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1 CD4+ T-LYMPHOCYTE, GAMMA-INTERFERON, INTERLEUKIN-4 AND ULTRASONOGRAPHIC SPLENIC SIZE IN HOMOZYGOUS SICKLE CELL ANAEMIA PATIENTS BY DR OMOTOLA TOYIN OJO M.B,B.S (IB) DEPARTMENT OF HAEMATOLOGY UNIVERSITY COLLEGE HOSPITAL, IBADAN. A DISSERTATION SUBMITTED TO THE NATIONAL POSTGRADUATE MEDICAL COLLEGE IN PART FULFILMENT OF THE REQUIREMENT FOR THE AWARD OF THE FELLOWSHIP OF THE NATIONAL POSTGRADUATE MEDICAL COLLEGE OF NIGERIA IN PATHOLOGY (FMCpath). MAY, 2011.
Transcript of CD4+ T-LYMPHOCYTE, GAMMA-INTERFERON, INTERLEUKIN-4 …
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CD4+ T-LYMPHOCYTE, GAMMA-INTERFERON, INTERLEUKIN-4
AND ULTRASONOGRAPHIC SPLENIC SIZE IN HOMOZYGOUS
SICKLE CELL ANAEMIA PATIENTS
BY
DR OMOTOLA TOYIN OJO
M.B,B.S (IB)
DEPARTMENT OF HAEMATOLOGY
UNIVERSITY COLLEGE HOSPITAL, IBADAN.
A DISSERTATION SUBMITTED TO THE NATIONAL
POSTGRADUATE MEDICAL COLLEGE IN PART FULFILMENT OF
THE REQUIREMENT FOR THE AWARD OF THE FELLOWSHIP OF
THE NATIONAL POSTGRADUATE MEDICAL COLLEGE OF
NIGERIA IN PATHOLOGY (FMCpath).
MAY, 2011.
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DECLARATION
I hereby certify that this Dissertation is the result of my own research and has not been
submitted to any other college.
______________________________________________
Omotola Toyin Ojo M.B,B.S (Ib)
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DEDICATION
To the wonderful men in my life Funso Awoyemi, Olayinka, Ninuolaoluwa and Eniifeoluwa
Ojo.
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ACKNOWLEDGEMENT
My profound gratitude goes to my meticulous and dedicated supervisor, Prof. W.A
Shokunbi, for her commitment in scrutinizing every aspect of this work.
My sincere gratitude to all my teachers Prof. Y. A Aken’Ova for her invaluable support and
motherly attention, Drs T.R Kotila, T. S Akingbola, F.A Fasola and J.A Olaniyi for their
commitment and eagerness to impart the knowledge.
I am greatly indebted to Prof.Olaleye, Dr Odaibo, Mr. Chukwuma and the entire staff of the
Department of Virology, University of Ibadan for allowing me the use of their laboratory.
My sincere gratitude to Dr G.O Arinola for his invaluable advice. Appreciation also to Drs
Lasisi, Agunloye and Dairo for their continual support.
To my brother- in-law Dr Otegbola Ojo my sincere appreciation and deep gratitude.
I appreciate all my senior colleagues: Drs A.O Akinpelu, A.A Ibijola and all the residents in
Haematology department: Drs Eteng, Ekanem, Fowodu, Olutogun, Yuguda, Oke, Busari and
Ogundeji for their contribution to the successful completion of this project. Other residents in
Laboratory Medicine, especially Drs Onwukamuche and Esan: thank you for your
encouragement.
To all staff of the department of Haematology and HDCU, especially Mr. Olomu, Mrs.
Fadimu, Matrons Akanbi and Kolade, may God bless you.
To my ever supporting and understanding husband and children, thank you. My loving
parents, Mr. and Mrs. Awoyemi, you are indeed a pillar of support for me. I appreciate my
siblings for their encouragement. My friends especially the Obimakindes, Afolaranmis and
Kemi Fagbami I say thank you for the support.
To God Almighty in whom all things are possible, I give all the honour and glory.
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CERTIFICATION
This is to certify that I have supervised OMOTOLA TOYIN OJO in the conduct of the study
entitled “CD4+ T-LYMPHOCYTE, GAMMA INTERFERON, INTERLEUKIN-4 AND
ULTRASONOGRAPHIC SPLENIC SIZE IN HOMOZYGOUS SICKLE CELL ANAEMIA
PATIENTS”
---------------------------------------------------------------------------------------------------------------
Prof. W.A Shokunbi
M.B;B.S, FMCPath (Nigeria), FWACP (Lab Med.)
Certificate in HIV prevention (Hokkaido),
Certificate in Molecular Cellular Biology of the Normal Immune Response (Ibadan, in
collaboration with Luxemberg)
Department of Haematology
University College Hospital
Ibadan.
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KEY TO ABBREVIATIONS
EDTA Ethylene diamine tetra acetate
ELISA Enzyme linked immunosorbent Assay
HbA Haemoglobin A
HbF Haemoglobin F
HbS Haemoglobin S
IFN-γ Interferon-gamma
IL Interleukin
SCA Sickle cell anaemia
SCD Sickle cell disease
TNF-β Tumour necrosis factor beta
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TABLE OF CONTENTS
Page
Title ...………………………………………………………………….......i
Declaration …………………………………………………………………......ii
Dedication………………………………………………………………………...iii
Acknowledgements …………………………………………………………….iv
Certification ………………………………………………………………...v
Key to abbreviations …………………………………………………………….vi
Table of contents ……………………………................................................vii
List of Table …………………………………………………………………..ix
List of figures ………………………………………………………………........xi
Abstract ………………………………………………………………………..xii
CHAPTER ONE ……………………………………..………………………..1
Introduction
CHAPTER TWO ……………………………………………………………….4
Literature review
CHAPTER THREE ………………………………………………………..19
Materials and Methods
3.1 Study site Description………………………………………………………..19
3.2 Study Population Size ……………………………………………………….19
3.3 Study Design ………………………………………………………….20
3.4 Sample size Estimation………………………………………………………20
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3.5 Sample Collection …………………………………………………………..21
3.6 Splenic Ultrasonography……………………………………………………22
3.7 Medical Record………………………………………………………………23
3.8 Data Management ………………………………………………………….23
3.9 Ethical Approval …………………………………………………………24
CHAPTER FOUR
Results ………………………………………………………………………….25
CHAPTER FIVE
Discussion ………………………………………………………..………..49
Conclusion ………………………………………………………...…………...54
Recommendations and Limitation of the study…………………………………55
REFERENCES ……………………………………………………………….56
APPENDICES
Consent form ………………………………………………………….............63
Questionnaire ……………………………………………………………...........65
Ethical approval by UI/ UCH Ethics committee ………………………...........69
Procedure for CD4 count…………………………………………………………70
Procedure for IFN-γ……………………………………………………………….73
Procedure for IL-4………………………………………………………………...77
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LIST OF TABLES
1-Socio-demographic characteristics of the HbS patients and the controls…........................26
2-Median comparison of the CD4 count, IFN-γ and IL-4 of the HbS patients and
Controls…………………………………… ………………………………………………..27
3- CD4 count of the HbS patients versus controls by age groups ………….....................30
4- IFN-γ profile of the HbS patients versus controls by age groups……………………….33
5- IL-4 profile of the HbS patients versus controls by age groups……………………….36
6-Spleen size distribution in HbS patients and the controls……………………………….37
7- Pattern of infectious complications in HbS patients…………………………………….38
8- Median comparison of CD4 count in relation to spleen size in HbS patients versus
Controls…………………………………………………………………………………….40
9- Median comparison of IFN-γ in relation to spleen size in HbS patients versus
Controls………………………………………………………………………………………42
10- Median comparison of IL-4 in relation to spleen size in HbS patients versus
Controls………………………………………………………………………………………44
11- Correlation coefficients of the relationship between spleen size and CD4, IFN-γ and IL-4
………….……………………………………………………………………………………45
12- Median comparison of CD4 count in SCA patients with and without previous pneumonia
………………………………………………………………………………………………..46
13-Median comparison of IFN-γ in SCA patients with and without previous pneumonia
……………………………………………………………………………………………….47
14- Median comparison of IL-4 in SCA patients with and without previous pneumonia
……………………………………………………………………………………………….48
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LIST OF FIGURES
1- Abdominal ultrasonography showing spleen measurement………………………...........22
2-CD4 count of the HbS patients versus controls by gender………………………………..28
3- IFN-γ level of the HbS patients versus controls by gender…………………………........31
4-IL-4 level of the HbS patients versus controls by gender………. …………….............34
5-Correlation between Spleen size and CD4 count in HbS patients………….......................39
6-correlation between Spleen size and IFN-γ in HbS patients……………………………...41
7-Correlation between Spleen size and IL-4 in HbS patients ………………………………43
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ABSTRACT
BACKGROUND
Sickle cell anaemia is an autosomal inherited disorder of haemoglobin resulting from the
homozygous inheritance of the sickle gene. Reports have shown that patients with sickle cell
anaemia (HbSS) have an increased susceptibility to infection leading to increased morbidity
and mortality. Autosplenectomy and the consequent absence of splenic function may explain
the propensity to pneumococcal infection. Impaired leucocytes function and loss of both
humoral and cell mediated immunity are some of the other mechanisms that have been
reported to account for the immunocompromised state in patient with sickle cell disease.
