ABSTRACT
Sickles cell aneamia (SCA) patients are prone to unpredictable recurrent episodes of vaso-occlusive crisis (VOC). VOC is the hall mark of clinical manifestation of sickle cell disease (SCD). Bone pain crisis (BPC) is the most common acute form of VOC, infection is the most common precipitating factor. Immunoglobulin are serum glycoprotein produced by plasma cell that help fight infection. They are important effectors of specific humoral immunity. This study was designed to measure the level of serum immunoglobulin (IgG, IgA, and IgM) in Nigerian sickle cell aneamia patients during BPC and steady state and to ascertain if it has any association on the severity of SCA when compared with other known prognostic factors such as leucocytosis. A total of 100 participants were recruited into the study: subject-50 SCA patients in BPC, 35 of the SCA patients were seen again in steady state four weeks after the initial BPC (served as Auto-control subjects). Control subjects-50 apparently healthy HbA individuals. The participants were included in the study after an informed consent was obtained. The mean serum IgG level during BPC 19.96g/l compared with 15.92g/l in steady state was statistically significant (p = 0.001). The mean serum immunoglobulin (IgG and IgA) in SCA patients during BPC were higher (19.81g/l, 5.23g/l) respectively compared with mean serum levels of 11.28g/l and 3.4g/l respectively among HBA control subject (P< 0.001). The mean total WBC count was 12,097/cmm during BPC and 9.097 in steady state. The mean haematocrit (HCT) in SCA during BPC was 0.24 and 0.25 during steady state. The mean neutrophil count was 5.5 x 10
/l during BPC and 4.5 x 10
/l during steady state. There was no significant correlation between the mean serum immunoglobulin levels, total WBC count and neutrophil count. When separated into BPC severity groups, 55% of the patient with severe bone pain crisis (pain score 7-10) had a significant higher serum IgG level. Markedly raised levels of IgG may be predictive of bone pain crisis severity in SCA.
CHAPTER ONE
INTRODUCTION
Sickle cell disease (SCD) is a group of genetic disorders characterized by the presence of sickled erythrocytes in the blood, chronic haemolytic anaemia, recurrent vaso-occlusion of the microcirculation. The vaso-occlusion cause painful crisis, multiple organ damage and failure. SCD The principal genotypes of SCD include: Homozygous sickle cell disease – referred to as Sickle cell anaemia (HbSS) Compound heterozygous disease such as: o Sickle cell haemoglobin C disease – HbSC o Sickle cell β
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– thalassaemia – HbS/β
thalassaemia o Sickle cell β + - thalassaemia – HbS/β + thalassaemia Rare genotypes include: o Sickle cell haemoglobin D Punjab – HbS/D Punjab o Sickle cell haemoglobin O Arab – HbS O Arab o Sickle cell haemoglobin Lepore Boston – HbS/HbS Lepore Boston [1]
Homozygous sickle cell disease also known as Sickle Cell Anaemia (HbSS) results from the inheritance of the sickle cell gene from both parents. The carrier state or sickle cell trait results from the inheritance of sickle cell gene from one parent and a normal (HbA) gene from the other parent. The carrier state is a benign condition and is not classified as SCD [ 1 ]
HISTORY Sickle cell disease is an age long disease known to the people of Africa for hundreds of years. In 1874, the first written account was published by Africanus Horton in his book “The diseases of tropical climates and their treatment” [2]. The first description of sickle cell disease in Western literature was by a Chicago Physician, James Herrick, who noted in 1910 that a patient from West Indies had anaemia characterized by unusual red cells that were “Sickled shape” [3]. Subsequent study of the disease led to the discovery of the molecular genetics of SCD by Linus Pauling and colleagues in 1949 and Ingram in 1956 [4,5]. First reported cure following bone marrow transplantation in 1984 [6]. In 1995 following a multicentre study, hydroxurea became the first drug proven to prevent complications of SCD [7].
EPIDEMIOLOGY OF SICKLE CELL DISEASE Sickle cell disease has a world wide distribution. The sickle cell gene has a high prevalence in Equatorial Africa but also widespread in parts of Sicily, Southern Italy, Northern Greece, Southern Turkey, the Middle East, Saudi Arabia, especially the Eastern province and Central India [8,9]. The sickle cell gene is most common in areas where malaria is endemic [10]. Individuals with sickle cell trait (HbAS) are somehow more resistant to lethal effect of malaria than people with normal haemoglobin genes (HbAA) [11]. The widely accepted theory is that, sickle haemoglobin containing erythrocytes (HbS) offers selective protection against plasmodium falciparum by inhibiting proliferation of plasmodium falciparum, sickling of the parasitized erythrocyte and removal within the reticulo-endothelial system.
