) and 25 healthy (haemoglobin AA) age-matched children visiting the Paediatric Clinic of Specialist Hospital, Sokoto. Blood sample was collected (from both subjects and controls) and tested for complete blood count, copper and selenium level. Results of these parameters were analyzed using the Statistical Package for Social Sciences (SPSS version 25.0, IBM California Inc. USA) for windows software version. Interviewer-administered questionnaire was used to capture socio-demographic data of the subjects.

3.4 Sample size determination

The sample size was determined according to Coch-ran (1999) using the formula:

$n=$ Minimum sample size

$z=$ Standard normal deviation and probability.

$p=$ Prevalence or proportion of value to be estimated from previous studies.

$q=$ Proportion of failure ( $=1-P$ )

$d=$ precision, tolerance limit, the minimum is 0.05.

Therefore $n=$ z ${}^{2}$ pq/d ${}^{2}$

Where $Z=$ 95% (1.96)

$P=$ 3% (0.03) [2].

$q=1-0.003(=0.97)$

$d=$ 5% (0.05)

Therefore $n=(1.96)^{2}(0.03)(0.97)/(0.05)^{2}$

$n=$ 45

3.5 Sample collection

Four milliliters (4 ml) of whole blood was collected from both the patients with sickle cell disease and those without sickle cell disease (control) by clean venipuncture after which 2 ml was dispersed into Ethelene diamine tetra-acetic acid ( $K_{3}$ EDTA) tube and used for full blood count (PCV, white blood cell count, platelet count, red blood cell count and red blood cell indices) and remaining 2 ml of blood was dispersed into a plain tube and the serum was obtained after centrifugation at 3000 rpm for ten minutes on a bench-top centrifuge and used for analysis of copper and selenium. These samples were tested in the Pathology Laboratory of Usmanu Danfodiyo University Teaching Hospital (UDUTH) Nigeria. The following laboratory investigations were carried out on $K_{3}$ EDTA anticoagulated blood and serum.

3.6 Full blood count

Full blood count was determined using the 5-part differential haematology analyzer (Mythic 22 CT, 2008, Orphee, Switzerland). The Mythic 22 CT, 2008 Haematological analyser count blood cells based on Impedance Principle. Impedance counting was developed by Wallace Coulter in 1956 [28]. The Coulter counter system is based on the principle that red cells and white cells are poor conductors of electricity compared to the diluents such as saline. When the diluent is displaced by cells, it causes a measurable change in resistance. The cells are allowed to pass through an aperture through which an electric current is flowing. Cell passing through the aperture displace the diluents; and being a bad conductor of electricity, the increase in resistance is counted as a voltage pulse which are converted to digital recordings. The cell suspension is drawn through the aperture with the help of a vacuum pump into a system of tubing.

3.7 Measurement of copper

Principle: At pH of 4.7 copper is released from the carrier protein and forms with 4-(3,5-Dibromo-2-pyridylazo)-N-ethyl-N-sulfopropylaniline a chelate complex. The increase of absorbance of this complex can be measured and is proportional to the concentration of total copper in the sample.

3.8 Measurement of selenium

Principle of the test: Selenium is measured in serum by Atomic Absorption Spectrometry in a procedure based on standard methods [29, 30]. Quantification is based on the measurement of light absorbance at 196.0 nm by ground-state atoms of selenium from a selenium electrodeless discharge lamp (EDL) source. Serum samples, human serum quality control pools, and serum calibration standards are diluted with a matrix modifier (nitric acid or nickel nitrate). The selenium content is determined by using Shimadzu Model AA-6300 (Japan) ROM version 1.09 transversely heated graphite furnace atomic absorption spectrophotometer.

3.9 Study instrument

Questionnaire: A semi-structured interviewer-ad- ministered questionnaire was administered to all consenting participants to obtain information from their guardian/parent on their socio-demographic, nutritio- nal and medical history.

Informed consent: Written informed consent will be obtained from the parents or guardian of all the study participants (subjects and controls).