OBJECTIVE:
In order to predict the susceptibility of HbS patients to infection, this study assessed cellular
immunity using CD4+ T lymphocyte count, serum IFN-γ and IL-4 levels in patients with
sickle cell anaemia. These levels were correlated with spleen sizes.
METHOD
The study was carried out at the University College Hospital, Ibadan, Nigeria after obtaining
Ethics committee approval. The study population comprised of 40 sickle cell anaemia
patients in steady state (asymptomatic for at least 4 weeks) compared with 40 age and sex-
matched healthy HbA control. Blood specimen was analyzed for CD4+ T cell by Flow
cytometry and the serum was analyzed for IFN-γ and IL-4 by Enzyme linked immunosorbent
assay (ELISA).
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RESULTS
There was a significant increase in the IFN-γ level in sickle cell anaemia patients in steady
state (median value 8.61pg/ml) compared with HbA controls (median value 5.58pg/ml)
(p=0.047).
There was no significant difference in the median values of CD4+ T cell count and IL-4
level between the HbS patients and the control subjects (CD4 count: p=0.704, IL-4:
p=0.418).
Correlation coefficient between spleen size with CD4 count (rho=-0.150, p=0.356), IFN-γ
(rho=-0.023, p=0.887) and IL-4 (rho=-0.179, p=0.269) showed no significant relationship.
Malaria (37.5%) was the most common infection in the patients followed by pneumonia
(15%).
CONCLUSION
This study showed that the median IFN-γ level in HbS patients was significantly higher than
in HbA individuals. This higher value of IFN- γ may contribute to inflammation and tissue
damage in HbS patients, thus worsening morbidity and mortality. It is hereby suggested that
IFN-γ may help determine clinical severity, similar to the usefulness of HbF level in
assessing severity of sickle cell disease.
CHAPTER ONE
INTRODUCTION
Sickle cell anaemia is an autosomal inherited disorder of haemoglobin resulting from the
homozygous inheritance of the sickle gene1,2. Sickle cell haemoglobin (HbS) is the
commonest abnormal haemoglobin and it has a worldwide distribution1. It has variable
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clinical expression some of which include recurrent haemolysis, vasoocclusive crises and
recurrent infections with their attendant sequelae. Few, however, run a mild course and are
discovered only by chance on routine haematological examination for other conditions2.
Vaso-occlusive crisis is the most common sickle cell crises and is the hallmark of the
disease2. Vaso-occlusive crisis presents as an acute onset episode of pain which is often
severe and can affect any tissue in the body3. Repeated infarction of the spleen leads to
fibrosis, calcification and functional autosplenectomy4. The role of spleen in host immune
defense is thereby impaired.
Reports have shown that patients with sickle cell anaemia (HbSS), particularly children, have
an increased susceptibility to infection leading to increased mortality4,5,6. Autosplenectomy
and the consequent absence of spleen function may explain the propensity to bacteraemic
pneumococcal illness, Haemophilus influenzae B, and Salmonella in this population7.
Opsonophagocytic defect due to an abnormality of the alternative complement pathway,
deficiency of specific circulating antibodies, impaired leucocytes function and loss of both
humoral and cell mediated immunity are some of the other mechanisms that have been
reported to account for immunocompromised state in patients with sickle cell disease. It has
been demonstrated in recent years that the risks for splenectomized patients to life-
threatening infections, especially pneumococcal meningitis, are the same for sickle-cell
disease with functional asplenia8.
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Thymus-derived (T) lymphocytes play an important role in cellular immunity. In the blood, T
lymphocytes constitute 60-70% of peripheral lymphocytes9. Also found in the paracortical
areas of lymph nodes and periarteriolar sheath of the spleen. About 60% of mature T cells
express CD4 (Helper) and 30% express CD8 (Cytotoxic). By secreting cytokines CD4+ T
lymphocyte influence the functions of virtually all other cells of the immune system,
including other T cells, B cells, macrophages, and natural killer cells10,11. Two functionally
distinct subsets of Helper T cells secrete cytokines which promote the following activities:
TH1 subset of CD4+ cells produce IL-2, IFN-γ and TNF-β with IFN- γ being the prototype,
which activate T cell and macrophages to stimulate cellular immunity and inflammation. TH1
cells also secrete IL-3 and GM-CSF to stimulate bone marrow to produce more leukocytes.
TH2 subset of CD4+ cells secretes IL-4, IL-5, IL-6 and IL-10 with IL-4 being the prototype,
which stimulate antibody production by B cells. The balance between TH1 and TH2 activity
may steer the immune response in the direction of cell-mediated or humoral immunity.
The role of lymphocytes in the immunocompromised state in HbS patients has not been fully
defined. Low numbers of antibody-producing cells in these patients have been described, but
results of helper T-lymphocyte measurements and the cytokines produced by its subsets have
been less consistent, with conflicting reports on the level of these populations5,12. There is
paucity of study on cellular-defence system in patients with HbS especially in this region,
hence the need for this study.
The goal of this study is to correlate the level of CD4+ T lymphocytes, Gamma-Interferon
(IFN-γ) and Interleukin-4 (IL-4) in patients with sickle cell in relation to their
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ultrasonographic splenic size. The information may be of prognostic value in the course of
sickle cell anaemia.
BROAD OBJECTIVE:
In order to predict the susceptibility of HbS patients to infection. This study assessed the
cellular immunity using CD4+ T lymphocytes count, serum INF-γ and IL-4 levels in patients
with sickle cell anaemia. These values were correlated with spleen sizes.
