|Year : 2019 | Volume
| Issue : 1 | Page : 13-17
Haematological values in steady-state sickle cell anaemia patients and matched heamoglobin AA Controls in a Rural Area of Eastern Gabon
Landry Erik Mombo1, Gaël Mabioko-Mbembo2, Cyrille Bisseye1, Kevin Mbacky3, Fatoumata Thiam3, Apollinaire Edou3
1 Laboratory of Molecular and Cellular Biology (LABMC), University of Science and Technology of Masuku (USTM), Franceville, Gabon
2 Laboratory of Molecular and Cellular Biology (LABMC), University of Science and Technology of Masuku (USTM), Franceville; Paul Moukambi Regional Hospital Centre of Koula-Moutou (CHRPM), Koula-Moutou, Gabon
3 Paul Moukambi Regional Hospital Centre of Koula-Moutou (CHRPM), Koula-Moutou, Gabon
|Date of Web Publication||12-Mar-2019|
Landry Erik Mombo
Laboratory of Molecular and Cellular Biology (LABMC), University of Science and Technology of Masuku (USTM), BP 943, Franceville
Source of Support: None, Conflict of Interest: None
Background: In Gabon, universal neonatal screening of sickle cell disease is not carried out in rural areas, often leading to late detection of the disease. However, complete blood counts are available in rural areas. Materials and Methods: We evaluated the haematological parameters of 45 homozygous steady-state sickle cell anaemia (SCA) patients and compared them with 45 sex- and age-matched Haemoglobin AA controls in Koula-Moutou, a rural area in Eastern Gabon. Results: Homozygous SCA patients had low erythrocyte values (red blood cells: 2.50 × 1012/L, haemoglobin: 7.20 g/dL and haematocrit: 20.70%) and high leucocyte values (white blood cells: 14.40 × 109/L, lymphocytes: 5.24 × 109/L and monocytes: 1.60 × 109/L). Most of the SCA patients had severe anaemia (67%), normochromia (76%), lymphocytosis (73%) and monocytosis (84%). A haemoglobin level of < 8.5 g/dL together with a leucocyte level above 9.5 × 109 cells/L was used as screening test to detect homozygous SCA patients, with sensitivity of 84.4% and specificity of 97.8%. Conclusion: The values for erythrocyte and leucocyte cell lines of SCA patients in steady state are clearly different from those of the matched HbA/A controls. This makes it possible to set up a tool to detect SCA based on the haemogram in a rural area that does not possess haemoglobin electrophoresis. This tool could be used by healthcare workers in the absence of universal newborn screening for SCA.
Keywords: Gabon, red blood cell, rural areas, sickle cell anaemia, white blood cell
|How to cite this article:|
Mombo LE, Mabioko-Mbembo G, Bisseye C, Mbacky K, Thiam F, Edou A. Haematological values in steady-state sickle cell anaemia patients and matched heamoglobin AA Controls in a Rural Area of Eastern Gabon. Niger Postgrad Med J 2019;26:13-7
|How to cite this URL:|
Mombo LE, Mabioko-Mbembo G, Bisseye C, Mbacky K, Thiam F, Edou A. Haematological values in steady-state sickle cell anaemia patients and matched heamoglobin AA Controls in a Rural Area of Eastern Gabon. Niger Postgrad Med J [serial online] 2019 [cited 2019 Jun 16];26:13-7. Available from: http://www.npmj.org/text.asp?2019/26/1/13/253980
| Introduction|| |
Sickle cell anaemia (SCA) is an inherited autosomale recessive disease, which manifests as chronic haemolytic anaemia, painful episodes of vaso-occlusive crisis and polysystemic organic damage. In Gabon, neonatal screening for sickle cell disease (SCD) is not universal despite the launch of a national program to curb the disease in 2007. Neonatal screening of 4,068 newborns detected 1.33% HbS/S and 0.14% HbS/C SCD in urban areas in Libreville. However, neonatal screening is unavailable in rural areas, and sickle cell patients are thus screened later in life during medical consultation.
SCD includes a set of quantitative and qualitative changes inside and outside the blood vessels that are responsible for the destruction of red blood cells (RBCs). In sub-Saharan African SCD patients, severe anaemia is associated with clinical complications such as leg ulcers, microalbuminuria or pulmonary hypertension.
