European Journal of General Medicine Medical Investigations Society ISSN: 1304-3897 Vol. 6, Num. 3, 2009, pp. 170-174

European Journal of General Medicine, Vol. 6, No. 3, July-September, 2009, pp. 170-174

Article

Immuno-Haematological characteristics of Nigerian sickle cell disease patients in asymptomatic steady state

Salawu L¹ , Orimolade EA, Durosinmi MA²

¹ Department of Haematology & Blood Transfusion, Obafemi Awolowo University Teaching Hospitals Complex, Ile-Ife, Nigeria
² Department of Orthopaedics & Traumatology, Obafemi Awolowo Uni- versity Teaching Hospitals Complex, Ile-Ife, Nigeria;

Correspondence Address: Lateef Salawu, Senior Lecturer and Consultant, Haematologist, Department of Haematology and Blood Transfusion, Obafemi Awolowo University Teaching Hospitals Complex, Ile-Ife, Nigeria
lsalawu2002@yahoo.co.uk

Code Number: gm09037

Abstract

Keywords: Immuno-haematological parameters, sickle cell disease, asymptomatic steady state, Nigeria

Introduction

Sickle cell disease (SCD) is an inherited disorder of haemoglobin that results in chronic haemolysis. Affected individuals are prone to frequent and some-times severe infections. Several factors predisposing SCD individual to infections have been reported, and included abnormalities of opsonins and antibody pro-duction, defects in the alternate complement path-way, leukocyte functions, and cell-mediated immunity ^[1],[2]. The spleen which is uniquely positioned to bring circulating antigen into close contact with anti-gen presenting cells of the reticuloendothelial system in the spleen is also lost early in life as a result of fibrosis resulting from recurrent vaso-occlusive crisis leading to defective function of the organ.

Significantly low serum levels of complement compo-nents C3 and C4 have been reported in SCD patients by several investigators ^[3],[4],[5] which are responsible for the low opsonization and chemotactic functions and which explains the high susceptibility of these patients to infections. Chudwin et al. ^[6] in their study found increased alternative pathway activation in sera from patients with SCD, an indication of im-paired host defense in them. The increased activation has been attributed to factors such as dense irrevers-ibly sickle cells, membrane spicules, vesicles derived from such cells, or membrane phospholipids ^[7],[8]. Complement component C1 esterase inhibitor (C1-INH) is an inhibitory protein in the classical pathway of complement system through the inhibition of C1r and C1s. This protein (C1-INH) also interact with C3b to inhibit binding of factor B (C3 activator) to C3b to form C3bBb (alternate pathway C3 convertase) in the alternate pathway.

Immunoglobulins are serum proteins that help fight infections and also involved in the activation of the complement system. Several investigators have re-ported varying serum concentrations for these im-munoglobulins in steady state asymptomatic SCD pa-tients. Dieye et al. ^[4] and Natta and Outschoorn ^[9] reported high levels of IgA and IgG, but reduced and normal serum IgM levels in their separate studies, respectively. While Adeodu et al ^[10] reported signifi-cantly high serum IgA and IgM; Mohamed et al ^[11] did not find any significant difference in the complement and immunoglobulin levels in their SCD subjects. The objective of this study is to investigate some of the immuno-haematological features of Nigerian SCD pa-tients in asymptomatic steady state.

Material and Methods

Study Population

Sickle cell disease patients in steady state that pre-sented at the Haematology and Paediatric clinics of the Obafemi Awolowo University Teaching Hospitals Complex (OAUTHC), Ile-Ife, between November, 2003 and March, 2005 were recruited into the study. Apparently healthy age- and sex-matched non-sick-le cell disease patients were enrolled as controls. Each patient was assessed clinically to confirm steady state. Some demographic data (age, gender, weight and height) of both the subjects and controls were also documented.

Laboratory Studies

Blood samples were taken, in appropriate bottles, for blood counts (packed cell volume, white blood cells and differentials, platelets, erythrocyte sedi-mentation rate), serum immunoglobulins (IgG, IgM, and IgA) and complement proteins (C1 esterase in-hibitor and C3 activator). Haematological parameters were estimated within six hours of sample collection while serum immunoglobulins and complement pro-tein samples were stored at temperature of -20oC and estimated in batches.

The packed cell volume, white cells and platelet counts were done on automated counter (ADVIA-60 Bayer Corporation, New York, United States of America) and ESR was determined by Westergren's technique ^[12]. C3 activator, C1-INH, IgA, IgG and IgM were estimated by the single radial immuno-diffusion method of Salimonu et al ^[13], using monospecif-ic antisera (Dade Behring Marburg GmbH, Marburg, Germany).

Statistical Analysis

Data are presented as means and standard devia-tions (means ± SD). Student's t-test was used to test the significance of differences between mean values. Spearman's correlation coefficient and multiple re-gression analysis were computed where necessary. Probability (p) value greater than 0.05 was consid-ered insignificant. SPSS (SPSS inc., release 11.0.1; 15 November 2001) statistical software was used for all statistical analyses.

Results

Demographic Variables

A total of sixty subjects were investigated. Their clinico-pathologic characteristics are as documented on [Table - 1]. They are made up of thirty SCD subjects in steady state and 30 age- and sex-matched appar-ently healthy control subjects. Of the thirty SCD sub-jects, 12 were males while the rest 18 were females, with a male to female ratio of 1: 1.5. The thirty age- and sex-matched non-SCD disease patients were made up of 14 males and 16 females, with a male to female ratio of approximately 1:1. In all, more females were investigated, with a male to female ra-tio of approximately 1: 1.03. This is not statistically significant. The mean age of the SCD group was 15.9 ± 7.5 years, while that of the control group was 13.6 ± 9.8 years. There was no significant difference in their mean ages, weights and heights.