Statistical analysis: The data generated from this study was analyzed using the statistical package for social sciences (SPSS) for windows software version (25.0), IBM California Inc. USA. The mean, standard error of mean value was determined as applicable. The differences between mean was determined using student t test. $P$ -values less than 0.05 were considered statistically significant. Pearson correlation was used in comparing the variables. A $p$ -value $\leqslant$ 0.05 was taken as statistically significant.

3.10 Ethical consideration

Ethical approval for this study was obtained from the Ethical Committee of Specialist Hospital Sokoto Ethical Committee.

3.11 Limitation of the study

The scope of the study cannot be fully exploited because most of the patients are in crisis stage before coming to the hospital.

4. Results

A total of 45 children with sickle cell disease in steady state and 25 age and gender-matched apparently healthy controls were enrolled for the study. The results are presented as mean $\pm$ standard error of mean. Haematological parameters were compared between sickle cell disease patients and controls. The mean WBC and PLT was significantly higher among sickle cell disease subjects when compared to controls individuals (15.7 $\pm$ 1.06 and 392.9 $\pm$ 29.3) and (7.4 $\pm$ 0.37 and 281.8 $\pm$ 12.55) respectively ( $p<$ 0.05). The mean RBC, HCT, HGB, MCH and MCHC was significantly lower among Sickle cell disease patients when compared to controls subjects (1.7 $\pm$ 0.08, 23.0 $\pm$ 0.68, 8.5 $\pm$ 0.24, 24.4 $\pm$ 0.34, 27.9 $\pm$ 0.45) and (3.2 $\pm$ 0.13, 39.6 $\pm$ 0.67, 12.5 $\pm$ 0.16, 30.8 $\pm$ 0.41 and 34.3 $\pm$ 0.28) respectively with ( $p<$ 0.05).

The mean Copper and Selenium value was significantly lower (40.4 $\pm$ 1.44 $\mu$ g/dl and 54.6 $\pm$ 1.60 ng/ml) among Sickle cell disease subjects compared to controls (75.6 $\pm$ 1.30 $\mu$ g/dl and 86.3 $\pm$ 2.30 ng/ml) ( $p<$ 0.05). The WBC, HGB, HGT and Copper values of Sickle cell disease subjects shows a weak positive correlation with age. There values increase as the age increases ( $p>$ 0.05). The RBC, MCV, MCH, MCHC, PLT, and Selenium values of sickle cell disease patients shows a negative correlation which decreases as the age increases (p $>$ 0.05).

Table 1
Socio-demographic characteristics of patients and controls

Characteristics	Patients		Controls
	$N=$	Percentage	$N=$	Percentage
	45	(%)	25	(%)
Age groups(years)
1–5	22	31.4	5	7
6–10	15	21.4	7	10
11–15	8	11.4	13	18.6
Gender
Male	19	27.1	12	17.1
Female	26	37.1	13	18.6
Ethnicity
Hausa	26	37.1	14	2
Fulani	14	20	8	11.4
Igbo	1	0.1	0	0
Yoruba	0	0	3	4.3
Other	4	5.7	0	4.3
Educational attainment of parents
Tertiary certificate	6	8.6	8	11.4
Secondary certificate	4	5.7	6	8.6
Primary certificate	26	37.1	6	8.6
No formal education	9	12.9	5	7.1
Household income of parents
5,000–15,000	2	2.9	0	0
16,000–25,000	7	10	1	1.4
26,000–35,000	12	17.1	10	14.3
36,000–45,000	11	15.7	9	12.9
Above 45,000	13	18.6	5	7.1

Key: $N=$ number, % $=$ percentage.

Table 1 shows the socio-economic and demographic characteristics of the subjects and controls and reveals that majority of the patients; 22 (31.4%) and control group; 5 (7%) were in the age range of 1–5 years. The distribution of patients and control based on gender shows slight increase in female 26 (37.1%) and 13 (18.6%) than in male respectively. The distribution of patients and control group based on ethnicity shows that majority of subjects and controls were Hausa; 26 (37.1%) and 14 (2%) respectively. The distribution of the subject’s parents based on educational status shows that 6 (8.6%) had tertiary education, 4 (5.7%) had Section 26 (37.1%) had primary education and 9 (12.9%) had no formal education as compared with control where 8 (11.4%) had tertiary education, 6 (8.6%) had secondary education, 6 (8.6%) had primary education and 5 (7.1%) had no formal education.

