Hemogram Response to Vasoclusive Crisis in Sickle Cell Patients in Enugu, South East, Nigeria

Vasoclusion crisis is a major cause of morbidity and mortality in patients with Sickle Cell Anemia (SCA). There are currently a paucity of data on the hemogram response to vasoclusive crisis in patients with SCA in the Enugu metropolis. The present study is designed to determine the hemogram response to vasoclusive crisis in SCA patients compared to steady state and controls. A total of 150 subjects comprising 75 confirmed SCA patients (35 males and 40 females) aged between 16 and 30 years from Sickle Cell Clinic and 75 apparently healthy age and gender-matched controls of the Enugu State University of Science and Technology Teaching Hospital Enugu State participated in the study. Sample size was calculated using single proportion method. Ethical clearance was obtained from the Ethical Review Board of the hospital. Informed consent was obtained from subjects. Blood sample (5.0 ml) was collected from each subject and 3.0 ml was dispensed into ethylene diamine tetracetic acid tubes for estimation of the hemogram using Mindray 530-BC hemoanalyser. The hemogram revealed significant decrease (p < 0.05) in Hemoglobin (Hb) during crisis (9.17 ± 0.91 g/dl) and during stead-state (11.63 ± 0.45 g/dl) and in hematocrit (Hct) during crisis (34.80 ± 3.03%) and during steady state (34.90 ± 1.37%) compared to controls Hb (13.5 ± 0.5 g/dl) and Hct (39 ± 1.5%). The total white blood cell count (TWBC) (8.3 ± 2.0 x 109/l) significantly increased (p < 0.05) in crisis compared to TWBC (5.2 ± 1.0 x 109/l) and (5.5 ± 0.5 x 109/l) for the steady-state and controls respectively while the Platelet (PLT) significantly increased (p < 0.05) in crisis (200 ± 55.05 x 109/l) compared to PLT (164 ± 45.39 x 109/l) and (150 ± 20.0 x 109/l) for the steady-state and controls respectively. This finding provides scientific data for changes in the hemogram in sickle cell patients during crisis and steady state.

Sickle Cell Disease (SCD) is the most common genetic blood disease globally [1]. Sub-Saharan Africa has the highest burden of the disease where currently 75% of all patients reside and this amount is estimated to increase to 85% year by the year 2050 with Nigeria particularly having the highest burden for the region [2,3]. It is a group of conditions resulting from the inheritance of abnormal allelomorphic genes controlling the formation of the beta (β)-chain of Hemoglobin (Hb) at least one of which is the sickle gene (Hbs) [3]. The common forms of the disease are HbS, HbSD and HbSβ - thalassemia [4]. The homozygous state (HbSS) termed Sickle Cell Anemia (SCA) is the most common and severest form of the disease which is caused by a point mutation (GAG to GTG)) in the sixth codon of the amino acid sequence of the beta globin (Hβ) [3,5]. This anomaly results in the replacement of the amino acid glutamic acid for valine with eventual formation of hemoglobin S (HbS) instead of Hemoglobin A (HbA) which causes the polymerization of the hemoglobin molecule leading to erythrocytes with less flexible sickle shape different from the normal biconcave disc shape of erythrocytes with HbAA. Patients with sickle cell anemia experience alternating periods of apparent good health (steady state) and acute vasoclusion (crisis state) which is triggered by conditions such as infection, dehydration and hypoxia [3,6,7]. Significant alterations in the hemogram has been reported for patients with sickle cell anemia during vasoclusive crisis [2].

The hemogram gives the profile of blood cells (red blood cells, white blood cells, platelets) in the body which are applied for the diagnosis and prognosis of diseases [8]. There is currently a paucity of data on the hemogram response to vasoclusive crisis in sickle cell anemia patients for the Enugu population. The present study is therefore designed to determine the hemogram of sickle cell anemia patients in vasoclusive crisis compared to steady state and health controls.

Study area

Enugu State is located in the South Eastern part of Nigeria. The state derived its name from its capital and largest city Enugu. It has an area of 7,161 km2 with a population of 3,267,837 comprising mainly the Igbo tribe of the southern Nigeria. It lies between longitudes 6°30’E and latitudes 5°15’N. It consists of three senatorial zones namely Enugu East, Enugu West and Enugu North senatorial zones. The Enugu State University of Science and Technology Teaching Hospital is the major tertiary health facility for the state and is located at the center of Enugu metropolis (Parklane) for easy accessibility to Enugu residents [9].

Study design

This was a cross sectional survey in a total of 150 subjects comprising 75 confirmed sickle cell anemia patients (35 males and 40 females) aged between 16 and 30 years from the Sickle Cell Clinic, Enugu State University of Science and Technology Teaching Hospital (ESUTH) Parklane, Enugu State, Nigeria and 75 apparently healthy (35 males and 40 females) age-matched controls participated in the study. Ethical clearance was obtained from the institutions Ethical Review Board with the registration number: ESUTHP/C-MAC/RA/034/Vol.4/39.

Sample size

The sample size was calculated using single proportion method.

$N=\frac{{\text{Z}}^{\text{2}}\left(\text{P}\right)\left(\text{1}-\text{P}\right)}{{\text{D}}^{\text{2}}}$

where

n = the desired sample size when the population is more than 10,000

z = standard variation usually set at 1.96 (which corresponds to 95% confidence interval)

p = population proportion of 10% which is 0.1.