Laboratory Characteristics

Expectedly, the haematocrit was significantly lower in SCD subjects (t= 12.34, p= 0.000) compared to the control subjects, while the white cells count was sig-nificantly higher in SCD subjects (t= 5.22, p= 0.000).However, unexpectedly the mean ESR value in the SCD patients was insignificantly higher compared with the control subjects, while the mean platelet count was slightly lower in the SCD subjects, though this is not significant. Serum levels of IgA and C3 activa-tor were significantly higher in SCD subjects (IgA: t= 2.47, p= 0.019; C3 activator: t= 2.79, p= 0.009). Serum C1 INH and IgM levels though higher in SCD subjects, were not significantly elevated, while serum IgG was slightly higher in the controls.

The ESR and PCV, as expected, showed significant negative correlation in both groups (S: r= -0.364, p= 0.048; C: r= -0.438, p= 0.016). In the SCD patients, a strong positive correlation was found between ESR and C3 activator (r= +0.449, p= 0.047), and between ESR and serum IgM levels (r= +0.531, p= 0.016). In the controls, the ESR showed significant positive cor-relation with white cells (r= +0.433, p= 0.017), while a significant negative correlation was also found be-tween the C3 activator and IgA (r= -0.643, p= 0.018).

Discussion

Sickle cell disease patients are prone to frequent and sometimes fatal infections as a result of some ab-normalities of the immune system, including those of the spleen, complement proteins, immunoglobulins, leukocyte functions and cellular immunity. This study was designed to investigate some aspects of im-muno-haematological characteristics of Nigerian SCD patients in steady asymptomatic state. Complement component C3 activator, also known as alternative pathway factor B, is a regulatory protein which com-bines with unstable C3b to form the more stable C3bBb that is capable of activating more C3 in the alternative pathway. Activation of the alternative pathway of complement is associated with a decrease in the serum level of C3 activator. In this study, sig-nificantly high serum levels of C3 activator was found in SCD patients compared to the controls. This is similar to the findings of Donadi et at. ^[14] in Brazil who reported elevated factor B and C3 complement component in asymptomatic SCD patients and those of Rao ^[15] that reported elevated C3b and fac-tor P, all indicating a defective alternative pathway of complement activation SCD patients. Complement component C1 esterase inhibitor (C1-INH) is an im-portant regulator of many plasma mediator pathways, including the complement system. Jiang et al. ^[16] in their study showed that C1-INH inhibits alterna-tive pathway activation by inhibiting the activities of factor B and the cleavage of C3 indirectly through C3b and that the removal of C1-INH restored remark-ably the activities of the pathway. Our study showed higher serum C1-INH in the SCD patients compared to the controls, which could also suggest a defec-tive alternative complement pathway in Nigerian SCD patients. Immunoglobulin A (IgA) was found to be sig-nificantly higher in our cohort of SCD patients similar to reports by other investigators ^{[5],[10],[14]}. High levels of IgA are associated with hepatic dysfunction, cholelithiasis, haemolysis and post-splenectomy state, all of which are features not uncommonly seen in SCD patients and which could not be ruled out as a cause of the high IgA seen our in patients.

Erythrocyte sedimentation rate (ESR) is an old and widely used laboratory indicator of inflammation. The rate of red cell sedimentation is dependent on several factors including plasma proteins such as fi-brinogen, alpha-2 macroglobulin and immunoglobulins ^[17]. In SCD, because of the abnormal red cell shape, red cell rouleaux formation is less thereby reducing erythrocyte sedimentation. The higher ESR found in our SCD subjects could probably result of anaemia and higher serum proteins including immunoglobulins and complement proteins, which can also influence red cell aggregation and which are found at higher concentrations in them. It is also noteworthy that ESR correlated positively with IgM but not IgG or IgA in this study. Immunoglobulin M is a pentamer with ten potential combining sites, thereby making it a better agglutinator of red cells to enhance sedimen-tation. In a study of correlates of inflammation in patients with rheumatoid arthritis, Verma et al. ^[18] similarly found IgM to correlate more with disease activity than IgG or IgA. The significant positive cor-relations found between ESR and C3 activator is a confirmation of it being classified as a positive acute phase protein ^[19]. In the control subjects, the posi-tive correlation between white cell count and ESR is a further confirmation that leukocytosis is one of the systemic responses to inflammation. It is of interest to note the strong negative correlation between IgA and C3 activator in the control subjects. Aggregated IgA is an activator of the alternative pathway, while the C3 activator is a regulator protein in the same pathway. Decreased values of C3 activator are found when the pathway is activated, and which probably explains the negative relationship between the two proteins.

High platelet count is the usual finding in most pa-tients with SCD in asymptomatic steady state, except in crisis situation such as vaso occlusive crisis ^[20]. The mean platelet count in our SCD patients was lower than the control, though the difference is not significant. Studies have shown that minor epi-sodes of microvascular occlusion do occur in the so called asymptomatic steady state ^[21] which though insufficient to cause the overt painful crisis, but may consume some platelets. This phenomenon could probably explain the lower platelet count in our SCD patients.

It could be concluded from this study that comple-ment dysfunction and abnormality of immunoglobulins are evident in Nigerian SCD patients and that in the so called asymptomatic steady state; inflammatory processes are ongoing as earlier reported and as shown by increase in some markers of inflammation assessed in these patients.

Acknowledgements: We thank our colleague in the department of Haematology for allowing recruiting some of their patients into the study.

References

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