Total monthly income of subjects parents indicated that 2 (2.9%) received 5,000–15,000, 7 (10%) rece-ived 16,000–25,000, 12 (17.1%) received 26,000–35,000, 11 (15.7%) received 35,000–45,000, 13(18.6%) received above N45,000 as compared to control where 0 (0%) received 5,000–15,000, 1 (1.4%) received 16,000–25,000, 10 (14.3%) received 26,000–35,000, 9 (12.9%) received 36,000–45,000, 5 (7.1%) received above 45,000 respectively.

Table 2 shows the mean $\pm$ SEM for copper and selenium values among Sickle Cell Disease subjects and controls. It shows that copper and selenium level were significantly lower among children with sickle cell disease as compared to controls ( $p<$ 0.05).

Table 3 shows the mean $\pm$ SEM haematological values among Sickle Cell Disease subjects and controls. Haematological parameters were compared between sickle cell disease cases and controls. Haematological parameters (RBC, HGB, HGT, MCV, MCH, MCHC) levels was significantly lower among subjects compared to controls ( $p<$ 0.05), while WBC and PLT levels was significantly higher among subjects compared to controls ( $p<$ 0.05).

Table 4 presents the correlation between the full blood count, copper and selenium against age of sickle cell disease subjects. The WBC, HGB, HGT and Copper values ( $+$ 0.212, $+$ 0.186, $+$ 0.107, $+$ 0.132) of Sickle cell disease subjects increases as the age increases without ( $p>$ 0.05). The RBC, MCV, MCH, MCHC, PLT, and Selenium values ( $-$ 0.046, $-$ 0.010, $-$ 0.091, $-$ 0.072, $-$ 0.061, $-$ 0.144) of sickle cell disease patients decreases as the age increases ( $p>$ 0.05). A positive statistically significant correlation was found between the haematological parameters (WBC, HGB, HGT and Copper) among the sickle cell disease subjects ( $p<$ 0.05).

Table 2

Mean copper and selenium value among sickle cell disease subjects and controls

	Group of	$N$	Mean	$t$ -value	$p$ -value
	patients		( $\pm$ SEM)
Copper level	Control	25	75.6 $\pm$ 1.30	16.255	0.000
( $\mu$ g/dl)	Test	45	40.4 $\pm$ 1.44
Selenium	Control	25	86.3 $\pm$ 2.30	11.526	0.000
level (ng/ml)	Test	45	54.6 $\pm$ 1.60

Key: $N=$ number, SEM $=$ standard error of mean, Level of significance is considered when $p<$ 0.05, $\mu$ g/dl $=$ microgram per deciliter, ng/ml $=$ nanogram per milliliter.

Table 3

Mean Haematological parameters value among sickle cell disease subjects and controls

	Group of	$N$	Mean	$t$ -value	$p$ -value
	patients		( $\pm$ SEM)
WBC (^10 ${}^{{9}}$ /L)	Control	25	7.4 $\pm$ 0.37	$-$ 5.666	0.000
	Test	45	15.7 $\pm$ 1.06
RBC (^10 ${}^{{9}}$ /L)	Control	25	3.2 $\pm$ 0.13	10.931	0.000
	Test	45	1.7 $\pm$ 0.08
HGB (g/dl)	Control	25	12.5 $\pm$ 0.16	11.613	0.000
	Test	45	8.5 $\pm$ 0.24
HGT (%)	Control	25	39.6 $\pm$ 0.67	16.010	0.011
	Test	45	23.0 $\pm$ 0.68
MCV (fl)	Control	25	85.9 $\pm$ 1.16	9.104	0.000
	Test	45	67.9 $\pm$ 1.32
MCH (pg)	Control	25	30.8 $\pm$ 0.41	11.676	0.000
	Test	45	24.4 $\pm$ 0.34
MCHC (g/dl)	Control	25	34.3 $\pm$ 0.28	9.991	0.004
	Test	45	27.9 $\pm$ 0.45
PLT (^10 ${}^{9}$ /L)	Control	25	281.8 $\pm$ 12.55	$-$ 2.740	0.008
	Test	45	392.9 $\pm$ 29.3