Although SCD pathology is mainly due to RBCs, white blood cells (WBCs) also participate in obstructing blood vessels, a central phenomenon of vaso-occlusive crises in SCD. The haematological parameters in a complete blood count (CBC), which determines WBC abnormalities, may be a useful tool to assess SCD severity. For instance, a high number of medical emergencies due to SCD are associated with leucocytosis.
In many studies, a decrease in haematological parameters such as RBC count and haemoglobin and an increase in WBCs and platelets are associated with an increase in the number of vaso-occlusive crises in homozygous SCD patients., In addition, haematological values are reported to differ between patients with HbS/S or HbS/β-thal and those with HbS/C.,, Haematologic parameters can also be used in collaboration with other parameters to evaluate the efficacy of hydroxycarbamide therapy, as shown by a study from Italy where improvement in haemoglobin, haematocrit, mean corpuscular volume (MCV), platelets and WBCs counts was observed among SCD patients on hydroxycarbamide.
Although haematological parameters in SCD patients and control groups have been examined in previous studies,, few studies have compared sickle cell patients to sex- and age-matched control populations in Africa. The aim of our study was to compare the haematological parameters of homozygous SCA patients and sex- and age-matched HbA/A controls in order to test whether CBC results are a useful guide for medical staff in a rural area of Gabon to refer patients for haemoglobin electrophoresis.
| Materials and Methods|| |
Ninety participants who presented at Paul Moukambi Regional Hospital Centre (CHRPM) for a routine medical consultation between July and December 2015 were included in the study. The study population was 44 females and 46 males, aged 1–26 years with an average age of 10 years. Of the 90 participants, 45 were SCA patients and 45 were sex- and age-matched controls. All SCA patients were in steady state with HbS/S confirmed by haemoglobin electrophoresis. None of the SCA patients was on hydroxycarbamide therapy. All sex- and age-matched controls were apparently healthy with HbA/A confirmed by haemoglobin electrophoresis.
The separation of the normal haemoglobins (A and A2) and the detection of the major haemoglobin variants (S or D and C or E) were performed by qualitative electrophoresis on alkaline agarose gels (pH 8.5) using Hydragel 15 Haemoglobin(e) kits (a semi-automated Hydrasys system from SEBIA– Paris, France) and following the manufacturer's instructions.
CBCs were made using venous blood samples collected in an ethylenediaminetetraacetic acid bottle and analysed within 24 h of collection using an automated haematology analyser (Mindray BC 3000 plus, Mindray Bio-Medical Electronics Co., Ltd, Shenzhen, People's Republic of China) following the manufacturer's instructions.
Our study was approved by the Institutional Ethics Committee of Paul Moukambi Regional Hospital Centre (CHRPM) in June 2015 in Koula-Moutou (Gabon). Adults and parents or guardians of children gave their written informed consent before being included in the study.
Statistical analysis was performed using (SPSS, IBM corp., New York, NY, USA). The median values of haematological parameters were compared between SCA and matched controls using Wilcoxon and Kruskal–Wallis rank sum tests. The difference was considered significant where P < 0.05.
| Results|| |
Red blood cell values in steady-state sickle cell anaemia patients and matched controls from rural areas
RBC count, haematocrit and haemoglobin levels were significantly lower in homozygous SCA patients than in matched controls and significantly higher for mean corpuscular haemoglobin (MCH) and MCH concentration values [Table 1]. In the erythrocyte parameters, only MCV showed no significant difference between homozygous SCA and matched controls (P = 0.778). Severe or moderate anaemia was significantly more likely to be present in SCA patients than in matched controls [Table 2] (96% vs. 35%, P < 0.001). The prevalence of microcytic anaemia was not significantly different between homozygous SCA patients (29%) and matched controls (26%) (P = 0.815). Hypochromia was significantly more prevalent in controls (69%) while normochromia (76%) was higher in homozygous SCA patients (P < 0.0001).