$\text{n}=\frac{\text{1}.{\text{86}}^{\text{2}}\left(0.\text{1}\right)\left(\text{1}-0.\text{1}\right)}{0.0{\text{5}}^{\text{2}}}=\frac{\text{3}.\text{861 x }0.\text{1 x }0.\text{9}}{0.00\text{25}}=\frac{0.\text{35744}}{0.00\text{25}}=\text{138}.\text{3}$

Subjects inclusion criteria

Sickle cell anemia patients 16 years of age and older in a period of stable clinical condition occurring at least one week before or three weeks after or vasoclusive crisis or three months after a hemolytic crisis requiring a blood transfusion served as the subjects for steady-state, patents in active occlusive pain served as the crisis group while healthy individuals with HbAA genotype who are 16years of age and older served as the control.

Subjects exclusion criteria

Individuals who are taking any drug as well as those who smoke or drink too much alcohol (14 units per week for females and 21 units per week for males) were excluded from the study (control exclusion criteria). Sickle cell anemia patients with any additional medical conditions such as hypertension or diabetes mellitus, or those who have had a blood transfusion within the last three months were excluded from the study.

Sample collection

Venous blood (5 ml) was collected from each subject who gave consent and dispensed into Ethylene Diamine Tetra Acetic Acid container for hemoglobin electrophoresis and hemogram determination.

Hemoglobin electrophoresis (Cellulose acetate method)

Principle: Hemoglobin, a negatively charged protein, migrates to the anode when exposed to an electric field in an alkaline medium which distinguishes it from other heme proteins. The rate of migration is directly proportional to the net charge in the molecule with different hemoglobin types observed as variant bands involving Hb A, F,S,C,D and E [10].

Procedure: A hemolysate is prepared from whole blood by mixing with water; the hemolysate is spotted to a cellulose acetate paper as bands using an applicator. The cellulose acetate paper is transferred into an electrophoretic chamber containing citrate buffer at PH8.4; the electrophoretic tank is set of 220 volts; switched on and allow for 15 minutes for band separation.

Hemogram (Mindray 530BC autoanalyzer method)

Principle: The mindray autoanalyzer works by electrical impedance in which cells in suspension passes through a small aperture with electrical current, voltage pulses are generated, the number of which corresponds to the number of cells and the amplitude of the pulses is proportional to the cell size [11].

Procedure: The machine is switched-on and allowed to warm boot for 10 minutes; a period during which it auto-rinses its diluents, after which it displays “setting sample analysis”. The new sample option in the sample analysis setting is selected and the sample ID number is inputted accordingly. The sample is appropriately mixed in a vortex mixer and introduced into the sample probe and the start button pressed. The machine then performs auto analyses within 30 seconds and displays the result on the screen after which it immediately displays new sample setting for the next sample to be analyzed.

Statistical analysis

Data was subjected to inferential statistics in Statistical Package for Social Science (SPSS) for window version 20 (IBM, Armok, NY, USA) using one-way analysis of variance at 95% confidence internal. Probability value less than 0.05 was considered statistically significant.

Table 1 showed the socio-demogaphic characteristics of the subjects. The mean age of the subjects was 18 ± 4 years. Majority of the subjects were within the age group of 16-20 years, 22% were within 21-25 years while 16.6% were within 26-30 years. Females formed 53.3% of the subjects while males formed 46.6%. Majority of the subjects attended high school, 34% attended grade school, 14.6% attended college while 5.3% were not educated. There was a significant decrease (p < 0.05) in the hemoglobin, hematocrit, and Red blood cell counts during crisis and steady states compared to controls. We also recorded a significant increase in the platelet count and the total white blood cell count during the steady and crisis state compared to the controls (Table 2).

Changes in hemogram may explain the complications observed in patients with sickle cell anemia. The results of the present study shows that patients in crisis state had lower hemoglobin, hematocrit and red blood cell counts but higher leukocyte and platelets counts compared to steady-state and the values in normal individuals. This is similar to the findings of various studies conducted in other populations such as Sudan, the Republic of Congo and a recent study in Enugu, Nigeria which had observed lower hemoglobin, hematocrit and red blood cell counts but higher leukocyte and platelet counts in sickle cell anemia patients during vasoclusive crisis compared to the steady state and normal controls [12-15].

The effects of infections and hemolysis could explain the lower values of hemoglobin, hematocrit and red blood cell in patients during crisis [14]. Similarly, the effects of infections and recurrent splenic vessel occlusion which makes patients more vulnerable to opportunistic infections may explain the increased leukocytes counts observed in patients during crisis while the increased platelets observed in patients during crisis may be explained by increased thrombopoiesis resulting from a negative feedback to thrombopoietin production which is a common feature of anemia of chronic diseases such as the sickle cell disease [13,16]. Increased leukocyte and platelet indices are known markers of inflammatory conditions such as sickle cell anemia. The present findings therefore showed that early diagnosis of the hemogram response in SCA patients may be very useful for the prognosis and better management of patients with SCA particularly during crisis. The hemogrm is a cheap but reliable laboratory parameter that has become a routine diagnostic variable for patients care. The small sample size as well as the use of single centre for the present study could be considered a limitation. Further large-scale surveys are needed to support the present findings.

The finding of the present study provides scientific data for alterations of the hemogram during vasoclusive crisis in sickle cell anemia which underscores the diagnostic and prognostic importance of the hemogram for the management of patients.

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