SUB-OBJECTIVES:
1. Determination of the levels of CD4 T lymphocytes, INF-γ and IL-4 in patients with sickle
cell anaemia.
2. Determination of the relationship between spleen size and CD4 T lymphocytes, IFN-γ and
IL-4 in patients with sickle cell anaemia.
3. Determination of the prevalence of infectious complications in patients with sickle cell
anaemia.
CHAPTER TWO
LITERATURE REVIEW
2.1 History
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Sickle cell disease (SCD) encompasses disorders in which the pathology can be attributed to
the inheritance of the HbS gene either in the homozygous state (HbSS) or as compound
heterozygous. Therefore, SCD include the homozygous sickle cell anaemia (βs/ βs), other
compound heterozygotes such as haemoglobin SC(βs/βc), haemoglobin S beta plus
thalassaemia(βs/β+thal), haemogobin S beta zero thalassaemia(βs/β0thal), the HbS gene with
other less common variants such as SD Punjab, SO Arab, S Lepore, SE, SHPFH and α-
thalassaemia .
Sickle cell anaemia is an inherited disorder of haemoglobin resulting from the inheritance of
two sickle genes1. It is one of the most common haemoglobinopathies. The symptoms related
to sickle cell crises were known in Africa, long before they were recognized in the western
hemisphere. Symptoms of sickle cell anaemia could be traced back to 1670 in one Ghanaian
family13. The clinical features were unknown until 1904 when a Chicago cardiologist, whose
intern Ernest Edward Irons found “peculiar elongated and sickle-shaped” cells in the blood of
an anaemic Grenadian dental student in the USA14. In Nigeria, age-old concepts of Ogbanje
(Ibo) and Abiku (Yoruba) provide a traditional explanation for excess mortality in children in
some families, presumably caused by sickle cell disease (SCD)1.
2.2 Epidemiology
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Sickle cell disease has a worldwide distribution. The sickle cell gene is known to be
widespread occurring in Equatorial Africa, Southern Italy, Northern Greece, Southern
Turkey, Sicily, the Middle East, the Eastern province of Saudi Arabia and India15,16.
The incidence of sickle cell disease is highest in Equatorial Africa, where frequency of
heterozygous inheritance is 20-40%16,17. The high incidence of SCD in Africa has been
linked to the similar geographical distribution shared by HbS gene with falciparum malaria
endemicity18. Individual with sickle cell trait (HbAS) have a selective advantage in region
with Plasmodium falciparum endemicity15,17. HbAS is associated with protection against
severe malarial anaemia, high density parasitemia, and cerebral malaria18,19. In Nigeria, the
incidence of the sickle cell trait (HbAS) is 30% in the Northern parts where malaria is
hyperendemic; and 24% in Southern region where malaria is holoendemic16,17. The
prevalence of sickle cell anaemia among Nigerian infants ranges from 2-3%15.
2.3 Pathophysiology
The clinical manifestations arise from the tendency of the Haemoglobin (HbS or sickle
haemoglobin) to polymerize and deform red blood cells into the characteristic sickle shape.
This property is due to a single nucleotide change in the β-globin gene leading to substitution
of valine for glutamic acid at position 6 from the N-terminal of the β-globin chain
(β6glu→val or βs). The homozygous state (HbSS or sickle cell anaemia) is the most common
form of sickle cell disease, but interaction of HbS with thalassaemia and certain haemoglobin
variants (HbC, HbD e.t.c.) also leads to sickling. The term sickle cell disease (SCD) is used
to denote all syndromes in which the pathology can be traced to sickling phenomenon.
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As red cell traverses the microcirculation, oxygen is released from oxyhaemoglobin
generating deoxyhaemoglobin. Deoxygenation of HbS leads to a conformational change that
exposes a hydrophobic patch on the surface of the βs-globin chain at the site of β6 valine.
Binding of this site to a complementary hydrophobic site on a β-subunit of another
haemoglobin S tetramer triggers the formation of large polymers. The polymers consist of
staggered haemoglobin tetramers that aggregate into 21-nm-diameter helical fibers, with one
inner and six peripheral double strands. The polymerization proceeds after a delay, the length
of which is extremely sensitive to the intracellular deoxyHbS concentration9. The rate and
extent of polymerization depend primarily on three independent variables: the cell’s degree
of deoxygenation, the intracellular haemoglobin concentration and the presence of HbF14,20.
The HbS polymers thus formed lead to cellular injury, distortion of the shape of red cells and
marked decrease in deformability with consequent increase in blood viscosity and vaso-
occlusion. Repeated or prolonged sickling progressively damages the red cell membrane and
this leads to increased mechanical fragility and premature destruction of the sickle cells by
the reticuloendothelial organs; which is a phenomenon of primary importance in the
pathophysiology of SCD. Membrane damage causes movement of potassium ions and water
out of the cell by the Gardos pathway and potassium–chloride co-transport, leading to
dehydration of red cells. The intracellular haemoglobin concentration rises (producing dense
cells), which shortens the gelation delay time (GDT) to sickle polymer formation1,2,9. Vaso-
occlusion is initiated and sustained by interactions among sickle cells, endothelial cells and
plasma constituent. Adhesive interaction between sickle cell and endothelial cells occurs as a
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result of injury to cell membrane causing the red cells to become abnormally adherent to the
vascular endothelium through vascular cell adhesion molecule 1 (VCAM-1), thrombospondin
and fibronectin9,15,.
Granulocytes interact with sickle cells and endothelial cells and are stimulated to release
injurious cytokines which mediate inflammation.
Reticulocytes which are prematurely released from bone marrow in haemolytic disease
display additional adhesive ligands that facilitate interactions between sickle cells and
endothelial cells4,9. These ligands are integrins which bind both fibronectin and VCAM-1.
VCAM-1 is expressed on the surface of endothelial cells after activation by inflammatory
cytokines e.g. Tumor necrosis factor20.
2.4 Clinical manifestation
The clinical features seen in sickle cell anaemia result mainly from vaso-occlusion and
chronic haemolytic process. The episodic acute severe pain resulting from vaso-occlusion is
the most common feature of sickle cell anaemia. The obstruction of blood vessels by the rigid
sickled erythrocytes may lead to ischaemia and necrosis of tissues supplied by those vessels.
Clinical symptoms vary tremendously between patients with SCD for several reasons. The
disease is more severe in patients with HbSS or HbS-β0-thalassaemia than in those with HbS-
Β+-thalassaemia or HbSC disease. The Arab–Indian haplotype produces a less severe disease
than the African haplotypes. The co-inheritance of one or two α-gene deletions also modifies
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the clinical picture12. The high HbF level observed in hereditary persistence of fetal
haemoglobin (HPFH) is associated with very mild disease. However, for poorly recognized
reasons, the disease severity varies enormously even within the subgroup of patients with
HbSS. In countries with inadequate healthcare, SCD is associated with high mortality in the
first 3 years of life as a result of sepsis and splenic sequestration. In the developed world, the
typical patient with SCD has moderately severe anaemia, leads a relatively normal life
interrupted by ‘crises’ as a result of vasoocclusion9. The four major crises types include vaso-
occlusive, hyperhaemolytic, sequestration and aplastic crises21.