|Table 1: Mean and median values of haematological parameters in steady-state sickle cell anaemia patients and matched controls living in rural areas (Koula-Moutou)|
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|Table 2: Distribution of blood cell anomalies in steady-state sickle cell anaemia patients and matched controls living in rural areas (Koula-Moutou)|
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Other blood cell values in steady-state sickle cell anaemia patients and matched controls from rural areas
The median WBC counts (lymphocyte, monocyte and granulocyte) were twice as high in SCA patients as in matched controls (P < 0.001) [Table 1]. Lymphocytosis was present in 73% of homozygous SCA patients but was observed in only 22% of controls (P < 0.001). Similarly, monocytosis and granulocytosis were significantly more prevalent in homozygous SCA patients than in matched controls (84% vs. 33%, P < 0.001; 51% vs. 11%, P < 0.001).
Median platelet count was significantly higher in homozygous SCA patients than in matched controls (P = 0.035), [Table 1]. However, the prevalence of abnormalities of the platelet lineage (thrombocytopenia and thrombocytosis) was not significantly different in homozygous SCA patients and matched controls (P = 0.258), [Table 2].
Haemogram method for sickle cell disease detection
The haemogram method tested in our study consists of the diagnosis of SCA through a threshold alert if haemoglobin is <8.5 g/dL and leucocyte is >9.5 × 109/L. As shown in [Table 3], the sensitivity, specificity and positive predictive values of this method were 84.4%, 97.8% and 97.4%, respectively.
|Table 3: Comparison of haemogram method and haemoglobin electrophoresis in the detection of sickle cell anaemia|
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| Discussion|| |
We evaluated the haematological parameters of steady-state homozygous SCA patients and controls matched by age and sex living in rural areas in eastern Gabon to determine a set of indices to screen for SCA.
Red blood cells in steady-state sickle cell anaemia patients and matched controls from rural areas
The haemolysis that occurs in homozygous SCA patients accounts for the reduction of the erythrocyte count found in this study, such as RBC (2.50 × 1012 cells/L), haemoglobin (7.20 g/dL) and haematocrit (20.70%). The erythrocyte values obtained in this study are lower than those observed in Nigerian and African-American homozygous SCA populations., This difference is also found when we compare the haemoglobin levels of Gabonese control populations with those of the Nigerian and African-American control populations., This reduction in haemoglobin levels in both SCA and controls could be due to nutritional deficiency and high prevalence of infectious diseases in rural areas.
Several studies have shown that erythrocyte counts vary across HbS/C, HbS/β-thal and HbS/S SCD genotypes, with lowest values for the latter.,, All sickle cell patients in our study were of HbS/S genotype, which may explain their lower erythrocyte values. However, the haemoglobin level for HbS/S cases (7.20 g/dL) is lower than that of sickle cell patients with the same genotype in the USA (Hb = 8.6 g/dL) and in Nigeria (Hb = 7.54 g/dL)., This could be explained by the absence of α-thalassemia among SCA patients in our study. The absence of α-thalassemia leads to a reduction in erythrocyte haematological values, for example, the haemoglobin level falls from 8.92 to 7.98 g/dL. However, an earlier study of heterozygous SCA patients in Gabon showed a high frequency of α-thalassemic traits determined indirectly, and another study in neighbouring Cameroon showed co-inheritance of the α-thalassemic variant in homozygous and heterozygous sickle cell patients., This suggests the probable presence of α-thalassemic variant in our homozygous SCA patients. Nevertheless, the erythrocyte values are low, supporting the hypothesis that nutritional deficiencies and infection pressure are greater problems in rural Gabon than in the USA and Nigeria.
A study conducted at Monkole Hospital Centre in Kinshasa (Democratic Republic of Congo) with 42 sickle cell patients of the Bantu ethnic group in conditions similar to those in our study, found haematological values similar to ours in homozygous SCA patients except for MCV and platelet counts. In our study, abnormalities of the blood count, such as microcytosis, macrocytosis, thrombocytopenia and thrombocytosis, did not differ in prevalence between homozygous SCA and matched controls. The difference observed between the two studies may be due to the use of matched controls for sex and age in our study and of unmatched controls in the Tshilolo et al.'s study because haemogram values depend on these 2 factors.