Thus people with sickle cell trait (HbAS) would have a better chance of surviving malaria attacks and passing the genes (sickle cell and normal haemoglobin gene) to the next generation. This phenomenon of survival advantage by individuals with HbAS is called balanced polymorphism and has ensured the selection and spread of sickle gene through generations. SCD is a major public health problem on a global scale and the commonest blood disorder that affect mankind [1,12]. The magnitude of the problem is pronounced in Nigeria with a population of about 140 million and annual growth rate of 3.2%, about 25% of adult Nigerians have sickle cell trait (HbAS), 2 – 3% have sickle cell anaemia (HbSS) [13]. WHO estimates the prevalence of sickle cell anaemia to be about 20 per 1000 live births annually which translates to about 150,000 children born annually with sickle cell anaemia in Nigeria, making the country the highest with sickle cell anaemia burden in the world [14].
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MOLECULAR GENETICS, PATHOPH YSIOLOGY OF SCD Sickle haemoglobin is a mutant haemoglobin arising from a point mutation substituting Thymine for Adenine in the sixth codon of the beta globin-chain at the DNA level. This causes coding for valine instead of glutamic acid in the 6 th position of the Hb beta-chain [4,5]. The resulting haemoglobin becomes polymerized and poorly soluble when the oxygen tension is lowered and the red cells containing this haemoglobin becomes distorted and rigid [15]. Homozygous SCD has a wide spectrum of clinical manifestations, the clinical manifestation which is protean, and is modulated by multiple genetic, environmental, cellular and humoral factors acting in concert. Factors such as different haemoglobin haplotypes, level of HbF, alpha-thalassaemia, leucocytosis, haemoglobin level and gender have been identified as determinants of disease severity [16,17,18,19].
The common and most distressing manifestation of SCD is the episodic and unpredictable nature of the vaso-oclusive crisis (VOC). VOC is a hallmark of the clinical manifestation of SCD [20]. The crisis occurs unpredictably over a life-time and accounts for the most common cause of emergency room visits and hospitalization. The most common acute VOC is the bone pain crisis (BPC). BPC is defined as painful episodes involving one or more sites of bones in the absence of preceding trauma or obvious infection. The commonest sites are the long bones of the limbs, the lumbar spine, and thoracic cage. The hands and the feet are not often affected after childhood. The psychological and social life of these individuals may be severely affected when the disease degenerates to a chronic debilitating condition. Earlier studies suggest the pathophysiology of SCD to be due to physical plugging of the blood vessels by poorly deformable and abnormally adherent sickle erythrocytes [21,22]. Recent findings suggest the involvement of three fundamental pathological processes which include: sickling of the erythrocytes, vaso-oclusion and susceptibility to infection [23,24,25]. The current model views the pathological process of vaso-occlusion as a result of interaction of erythrocytes, leucocytes, platelets, plasma proteins and vascular endothelium [25]. The pathogenesis is characterized by ischaemia-reperfusion injury, infarction and inflammation [16,26,27]. Malaria, bacterial and viral infection, are the most common precipitating factors of VOC and leading cause of mortality in SCD [28]. SCD is associated with increased susceptibility to infection because of a defect in the alternative pathway of complement activation and opsonization [21,23,29], reduced ability of neutrophils to kill pathogenic organisms [21,30,31], and functional hyposplenism further decreasing macrophage function and immune response to organisms in the blood [21,32].