Vaso-occlusive crisis (VOC):
VOC is responsible for most of the severe complications of SCA and can occur throughout
the microvasculature3. It is the most common sickle cell crisis and in fact, the hallmark of the
disease2. Vaso-occlusive crisis presents as an acute episode of pain which is often severe and
can affect any tissue in the body3. Most common sites are the bones (back and extremities),
chest and abdomen2,3,22. The frequency of the crisis varies from almost daily to less than one
a year and can last for days or even weeks2,3. Multiple areas are often involved
simultaneously and symmetrical involvement of the extremities is common. In early
childhood, one of the manifestations of sickle cell anaemia is dactylitis23. Nearly one-half of
children with SCA suffer from this painful disorder2. Dactylitis occurs almost entirely in first
four years of life with peak incidence at about one year of age1,2.
The acute chest pain syndrome is another complication of Vaso-occlusive crisis in SCA. It is
frequent, affecting about 40% of all people with SCA and may be fatal3. The occurrence in
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children is less3. Repeated infarctions of the spleen lead to fibrosis, calcifications, and
autosplenectomy. Thus, the spleen is palpable in less than 10%-20% of adults1,2.
Sequestration crisis:
This occurs particularly in infants and young children although it can occur in adults with
splenomegaly2. It is characterized by sudden massive pooling of red cells especially in the
spleen. A major acute sequestration crisis is considered to be one in which the haemoglobin
(Hb) level is less than 6g/dl and has fallen more than 3g/dl when compared with the baseline
value. Minor acute sequestration crisis is one in which the haemoglobin level is higher than
6g/dl2. In a study of children with SCD born in Los Angeles in the 1960s and 1970s, such
crisis was responsible for10-15% deaths in the first 10 years of live2.
Aplastic Crisis:
This is usually a single lineage aplasia affecting the erythroid cell line. Depression of
erythropoiesis is generally associated with infections. The B19 strain of Parvovirus appears
to be by far the most important cause of this type of crisis2. Single lineage erythroid aplasia is
the commonest presentation but bilineage depression may occur and rarely trilineage
aplasia2.
Haemolytic Crisis:
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The red cell life span is shortened and an increased rate of haemolysis above the steady state
is termed haemolytic crisis2. It is characterized by an increasing jaundice associated with a
falling Hb and an elevated reticulocyte count.
2.5.1 THE SPLEEN IN SICKLE CELL ANAEMIA
The spleen is one of the organs most often affected in sickle cell anaemia and this has been
linked to its prominent function as a reticuloendothelial organ. An overview of normal
splenic anatomy and function will be outlined in order to appreciate the splenic changes in
sickle cell anaemia.
i)Anatomy of normal spleen
The spleen is located in the left hypochondrium between the fundus of the stomach and the
diaphragm. It has a normal weight of 100-250 g and it measures 12cm in length, 7cm in
width, and 3cm in thickness. It is enclosed within a thin, glistening connective tissue capsule
that appears slaty gray through which the dusky red, friable parenchyma of the splenic
substance can be seen10. Blood vessels enter and leave the spleen through the hilus which is
on its medial surface. The splenic pulp has reticular cells and fibers which form filtration bed
for the blood. Essentially, it consists of a connective tissue framework, vascular channels,
lymphatic tissue, lymph drainage channels and cellular components of the haemopoietic and
reticuloendothelial systems10.
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Histologically, the white pulp is composed of periarteriolar lymphatic sheath and lymphatic
nodules. The periarteriolar lymphatic sheath contains primarily T- lymphocytes while the
lymphatic nodules contain B-lymphocytes. The white pulp is the main site of splenic
immunologic activity. It contains 25% of the T-lymphocyte pool and 10-15% of the B-
lymphocyte pool9. The red pulp is traversed by numerous thin walled vascular sinusoid
separated by splenic cords. The endothelial lining of the sinusoid is discontinuous providing
passage of blood cells between sinusoid and cords, and the red cell must be extremely pliable
to pass between the slits. The splenic cords are sponge-like, consisting of macrophages which
are loosely connected through dendritic processes; hence create both physical and functional
filtration beds.
The vascular system is made up of the splenic artery and its branches of trabecular arteries,
which extend from the capsule and terminates in the red pulp as arteriolar capillaries. Blood
traversing the red pulp takes two routes to reach the splenic veins24. Some of the capillary
flow is into splenic cords and then into surrounding splenic sinusoid before reaching the vein,
this is called the open circulation. The other blood channel is by direct passage of blood from
the capillaries to the veins and this is called the closed circuit system.
ii) Functions of the spleen
(a) Splenic clearance
The major function of the spleen is the filtration of undesirable particles 25(e.g. defective red
cells, microorganism, cellular debris) from the blood and destruction by phagocytosis in the
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splenic cords (i.e. culling). The spleen also has ability to excise particles from intact cell
without destroying it (i.e. pitting), thus cellular inclusion bodies such as Heinz bodies,
Howell Jolly bodies, intracellular organisms are removed from the red cells by this process.
(b) Defence against infection
The spleen is a major secondary organ in the immune system. The reticular network in the
periarterial lymphatic sheaths traps antigen, permitting it to come in contact with effector
lymphocytes. There is a constant flow of both T and B cells through the spleen; T cells are
the more mobile cells and stay in the spleen for a few hours, while B cells settle in the
follicles where they release immunoglobulins9.
(c) The spleen is a source of lymphoreticular cells and sometimes haematopoietic cells.
Splenic haemopoeisis normally caeses before birth, but in some haematological disorders
such as myeloproliferative disorders e.g. myelofibrosis; chronic anaemia extramedullary
splenic haemopoesis may be reactivated.
(d) Splenic reservoir
The spleen acts as reservoir and storage site for red cells, leucocytes and platelets because of
its rich vascularisation and phagocytic function. In humans, the normal spleen harbours 30-
40mls of erythrocytes and approximately 30-40% of the total body platelets, but with
splenomegaly this reservoir is greatly increased. The enlarged spleen may also trap sufficient
amount of white cells to induce leucopenia10.
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2.5.2 THE SPLEEN IN SICKLE CELL ANAEMIA
The spleen in the sickle cell anaemia patient is usually normal in size and function at birth.
However, the spleen in the HbS patient undergoes a sequence of changes with age. The
progressive replacement of foetal haemoglobin (HbF) with Haemoglobin S (HbS) heralds the
onset of haemolysis (usually from age 2 months) with resultant increase in splenic activity
and size26,27. The spleen becomes palpably enlarged by the fifth month of life and in some
children as early as one month of age. In a representative sample of patients followed from
birth, splenomegaly was observed in 37% by age 6 months, 65% by age 12 months and 77%
by age 24 months27. The development of splenomegaly is mainly due to the complex
structure of the spleen which encourages stasis, anoxia and entrapment of sickled
erythrocytes within the splenic pulp and the sinuses. The erythro-stasis leads to marked
congestion of the red pulp and sequestration of blood28. The spleen begins to regress in size
following recurrent vaso-occlusion and multiple infarctions and subsequently becomes a
siderotic mass at which time it is no longer palpable29. This process is termed
Autosplenectomy and it occurs between the ages of 6-8 years9,29. Nevertheless splenomegaly
persists in some patients up to adolescence and even adulthood9,27,28. The persistence of
spleen has been attributed to high levels of HbF, and presence of homozygous alpha
thalassaemia, which is associated with less intravascular sickling, and therefore a more
normal perfusion of the splenic vascular bed29. Persistence of splenomegaly has also been
linked to malaria endemicity in which there is hyperplasia of the reticuloendothelial cells of
the spleen from the chronic antigenic stimulation by the malaria parasite,30,31,32. In children
splenic function is impaired i.e. functional hyposplenia33, even though splenomegaly is
26
present. Extensive erythrophagocytosis causing blockade of the mononuclear phagocyte
system has been postulated as a reason for the impaired splenic function10.