Other blood cell counts in steady-state sickle cell anaemia patients and matched controls from rural areas
Because of SCA and underlying bacterial infections, leucocyte counts were higher in homozygous sickle cell patients than in controls, with 14.40 × 109/L for WBCs; 5.24 × 109/L for lymphocytes; 1.60 × 109/L for monocytes and 7.51 × 109/L for granulocytes. However, leucocyte values in homozygous SCA patients in rural areas were still very high compared with leucocyte counts of 12.72 × 109/L observed in homozygous sickle cell patients in urban areas in Nigeria. This may be explained by a high prevalence of infectious disease in rural areas.
The leucocytosis observed in homozygous SCA patients in our study affects all types of WBCs (lymphocytosis, monocytosis and granulocytosis). This was not the case for leucocytosis found in African-American homozygous sickle cell patients where the lymphocyte was normal. The 2-fold higher monocyte counts and monocytosis observed in sickle cell patients can be explained by monocyte activation in these individuals. Indeed, a recent study suggests that haem-linked iron, which is released during intravascular haemolysis in sickle cell patients, contributes to the inflammation and activation of monocytes through toll-like receptors signalling increment.
Although the median platelet count was higher in homozygous SCA patients than in controls in our study, there was no association between thrombocytosis and steady-state SCA. The precise role of platelets in thrombocytosis, inflammation and haemolysis is only partially understood. Nevertheless, circulating platelets in patients with sickle cell crisis can stimulate endothelial cell activation. This stimulation is performed by direct contact or even adhesion of platelets to endothelial cells and through the NF-kB-dependent signalling pathway, thus contributing to the vaso-occlusive process. Thrombocytosis was not associated with steady-state SCA in our study, although it is associated with acute sickle cell crisis. A very high platelet count that is synonymous with thrombocytosis suggests the additional inflammatory mechanism in which platelets of sickle cell patients secrete significant amounts of cytokines and many cytokine messenger RNAs. This second mechanism, which affects vasculopathy during SCA, is rare in steady-state sickle cell patients because of the lower level of platelets in this phase.
Haemogram method for sickle cell anaemia detection
The diagnosis of SCD in African populations is generally late at a mean age of 7 years. The haemogram could be a useful tool in the diagnosis of SCA, particularly in rural areas, through a threshold alert if haemoglobin <8.5 g/dL and leucocyte >9.5 × 109/L. Using this method, 84.4% of SCA patients would have been referred for SCA diagnosis during our study. Moreover, for future studies, a positive predictive value of 97.4% may constitute a good indicator for SCD electrophoresis screening of patients. With a specificity of 97.8%, this method avoids the selection of individuals with solely uncomplicated Plasmodium falciparum malaria in Sub-Saharan Africa, which present mean haemoglobin of 8.9 g/dL and mean leucocyte count of 8.8 × 109/L. The haemoglobin threshold of 8.5 g/dL proposed in this study corresponds to the threshold observed in 504 sickle cell children starting hydroxycarbamide therapy in a study conducted in Italy. However, this haemogram method is only useful to refer presumed sickle cell patients with haemoglobin HbS/S.
The main limitation of our study (red or WBC counts and haemogram method of detection of SCA) was the small sample size. To be definitively adopted, the proposed haemogram method should be evaluated in a larger population and for a longer period.
| Conclusion|| |
In our study in rural Gabon, steady-state homozygous SCA patients had very low erythrocyte values, whereas they had a very high number of leucocytes. These SCA patients mainly presented severe anaemia, normochromia, lymphocytosis and monocytosis. Haemoglobin levels <8.5 g/dL and leucocytes >9.5 × 109/L could be used to trigger SCA screening in rural Gabon. This tool could be used in the absence of neonatal SCD screening in poorly served areas.
The authors thank Mr. Lionel Kevin Makosso and Mr. Stephane Meyet Me Bie (USTM, Franceville, and CHRPM, Koula-Moutou, Gabon), and all technicians of Medical Analysis Laboratory (CHRPM, Koula-Moutou, Gabon) for technical help, Pr. Joanna M. Setchell (Durham University, Durham, UK) for critically reading the manuscript and Pr. Jacques Elion (National Blood Transfusion Institute, Paris, France) for general support.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3]