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Autosplenectomy results from repeated infarction of splenic tissue leading to absence of the spleen in most adult. Chronic haemolysis also increases susceptibility to Salmonella and other organisms possibly by making iron available for bacterial metabolism [21]. Post infarctive tissue necrosis provides a good medium for the thriving of bacteria leading to complications like osteomyelitis [21]. Humoral immunity is unimpaired in SCD and immunoglobulin levels tend to be high in response to frequent infection [21,33,34]. Infection precipitates VOC by increasing the interaction of leucocytes with vascular endothelium. Acute inflammatory reaction to infection or tissue injury increases the local and circulating levels of tissue necrosis factor alpha (TNF-α) and interleukin-Iβ (IL-Iβ) which activates leucocytes to express more adhesion molecules (α2Mβ2 & L-selectin) [35], and vascular endothelial cells express more ligands (Intercellular adhesion molecules – I-ICAM-I, Vascular adhesion molecules –VCAM-I) for the adhesion molecules on erythrocytes and leucocytes. This increases aggregation of blood cells to each other and their adhesion to the vascular endothelium leading to vaso-oclusion in the post capillary venule. Vaso-oclusion can occur in various parts of the body leading to ischaemic-reperfusion injury, infarction, inflammation, generalized painful crisis and organ damage, thus making SCD a multi organ disease. There is evidence that patients with SCD have a persistent state of low grade inflammation [26] as evidenced by elevations of markers of inflammation such as TNF-α, IL-1β, C-reactive protein during steady state and a significantly higher level during painful VOC [26,33,36,37,38]. Acute phase response occurs as a result of release of cytokines (IL-Iβ, TNF-α) from monocytes and macrophages at the site of tissue injury. Acute phase reactants:
C-reactive protein, serum amyloid A are elevated during steady state and higher during VOC [24]. Cytokines (IL-Iβ, TNF-α) and cell adhesion molecules – VCAM-I, Selectins fibrinogen, Vaso-active substances, platelet activating factor (PAF) and endothelin-1 (ET-I) are also elevated [36, 37,39]. Immunoreactive substances (IgG, IgA and IgM and HLA class 1 heterodimer) have been observed to be elevated in steady state and during VOC [33,34,40]. The abnormal levels of these acute phase reactants, cytokines and other mediators of inflammation probably play a role in the development of acute complications such as acute chest syndrome and chronic organ damage that occur in SCA.
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It is almost a century now since SCD was described by James Hierrick [3] and almost half a century since the molecular genetics of the disease was discovered by Pauling Itano and Ingram [4,5], yet our knowledge of the pathophysiology of SCD is not fully clear. The multiple complex ways in which the mutation produces painful VOC and chronic organ damage in different parts of the body is still being investigated. Several markers of clinical severity of the disease such as HbF, α-thalassaemia, Hb level, leucocytosis have been documented [16,17,18,19]. Therapeutic medical advances have been made: Hydroxyurea therapy with proven efficacy to reduce the frequency of painful crisis in selected individuals [7]. Further suggestion has been made for targeted therapy for the reduction of candidate adhesion molecules like P-Selectin and L-Selectin [35]. The goal is to improve the quality of life and survival of patients with SCD.
Considering the episodes and unpredictable nature of VOC, efforts are being made to identify the prodromal phase of sickle cell crisis by measuring the levels of
various acute phase reactants, immunoreactive substances and other markers of inflammation and probably use the markers as predictors of clinical severity of the disease. Immunoglobulins are glycoprotein molecules that are produced by plasma cells in response to an immunogen and function as antibodies [33]. They are important effectors of specific humoral immunity. Elevation of one or more of the different sub-classes of immunoglobulin have been reported in SCD [33,34,40]. This is attributed to the reticuloendothelial stimulation from haemopoeitic stress and recurrent infection [33]. The objective of this study therefore is to measure the level of serum immunoglobulin (IgG, IgA, IgM) in SCA patients during steady state and bone pain crisis, ascertain if serum immunoglobulin levels have any association on the severity of sickle cell anaemia when compared with other known prognostic factors such as leucocytosis. The serum immunoglobulin levels may serve as a useful prognostic index or severity index, to identify high risk patients and guide timely and appropriate therapeutic intervention that will benefit the patient and improve longevity.
OBJECTIVE AND SUB-OBJECTIVES Broad Objective To determine serum immunoglobulin (IgG, IgA, IgM) levels in Nigerian patients with sickle cell anaemia during bone pain crises and in steady state with a view of assessing its prognostic value.
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SUB OBJECTIVE To measure the serum immunoglobulin levels in SCA patients and HbA individuals. To compare the serum immunoglobulin levels in sickle cell anaemia patients with that of HbA individuals (control subjects). To compare serum immunoglobulin levels in bone pain crisis and steady state in sickle cell anaemia patients. To correlate serum immunoglobulin levels with total white blood cell and neutrophil count in sickle cell anaemia patients during bone pain crisis and steady state.
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