2.5.3 CLINICAL CONSEQUENCES OF SPLENIC CHANGES IN
SICKLE CELL DISEASE
Some of the complications associated with the splenic changes include acute splenic
sequestration, hypersplenism and increased susceptibility to infection.
I ) Acute Splenic Sequestration
It is characterized by acute splenic enlargement, increased intrasplenic pooling of red cells
and rapid decline in haemoglobin level. There is usually a hypercellular marrow and
reticulocytosis. It may result into death from peripheral circulatory failure and it is an
important cause of mortality in children29.
ii) Hypersplenism
It is characterized by chronically enlarged spleen, excessive destruction of red cells, platelets
and white cells. There is reticulocytosis with bone marrow hyperplasia.
27
iii) Infection
It is well established that sickle cell patient are susceptible to infection and this has been
associated with loss of reticuloendothelial function of the spleen29. The onset of
splenomegaly at the age of 5 months in sickle cell anaemia is associated with a decline in
immune function of the spleen29,34. This disparity between splenic size and splenic function is
termed functional hyposplenia, a concept originated by Pearson et al (1979)33.
Studies have shown that the leading cause of death especially in Africa is infection. In a
study by Ikemefuna and Emodi35, infections were the commonest cause of admission
(69.6%) in children with SCA. Patients with sickle cell anaemia (SCA), particularly children,
have an increased susceptibility to infection which leads to mortality due to the disease36.
Overwhelming infections caused by encapsulated bacteria (such as salmonella spp.) and
Plasmodium falciparum are an important cause of morbidity and death in patients with
sickle cell disease37,38. Bacterial infections afflicting these patients include fulminant
meningitis and septicaemia caused by Streptoccocus pneumoniae and H. influenzae type b,
and non-typhoid salmonellosis.
Children less than five years of age are at greatest risk for meningitis and septicaemia, while
Salmonella osteomyelitis is probably common to all age groups39,40,41. The most important
contributing factors to this increased susceptibility to encapsulated bacteria are: a state of
functional asplenia, an opsonophagocytic defect due to an abnormality of the alternative
complement pathway, a deficiency of specific circulating antibodies, impaired leukocyte
function and loss of both humoral and cell mediated immunity.
28
Devitalisation of gut and bone due to repetitive vaso-occlusive crises, saturation of the
macrophage system with red cell breakdown products of chronic haemolysis, and underlying
splenic and hepatic dysfunction all predispose to salmonella infections. Seventy per cent of
septicaemias and meningitis among under-fives with sickle cell disease is caused by
Streptoccocus Pneumoniae39. It has been demonstrated in recent years that the risks for
splenectomized patients of life-threatening infections, especially pneumococcal meningitis,
are the same as in sickle-cell disease with functional asplenia36,37. The role of lymphocytes in
the immunocompromised state in SCA has not been fully defined. Low numbers of antibody-
producing cells in patients with SCA have been described, but results of T-lymphocyte subset
measurements have been less consistent, with conflicting reports on the level of T-
lymphocytes subets8,12 .
2.5.4 Importance of T –lymphocyte
Thymus-derived (T) lymphocytes play an important role in cellular immunity. In the blood, T
lymphocytes constitute 60-70% of peripheral lymphocytes. Also found in the paracortical
areas of lymph nodes and periarteriolar sheath of the spleen. About 60% of mature T cells
express CD4 (Helper) and 30% express CD8 (Cytotoxic). By secreting cytokines CD4+ T
lymphocyte influence the function of virtually all other cells of the immune system, including
other T cells, B cells, macrophages, and natural killer cells10,41,42.
Helper T cells have 2 important functions:
(i) To stimulate cellular immunity and inflammation
(ii) To stimulate B cells to produce antibody
29
Two functionally distinct subsets of T cells secrete cytokines which promote the following
activities:
TH1 subset of CD4+ cells produce IL-2, IFN-γ and TNF-β, which activate T cell and
macrophages to stimulate cellular immunity and inflammation. TH1 cells also secrete IL-3
and GM-CSF to stimulate bone marrow to produce more leukocytes. TH2 subset of CD4+
cells secretes IL-4, IL-5, IL-6 and IL-10 which stimulate antibody production by B cells. The
balance between TH1 and TH2 activity may steer the immune response in the direction of
either cell-mediated or humoral immunity.
The CD8+ T cells mediate their functions primarily by acting as cytotoxic cells. They play an
important role in resistance to viral infection, graft rejection and tumour immunity.
INTERFERON-GAMMA (IFN-)
IFN- is a glycoprotein produced by activated CD4 and CD8 T cells and by NK cells. IFN-
is a prototype of TH1 subsets of CD4+ T lymphocytes. IFN- produced by natural killer (NK)
cells may function as a mediator of innate immunity and contribute to septic shock.
Functions of IFN- :-
1) Activation of mononuclear phagocytes. IFN- directly induces synthesis of the
enzymes that mediate the respiratory burst, allowing human macrophages to kill
phagocytosed microbes.
2) IFN- acts on T-lymphocytes to promote their differentiation.
3) IFN- acts on B cells to promote switching to the lgG2a and IgG3 subclasses which
are precisely those that bind to FCγRs on phagocytes and NK cells. These subtypes of
lgG are also most potent complement activating lgGs.
30
4) IFN- stimulates the cytolytic activity of NK cells
5) IFN- activates neutrophils, up-regulating their respiratory burst.
Interleukin-4 (IL-4)
IL-4 is a cytokine and its principal cellular source is CD4⁺ T lymphocytes, specifically of the
TH2 subset. Other sources of IL-4 include activated mast cell, basophils and CD8⁺ T
lymphocyte
Functions of IL-4:-
1) IL-4 is a growth and differentiation factor for T cells, in particular for cell of the TH2
subset.
2) IL-4 stimulates the expression of certain adhesion molecules such as VCAM-1 on
endothelial cells, resulting in increased binding of lymphocytes, monocyte and
eosinophil.
3) IL-4 activates B cells which start expressing MHC class II antigens, and ultimately
influence them to secrete antibodies.
31
CHAPTER THREE
MATERIALS AND METHODS
3.1 STUDY SITE DESCRIPTION
The study was carried out at the Haematology clinic of the University college Hospital
(UCH), Ibadan. Ibadan is the capital of Oyo state, situated in the South West of Nigeria in
West Africa. It is a cosmopolitan city, renowned for its large size, where almost all the
Nigerian ethnic groups coexist but with preponderance of Yorubas, and with population of
2.5 million43.
3.2 STUDY POPULATION
The study population consisted of 40 HbS patients in steady state and 40 individuals with
haemoglobin A (HbA) confirmed by haemoglobin electrophoresis at pH 8.4-8.6 using
cellulose acetate membrane. Forty-eight HbS patients and 58 control populations were
recruited however only 40 HbS patients and 42 controls completed the study. The study was
conducted between May 2010 and July 2010.