Values are mean $\pm$ standard error of mean, Level of significance is considered when $p<$ 0.05. Key: $N=$ number of subjects; WBC $=$ white blood cell count, RBC $=$ red blood cell; HGB $=$ haemoglobin; HGT $=$ haematocrit; MCV $=$ mean cell volume; MCH $=$ mean corpuscular haemoglobin; MCHC $=$ mean corpuscular Haemoglobin concentration; PLT $=$ platelet.

Table 4

Correlation of age with haematological parameters, copper and selenium levels in sickle cell disease subjects

Parameters	$p$ -value	$r$ (regression)
WBC-test	0.358	$+$ 0.212
RBC-test	0.522	$-$ 0.046
HGB-test	0.522	$+$ 0.186
HGT-test	0.445	$+$ 0.107
MCV-test	0.336	$-$ 0.010
MCH-test	0.459	$-$ 0.091
MCHC-test	0.263	$-$ 0.072
PLT-test	0.173	$-$ 0.061
COPPER-test	0.391	$+$ 0.132
SELENIUM-test	0.289	$-$ 0.144

r $=$ regression, positive values means both parameters increases and decreases at the same time while negative value means one increases and the other decreases or vice-versal). Positive correlation (0.1–0.3 weak, 0.4–0.6 moderate, 0.7–0.9 strong). Negative correlation ( $-$ 0.3– $-$ 0.1 weak, $-$ 0.6– $-$ 0.4 moderate, $-$ 0.9– $-$ 0.7 strong). Key: $N=$ number of subjects; WBC $=$ white blood cell count, RBC $=$ red blood cell; HGB $=$ haemoglobin; HGT $=$ haematocrit; MCV $=$ mean cell volume; MCH $=$ mean corpuscular haemoglobin; MCHC $=$ mean corpuscular haemoglobin concentration; PLT $=$ platelet.

5. Discussion

Sickle cell disease (SCD) is an inherited chronic condition that arises from a point mutation in the DNA coding for the synthesis of $\beta$ -globin chain. It results in the production of sickle haemoglobin which has a high tendency to polymerize and deform red cells, leading to chronic haemolysis [31].

The sickle cell disease and $\beta$ -thalassaemia are the most common monogenic diseases and one of the worlds’ s major health problems. According to data from the World Health Organization [4]. It is estimated that 7% of the worldwide population are carriers for haemoglobinopathies, and 50% of them have gene for sickle cell anaemia [32]. Over 300,000 infants with sickle cell disorders are born annually [33]. The prevalence of this disease ranges from 0, 1/1,000 in non-endemic countries to 20/1,000 in several parts of African continent [12] and is spreading around the world. Haemoglobin disorders were originally endemic in 60% of 229 countries, potentially affecting 75% of births, but now are sufficiently common in 71% of countries among 89% of births, either in the whole population or among minorities [33]. The highest concentration of the gene occurs in the Black population of sub-Saharan Africa, where are groups with the gene that affects up to 40% of the population [34], and it is even more common in West Central Africa [32].

It is estimated that around 85% of births affected with SCD occur in Africa, and estimates suggest that 50–80% of these patients will die before adulthood. Sub-Saharan Africa contributes significantly to the global mortality of children aged less than 5 years with mortality rates of 100–250 per 1000. It is now well established that invasive bacteria disease is the leading cause of this childhood mortality [35]. More than 230, 000 children are born in Africa with sickle cell disease (SCD) each year: approximately 85% of all affected births worldwide [36].

Nutritionally essential trace metals and nonmetal are not antioxidants on their own but they are integral parts and are necessary for the proper functions of antioxidant enzymes like catalase, glutathione peroxidase and reductase, and superoxide dismutase [25]. Thus, it is necessary that the level of these nutritionally essential trace metals and nonmetals, which are important to these antioxidant enzymes, be assessed in sickle cell disease patients.