Inclusion criteria
1. All consenting patients with sickle cell anaemia between age 15 and 40 years.
2. All consenting individuals whose haemoglobin electrophoresis is A; age and sex matched
for study population.
32
3. All participants were seronegative for HIV.
4. No history of blood transfusion in the preceding 3 months of the study.
Exclusion criteria
1. Patients with HbS who had chronic infection such as osteomyelitis, chronic leg ulcers,
tuberculosis at the time of the study.
2. Patients who have had crises in the preceding 1 month of the study.
3. Patients with other haemoglobinopathy apart from HbSS.
3.3 STUDY DESIGN
A comparative cross sectional design was employed for use in this study.
3.4 SAMPLE SIZE ESTIMATION44
Quantitative sample size determination formula
n= (Zα+ Zβ)² (σ1- σ2)2
(µ 1- µ2)²
n—Minimum sample size
Zα—Standard normal deviate of α at 95% confidence interval =1.96
Zβ --Standard normal deviate of β =1.28 (Power of 90%)
σ1– Standard Deviation of IL-4 in HbS individual =21.02 45
σ2– Standard Deviation of IL-4 in HbA individual =11.29 45
µ1- µ2 = Anticipated difference in IL-4 mean between HbA individuals and HbS
individuals=20
33
n= (1.96 +1.28)2(11.3+21)2
20²
n=10952.04
400
= 27.38
≈ 27
A minimum sample of 27 HbS patients and 27 individuals with hemoglobin A (HbA) was
calculated.
3.5 SAMPLE COLLECTION
Informed consent was obtained from the subjects or caretakers (if below 18years) and
questionnaire was administered.
Antecubital fossa was cleaned with methylated spirit and 6mls of venous blood was
collected, 3mls each was transferred into two specimen bottles, one containing sodium
ethylene-diamine tetra acetate (EDTA) crystals and the other was transferred into a plain
tube.
Blood specimen in EDTA bottle was used in the analysis for CD4/CD45 lymphocytes using
Flow cytometry (Appendix IV: Principle of flowcytometry) and the serum obtained from the
clotted sample (i.e. specimen from the plain bottle) was used in the analysis of IFN-γ
(Appendix V: Principle of ELISA for IFN-γ) and IL-4 (Appendix VI: Principle of ELISA for
IL-4) by Enzyme linked immunosorbent assay (ELISA).
34
3.6 SPLENIC ULTRASOUND:
Patients and control groups had abdominal Ultrasound done for determination of splenic size
by the Radiologist, in the Department of Radiology, University College Hospital, Ibadan.
Ultrasound Scan using a 3.5-5MHz curvilinear transducer was done on the patients in supine
or right lateral position for complete visualization of the spleen. The splenic length was
measured in centimeter on longitudinal scan from the dome of the diaphragm to the inferior
splenic margin as in figure 1
Figure 1: abdominal ultrasonography showing Spleen measurement
35
3.7 MEDICAL RECORD:
In order to determine the prevalence of infectious complications in the patients, HbS patients’
case notes were reviewed by the principal investigator over the last 12 months preceding the
study.
3.8 DATA MANAGEMENT AND ANALYSIS
Questionnaire was edited and checked for errors. Data was entered using Statistical Package
for the Social Sciences (SPSS) version 17. Exploratory analysis was first carried out to check
for error in data entry and the normality of the distributions of the continuous quantitative
variables such as age, CD4 T lymphocyte, IFN-γ and IL-4.
The categorical data were summarized with frequencies and percentages, while the
quantitative data were summarized with median because these observations did not follow
the normal (Gaussian) distribution; the quantitative variables were also pictorially presented
in Box and Whisker plots.
Spleen status was determined by ultrasonography. The spleen was considered non-
functioning and atrophic if not visualized. Spleen size of 6-12cm was taken as normal and a
length exceeding 12cm was considered to be evidence of splenomegaly.8,46
Mann Whitney U was employed to test the difference in levels of CD4 T lymphocyte,
Interferon gamma and IL-4 between HbS patients and HbA individuals. Also, the differences
between those HbS patients with infectious complications and those without. Kruskal Wallis
36
was employed to test more than two median values. Chi-square test was used to investigate
association between categorical variables
The inferential analyses were carried out using non parametric methods such as median test
and Spearman rank correlation to test for the significance of the relationship and association
between variables. All the statistical tests were two tailed and were done at 5% level of type
1 error.
3.9 ETHICAL CONSIDERATION
Ethical approval was obtained from U.I/ UCH Ethics Committee, University College
Hospital Ibadan (Appendix III).
37
CHAPTER 4
RESULTS
There were 80 individuals who participated in the study comprising of 40 sickle cell anaemia
subjects and 40 HbA subjects in the control individuals.
4.1 Socio-demographic characteristics of the study subjects (Table 1)
The median age of the SCA patients was 25.5 years which is similar to that of the control
with median age of 27 years (p=0.345).The age range was between 16years and 40years for
the patients and 18years to 38years for the control.
There was slightly higher number of female in both HbS patients and the control population.
There were 22 (55%) females in HbS group and 25 (62.5%) in control while there were 19
(45%) males in the study and 15 (37.5%) in the control group. However, there was no
statistically significant difference in the proportion of males and females in both the study
and the control groups (X2 = 0.464, p= 0.496).
38
Table 1: Socio-demographic characteristics of the HbS and HbA Subjects
Variables HbS
n=40
No (%)
HbA
n=40
No (%)
Total
n (%)
p value
Age(years)
<20 13 (32.5) 5 (12.5) 18 (22.5)
21-30 18 (45) 22 (55) 40 (50) 0.354
31-40 9 (22.5) 13 (32.5) 22 (27.5)
Sex
Male 18 (45) 15 (37.5) 33 (41.3)
Female 22 (55) 25 (62.5) 47 (58.7) 0.496
39
4.2: Comparison of CD4 count, IFN-γ and IL-4 between HbS patients and
Controls.
Table 2 presents the median and mean estimates CD4 count, IFN-γ and IL-4 of the HbS
patients and control HbA subjects. The two groups had similar median CD4 count (p=0.704)
and IL-4 level (p=0.418). However the HbS patients had significantly higher median IFN-γ
level (8.6pg/ml) than the control HbA subjects (5.58pg/ml) (p=0.047).
Table 2: Median and mean comparison of CD4 count, IFN-γ and IL-4 between HbS
patients and Controls.
HbS patient
n=40
Control
n=40
Variables Median
Mean±SD
Median
Mean±SD
z p value*
CD4
count(cells/µL)
939
958±502
937
978±321
-0.380 0.704
IFN-γ(pg/ml) 8.61
9.84±7.82
5.58
7.24±6.83
-1.983 0.047
IL-4(pg/ml) 6.74
6.85±3.56
7.67
7.63±4.23
-0.809 0.418
*Mann- Whitney U test
40
Fig2: CD4 count profile of the HbS patients and the control HbA subjects
by gender
4.3: CD4 count profile of the HbS patients and the control HbA subjects
by gender.
As shown in Figure 2, male HbS patients had higher median CD4 count, 987cells/µL (203-
2000 cells/µL, mean±SD-934±503cells/µL) than their counterparts in the control group,
879cells/µL (470-1276 cells/µL, mean±SD-849±245cells/µL); however the difference was
not significant (p=0.373). Meanwhile, the female HbS patients had lower median CD4 count
884cells/µL (274-2141cells/µL, mean±SD-975±512cells/µL) than the female control subjects
41
1021 cells/µL (536-1809cells/µL, mean±SD-1056±340 cells/µL), although the difference
was not significant (p=0.654). Male HbS patients had higher median CD4 count than female
HbS patients but no significant difference (p=0.840). The female HbA control had higher
median value of CD4 count than the male HbA control however there was no significant
difference (p=0.057)
42
4.4: CD4 count profile of the HbS patients and the control HbA subjects
by age groups
Table 3 shows that among the HbS in the age group less than 20years, the median CD4 count
1170cells/µL was lower than the control subjects 1224cells/µL. These values are not
statistically different from each other (z=-0.336, p=0.737).This same pattern were observed
in age groups 21-30years and 31-40years. Overall, the total CD4 count did not differ between
HbS (median= 939cells/µL) and HbA controls (median=937 cells/µL), p=0.704
Table 3: CD4 count profile of the HbS patients and the control HbA subjects by age
groups
Variable n (%) HbS n (%) HbA z p-value
Age(years) Median
Mean±SD
cells/µL
Median
Mean±SD
cells/µL
<20 13(32.5) 1170
1212±468
5(12.5) 1224
1232±472
-0.336 0.737
21-30 18(45) 875
859±477
22(55) 880
889±301
-0.597 0.550
31-40 9(22.5) 450
489±232
13(32.5) 926
1002±408
-1.169 0.243
Total 40 939 40 937 -0.380 0.704
43
Fig 3: IFN-γ profile of the HbS patients and the control HbA subjects HbS
patients and the control HbA subjects by gender
4.5 IFN-γ profile of the HbS patients and the control HbA subjects by
gender
Figure 3 shows that the female HbS patients had significantly higher median IFN-γ
8.83pg/ml (0.40-35pg/ml, mean±SD-9.91±8.68pg/ml) than the control HbA subjects
5.98pg/ml (0.08-31.34pg/ml, mean±SD-8.17±7.99pg/ml) p=0.013. Male HbS patients had
relatively higher median IFN-γ 7.76pg/ml (0.46-19.93pg/ml, mean±SD-9.76±6.89pg/ml)
than their male counterparts in the control subjects 5.15pg/ml (0.46-16pg/ml, mean±SD-
44
5.71±4.07 pg/ml) though not stastically significant. However, there were outliers among the
female subjects and the males in the control group. Female HbS patients had higher median
IFN-γ level than male HbS patients but no significant difference (p=0.819). The female HbA
control had higher median value of IFN-γ than the male HbA control however there was no
significant difference (p=0.543)
45
4.6 IFN-γ profile of the HbS patients and the control HbA subjects by age
groups
As shown in Table 4 in HbS patients of age group less than 20 years, the median IFN-γ value
9.91pg/ml was higher than the control subjects 9.04pg/ml. These values are not statistically
different from each other (z=-0.202, p=0.840). In age group 21-30 years the median IFN-γ
value for HbS subjects 8.83pg/ml was higher than that in the control subjects 3.42pg/ml and
the difference was statistically significant(z=-2.404, p=0.016). In age group 31-40 years, the
control subjects had higher median IFN-γ value (5.58pg/ml) than HbS patients (3.85pg/ml)
but there was no significant difference (z=-1.539, p=0.124). Overall IFN-γ is significantly
higher among HbS (median=8.61pg/ml) compared to HbA controls (median=5.58), p=0.047
Table 4: IFN-γ profile of the HbS patients and the control HbA subjects by age.
Variable n(%) HbS n(%) HbA z p-value
Age(years) Median
Mean±SD
Pg/ml
Median
Mean±SD
Pg/ml
<20 13(32.5) 9.91
9.49±5.61
5(12.5) 9.04
8.90±5.20
-0.202 0.840
21-30 18(45) 8.83
8.93±5.95
22(55) 3.42
4.42±2.20
-2.404 0.016
31-40 9(22.5) 3.85
4.62±2.26
13(32.5) 5.58
6.61±6.50
-1.539 0.124
Total 40 8.61 40 5.58 -1.983 0.047
Fig 4: Interleukin-4 values of the HbS patients versus controls (HbA
subjects) by gender
46
4.7: IL-4 profile of the HbS patients and the control HbA subjects by
gender.
As shown in Figure 4, male HbS patients had lower median IL-4 value, 6.13pg/ml (0.83-
11.79 pg/ml, mean±SD-6.07±2.88pg/ml) than their counterparts in the control group,
6.74pg/ml(0.46-16 pg/ml, mean±SD-7.45±4.38pg/ml) however the difference was not
significant (p=0.435). The female HbS patients also had lower median IL-4 value 7.05pg/ml
(0.31-15.04pg/ml, mean±SD-7.49±3.98pg/ml) than their counterparts in the control group,
8.6 pg/ml (1.38-19.01 pg/ml, mean±SD-7.83±4.21 pg/ml), the difference was also not
significant (p=0.282). Female HbS patients had higher median IL-4 than male HbS patients
47
but no significant difference (p=0.286). The female HbA control had higher median value of
IL-4 than the male HbA control however there was no significant difference (p=0.868)
48
4.8: IL-4 profile of the HbS patients and the control HbA subjects by age
groups. As shown in Table 5 among the HbS patients in the age group less than 20 years, the
median IL-4 value 6.74pg/ml was lower than the control subjects 11.15pg/ml. These value
statistically differed from each other (z=-2.156, p=0.031). In age group 21-30years the
median IL-4 value in the HbS patients (7.36pg/ml) was higher than that in the control
subjects 6.12pg/ml but the difference was not statistically different from each other (z=-
0.395, p=0.693). Among the HbS in the age group 31-40 years, the median IL-4 value
6.12pg/ml was lower than the control subjects (7.98pg/ml). These values were not
statistically different from each other (z=-0.818, p=0.413). Overall, the total IL-4 value did
not differ between HbS (median= 6.74pg/ml) and HbA controls (median=7.74 pg/ml),
p=0.418
Table 5: IL-4 profile of the HbS patients and the control HbA subjects by age groups.
Variable n (%) HbS n(%) HbA z p-value
Age(years) Median
Mean±SD
Pg/ml
Median
Mean±SD
Pg/ml
<20 13(32.5) 6.74
7.45±4.38
5(12.5) 11.15
10.89±5.87
-0.202
0.031
21-30 18(45) 7.36
7.76±3.11
22(55) 6.12
7.17±3.92
-0.395
0.693
31-40 9(22.5) 6.12
7.17±3.92
13(32.5) 7.98
8.32±3.39
-0.818
0.413
Total 40 6.74 40 7.67 -0.809 0.485
49
4.9: Splenic size distribution
I) The spleen was not visualized (measurable) by ultrasonography (autosplenectomy) in 7 (20
%) and less than 6cm in 1 (2.5%) by ultrasonography (partial autosplenectomy) of the SCA
patients
2) The spleen was visualized (measurable) by ultrasonography in 32 (80%) of the SCA
patients. 26 (65%) with median of 8.45cm (range 6-11.8cm) had normal spleen size (6-12cm)
and 6 (15%) with median of 15.05cm (range 13.5-16.4cm) had spleen size greater than 12cm
3) The spleen was visualized (measurable) by ultrasonography in all the control and was
normal in size with median of 8.5cm (range 6-11.1cm).