In this study, we observed a significantly low mean serum copper level was observed from the comparison of sickle cell disease group with control group. Our finding agrees with the reports [36, 37, 38]. Low level of copper has been noted in sickle cell anaemia patients. However, it is contrary to previous reports [39, 40, 41]. which revealed a significantly elevated level of copper in sickle cell patients. Trace elements are important in red blood cell maintenance, body growth and development and their deficiency have been observed in sickle cell disease [42, 43]. There is increased turnover of haemopoietic cells due to chronic haemolysis and cell death leading to tremendous red marrow expansion. These conditions lead to hyper-metabolic rate and increase in nutrient and energy demand [44]. Copper is known to be essential in the proper functioning of different metal enzymes which include ceruloplasmin involved in iron metabolism [45]. Deficiency of copper is known to cause anaemia [46]. Studies have suggested that the copper containing enzyme, ceruloplasmin may have specific role, probably related to its function in mobilization of stored iron in the liver which makes iron available for haemoglobin synthesis [47]. However, it has been observed that in copper deficiency induced anaemia, in spite of elevated iron level in the liver, the rate of haemoglobin synthesis remain significantly reduced [48].

In this study, we observed a significantly low mean serum copper level was observed from the comparison of sickle cell disease group with control group ( $p=$ 0.000). Selenium (Se) plays a key role in the maintenance of normal health in human populations [49]. It has been demonstrated that, when taken as a supplement, Selenium modulates the cellular response to oxidative stress, inducing a faster restoration of the endogenous antioxidative defense system against the production of reactive oxygen species [50]. Glutathione peroxidase controls the intercellular level of hydrogen peroxide, reducing the formation of reactive oxygen species that can induce lipid peroxidation with consequent damage to the cellular membranes [51]. Deficiency of Se, Zn and Cu decreased activities of superoxide dismutase and glutathione peroxidase [52]. In this study, a significantly low mean serum selenium level was observed from the comparison of sickle cell disease group with control group and this agrees with the report of previous authors [37, 38, 52]. The antioxidant properties of selenoproteins help prevent cellular damage from free radicals, regulate thyroid function and play a role in immune system [53, 54].

There is evidence that selenium deficiency may contribute to development of a form of heart disease, hypothyroidism, and a weakened immune system [55, 56]. According to Singh [57] selenium deficiency may impair utilization of iodine because it is a key component of the enzyme, which is required to convert thyroxine to triidothyronine. It can make the body more susceptible to illness caused by other nutritional, biochemical or infectious stresses [58].

In this study, the mean haemoglobin (Hb) and mean Haematocrit (Hct) values were significantly lower among the Sickle cell disease patients compared to controls ( $p=$ 0.000). Our finding agrees with the work of previous authors [59]. Our finding partially agrees with previous report in Nigeria [60]. This could be as a result of chronic haemolysis [61], low erythropoietin response and shortened red cell survival [62]. Patients with sickle cell disease (SCD) generally have a background rate of red cell sickling, which drastically shortens the life span of red cells leading to a chronic haemolytic anaemia and jaundice even in steady state [63]. This explains the low Hb and Hct observed in this study. Sickle cell disease is a group of common inherited red blood cell conditions where the red blood cells form a sickle or crescent shape. The sickling of red blood cells causes the cells to breakdown prematurely, reducing the number of red blood cells in the body. This leads to chronic anaemia sometimes referred to as sickle cell anaemia and may cause tiredness, lethargy, pale skin (pallor) and delayed growth and development in children. Sickle cell disease is a group of common inherited red blood cell conditions where the red blood cells form a sickle or crescent shape. The sickling of red blood cells causes the cells to breakdown prematurely, reducing the number of red blood cells in the body. This leads to chronic anaemia sometimes referred to as sickle cell anaemia and may cause tiredness, lethargy, pale skin (pallor) and delayed growth and development in children. Sickle cell disease is a group of common inherited red blood cell conditions where the red blood cells form a sickle or crescent shape. The sickling of red blood cells causes the cells to breakdown prematurely, reducing the number of red blood cells in the body. This leads to chronic anaemia sometimes referred to as sickle cell anaemia and may cause tiredness, lethargy, pale skin (pallor) and delayed growth and development in children. Sickle cell disease is a group of common inherited red blood cell conditions where the red blood cells form a sickle or crescent shape. The sickling of red blood cells causes the cells to breakdown prematurely, reducing the number of red blood cells in the body. This leads to chronic anaemia sometimes referred to as sickle cell anaemia and may cause tiredness, lethargy, pale skin (pallor) and delayed growth and development in children. Sickle cell disease is a group of common inherited red blood cell conditions where the red blood cells form a sickle or crescent shape. The sickling of red blood cells causes the cells to breakdown prematurely, reducing the number of red blood cells in the body. This leads to chronic anaemia sometimes referred to as sickle cell anaemia and may cause tiredness, lethargy, pale skin (pallor) and delayed growth and development in children. Sickle cell disease is a group of common inherited red blood cell conditions where the red blood cells form a sickle or crescent shape. The sickling of red blood cells causes the cells to breakdown prematurely, reducing the number of red blood cells in the body. This leads to chronic anaemia sometimes referred to as sickle cell anaemia and may cause tiredness, lethargy, pale skin (pallor) and delayed growth and development in children. Sickle cell disease is a group of common inherited red blood cell conditions where the red blood cells form a sickle or crescent shape. The sickling of red blood cells causes the cells to breakdown prematurely, reducing the number of red blood cells in the body. This leads to chronic anaemia sometimes referred to as sickle cell anaemia and may cause tiredness, lethargy, pale skin (pallor) and delayed growth and development in children.

The mean WBC count observed in this study was significantly higher in SCD patients. Our finding agrees with the work of other authors [64, 65, 66]. This is expected because of the basic mechanisms which cause an increase concentration of neutrophils in venous blood of SCD patients which include demargination of intravascular neutrophils, accelerated release from the bone marrow and reduction in the rate at which neutrophils leave the blood [66].

Sickle cell disease (SCD), an inherited disorder of the haemoglobin (Hb) structure and synthesis, causes chronic haemolysis, frequent infections, and a variety of other acute and chronic complications that can lead to organ damage, severe pain, disability, and premature death [67, 68]. The main cause of sickle cell-associated morbidity and mortality is (micro) vascular occlusion. Direct adherence of sickle red blood cells (RBC) to the endothelial cell surface plays a major role in this process. This results in delayed passage with concomitant enhancement of RBC sickling, ultimately leading to tissue hypoxia and infarction [69, 70]. Increasing evidence suggests that white blood cells (WBC), especially neutrophils, may be involved in the initiation and propagation of vaso-occlusive crisis in SCD [71]. Elevated total WBC counts are common in SCD patients and WBC counts of more than 15,000 cells/ $\mu$ L are associated with an increased risk of early death in SCD [71]. Adhesion of (activated) neutrophils to endothelium in patients with SCD may lead to endothelial damage as described in other vascular diseases. Because the neutrophil is larger and more difficult to deform than the red cell, its attachment to the endothelium, particularly in the microcirculation, would impede passage of RBC and WBC, which could increase the risk for vaso-occlusive crises [72].

The mean platelet count of the SCD patients was significantly higher than control individuals. The observation agrees with several other reports [73, 74]. This is expected due to the decrease or absent splenic sequestration of platelets [75], the increase in erythropoietin which has structural homology with thrombopoietin [76]. An association between stroke in sickle cell disease and platelet count $>$ 450,000/ $\mu$ l has been reported [77]. Qualitatively, poor platelet aggregation responses to epinephrine and ADP were also reported in sickle cell disease [78].

The mean cell hemoglobin concentration (MCHC) was significantly low among the subjects compared to controls. Our finding is comparable to other studies [79]. This may be due to co-existing iron deficiency anaemia and other unknown factors such as a-thalassemia which is frequent and often associated to SCD [80].

It is said that MCV is high in sickle cell disease patients because of the increasing need of erythropoiesis due to chronic haemolysis leading to macrocytosis. It is also related to a folic acid deficiency. However, MCV was low in our study as previously observed [81]. Low MCV in these studies may be due to co-existing iron deficiency anaemia.

Finding from this study is enough justification for the clinical and haematological monitoring of SCD patients. There is need for the evidenced based management of children with SCD in Nigeria including penicillin prophylaxis has been shown to reduce infection [82], folate to stimulate erythrocyte production [83], vaccination against Streptococcus to reduce the infection rate from this pathogen [84], prevention of red cell dehydration, due to ion and water loss via the potassium selective pathways [85], hydroxyurea, transfusion/chelation therapy, and haematopoietic stem cell transplant [86, 87, 88].

6. Conclusion and recommendations

This study shows that WBC and PLT is significantly higher among sickle cell disease subjects compared to controls. RBC, HCT, HGB, MCV, MCH and MCHC is significantly lower among sickle cell disease subjects compared to controls. Sickle cell disease is associated with a lower levels of trace elements (copper and selenium) compared to controls. Finally, the result shows a weak positive correlation between the age and some haematological parameters (WBC, HGB, HGT and copper and a negative correlation between the age and some haematological parameters (RBC, MCV, MCH, MCHC, PLT and selenium) value. We recommend that routine laboratory investigation for trace elements (copper and selenium) and haematological parameters be included in the care plan for sickle cell disease children. We recommend government should assist in providing the equipment and the reagents for the routine testing of these parameters in hospital laboratories in the area.

Footnotes

Acknowledgments

We are grateful to the staff of Sokoto Specialist Hospital for their collaboration that facilitated the success of this study. We sincerely thank all the subjects and control individuals that participated in this study as participants. Our appreciation also goes the staff of the Haematology Department of Usmanu Danfodiyo University and Usmanu Danfodiyo University Teaching Hospital for their assistance with laboratory testing of the blood samples from the study participants. The study was sponsored from contribution from all authors.

Conflict of interest

The authors declare that there is no conflict of interest associated with this paper.

Authors contribution

Osaro Erhabor was involved in the development of the concept, study design, data collection, analysis, and writing of the paper. Kevin Ogar helped to develop the concept, supervised the study, and critically revised the paper. Erhabor Tosan participated in the study design and data interpretation, revised the paper and approved the final version. Amos Dangana participated in the acquisition of the data, revised the paper and approved the final version. All authors read and approved the final version of the paper.

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Some haematological parameters,copper and selenium level among children of African descent with sickle cell disease in Specialist Hospital Sokoto,Nigeria

Abstract

BACKGROUND:

OBJECTIVES:

METHOD:

RESULTS:

CONCLUSION:

Keywords

1. Introduction

2. Materials and method

2.1 Study area

2.2 Study population

3. Study subjects/selection

3.1 Inclusion criteria

ii. i. Children aged 1–15 years with confirmed sickle cell disease and who are in stable state visiting Specialist Hospital Sokoto, Nigeria. ii. Children whose parents and guardian gave a written informed consent for their ward to participate in this study. 3.2 Exclusion criteria

ii. i. Sickle cell disease children < 1 or > 15 years and children whose parents/guardian decline to give a written informed consent for their ward to participate in this study were excluded from participation in the study. ii. Non-sickle disease children. 3.3 Study design

3.4 Sample size determination

3.5 Sample collection

3.6 Full blood count

3.7 Measurement of copper

3.8 Measurement of selenium

3.9 Study instrument

3.10 Ethical consideration

3.11 Limitation of the study

4. Results

Table 1 Socio-demographic characteristics of patients and controls

6. Conclusion and recommendations

Footnotes

Acknowledgments

Conflict of interest

Authors contribution

References

ii. i.
Children aged 1–15 years with confirmed sickle cell disease and who are in stable state visiting Specialist Hospital Sokoto, Nigeria.
ii.
Children whose parents and guardian gave a written informed consent for their ward to participate in this study.

3.2 Exclusion criteria

ii. i.
Sickle cell disease children $<$ 1 or $>$ 15 years and children whose parents/guardian decline to give a written informed consent for their ward to participate in this study were excluded from participation in the study.
ii.
Non-sickle disease children.

3.3 Study design

Table 1
Socio-demographic characteristics of patients and controls