Table 6: Spleen size distribution in HbS and HbA subjects
Spleen
size(cm)
HbS
n(%)
Median
Mean±SD
Range
HbA
n(%)
Median
Mean±SD
Range
0-6 8(20) - - - - -
6-12 26(65) 8.45
8.71±1.86
6-11.8 40(100) 8.5
8.57±1.09
6-11.1
Above 12 6(15) 15.05
14.95±1.32
13.5-16.4 - - -
Total 40(100) 8.2
7.82±4.88
0-16.4 40(100) 8.5
8.57±1.09
6-11.1
50
4.10 Pattern of infectious complications in HbS patients
Six (15%) HbS patients had radiological confirmed pneumonia and 34 (85%) did not have
pneumonia. Fifteen (37.5%) HbS patients had laboratory confirmed malaria while 25
(62.5%) did not have malaria within one year of study.
Table 7: Pattern of infectious complications in HbS patients*(n= 40)
INFECTIOUS COMPLICATION IN THE LAST ONE
YEAR
FREQUENCY
Malaria 15(37.5%)
Pneumonia 6(15%)
*In some patients there was more than one complication
51
4.11: Relationship between CD4 cell and Spleen size in HbS patients
The scatter plot of the relationship between the spleen size of the HbS patients and their CD4
cell count is shown by figure 5. There was a negative correlation (r= -0.150) between CD4
count and splenic size, however, the relationship is weak, and also not stastically significant
(p=0.356).
Figure 5: Correlation between Spleen size and CD4 count in HbS patients.
52
As shown in table 8 the median of CD4 cell count was highest in HbS with normal spleen
size 1030cells/µL (mean±SD-1030±565cells/µL) and lowest in HbS with splenomegaly
826cells/µL (mean±SD-762±370cells/µL). The median in HbS with autosplenectomy was
923cells/µL (mean±SD-865±324 cells/µL). There was no significant difference in the median
CD4 cell count of the HbS patients with autosplenectomy, normal spleen size and
splenomegaly when compared with the control (z=0.636, p=0.525, z=-0.209, p=0.773 and
z=-1.236, p=0.209 respectively). Within the groups of HbS, there was no significant
difference in the median of the CD4 cell count in the 3 groups (z=1.238, p=0.539).
Table 8 Median Comparison of CD4 Count in relation to spleen size in both HbS and
HbA individual
Spleen
size(cm)
n(%) HbS
CD4(cells/µL)
n(%) HbA
CD4(cells/µL)
z p
0-6 8(20) 923 - - - -
6-12 26(65) 1030 40(100) 937 -1.238 0.773
Above 12 6(15) 826 - - - -
53
4.12 Relationship between Spleen size and IFN-γ
Figure 6 shows the scatter plot of the relationship between the spleen size of the HbS patients
and their IFN-γ. There was a negative correlation (r= -0.023) between IFN-γ and splenic size,
however, the relationship is weak, and also not stastically significant (p=0.887).
Figure 6: Correlation between spleen size and IFN-γ in HbS patients.
The median of IFN-γ was highest in HbS with autosplenectomy 9.48pg/ml (mean±SD-
12.07±9.56 pg/ml) and similar in HbS with splenomegaly 8.40pg/ml (mean±SD-9.26±8.04
pg/ml) and HbS with normal spleen size 8.18pg/ml (Mean±SD--9.28±8.04 pg/ml). There was
54
no significant difference in the median value of IFN-γ of the HbS patients with normal spleen
size and splenomegaly when compared with the median value of IFN-γ in the control
5.58pg/ml (z=-1.379, p=0.168; z=-0.653, p=0.514 respectively) (Table11).There was
significant difference in median value of IFN-γ in HbS patients with autosplenectomy
compared with the control (z= -2.243, p=0.025). Within the groups of HbS, there was no
significant difference in the median of the IFN-γ in the 3 groups (z=0.813, p=0.666).
Table 9 Median comparison of IFN-γ in relation to splenic size in both HbS and HbA
individual.
Spleen
size(cm)
n(%) HbS
IFN-
γ(pg/ml)
n(%) HbA
IFN-
γ(pg/ml)
z p
0-6 8(20) 9.48 _ _ _
6-12 26(65) 8.18 40(100) 5.58 -1.379 0.168
Above 12 6(15) 8.40 _ _ _
55
4.13 Relationship between splenic size and IL-4
As shown by the Scatter plot in Figure 7, there was a negative correlation (r= -0.179)
between the spleen size of the HbS patients and their IL-4, however, the relationship is weak,
and also not stastically significant (p=0.269).
Figure7: Correlation between spleen size and IL-4 in HbS patients.
56
The median of IL-4 was highest in HbS with autosplenectomy 9.89pg/ml (mean±SD-
9.36±3.64 pg/ml) and similar in HbS with splenomegaly 6.74pg/ml (mean±SD-5.98±3.23
pg/ml) and HbS with normal spleen 6.43pg/ml (mean±SD-6.28±3.43 pg/ml). There was no
significant difference in the median value of IL-4 of the HbS patients with autosplenectomy,
normal spleen size and splenomegaly when compared with the median value of IL-4 in the
control (z= -1.233, p=0.218, z=-0.132, p=0.187 and z= -0.915, p=0.360 respectively)
(Table12). Within the groups of HbS, there was no significant difference in the median of the
IFN-γ in the 3 groups (z=3.973, p=0.140).
Table 10: Median comparison of 1L-4 in relation to spleen size in both HbS and HbA
individual.
Spleen
size(cm)
n(%) HbS
IL-4
(pg/ml)
n(%) HbA
IL-4
(pg/ml)
Z p
0-6 8(20) 9.89 _ _ _ _
6-12 26(65) 6.43 40(100) 7.67 -1.321 0.187
Above 12 6(15) 6.74 _ _ _ _
57
Table 11 Correlation coefficients of the relationship between spleen size and CD4,IFN-γ
and IL-4
HbS patients(n=40) Control HbA (n=40)
r p-value r p-value
CD4 -0.150 0.356 0.290 0.069
IFN-γ(pg/ml) -0.023 0.887 0.013 0.937
IL-4(pg/ml) -0.179 0.269 -0.002 0.991
r: Spearmann correlation coefficient
4.14 Disaggregating the correlation analysis between spleen size and the other
immunological parameters, Table 11 presents the estimates of coefficients of the relationship
between the spleen size and the parameters. Yet, none of the parameters had significant
relationship with the spleen size in the HbS patients (p>0.05).
58
4.15: Association between pneumonia infection and CD4 count in SCA
patients
Table 12 shows median comparison of CD4 count in SCA patient with and without previous
pneumonic complication in the past 1 year. SCA patients with previous pneumonic
complication had lower CD4 count 764cells/µL (mean±SD-855±378 cells/µL) than SCA
patients without previous pneumonic complication (987cells/µL, mean±SD-974±523
cells/µL). The relationship between pneumonia and CD4 count was not significant
(p=0.705).
Table 12: Median Comparison of CD4 count in SCA patients with and without previous
pneumonia
Pneumonia n(%) CD4(cells/µL) z p
Yes 6(15) 764 -0.379 0.705
No 34(85) 987
Total 40(100) 940
59
4.16 Association between pneumonia infection and level of IFN-γ in SCA
patients
Table 13 shows median comparison of IFN-γ level in SCA patient with and without previous
pneumonic complication in the past 1 year. SCA patients with previous pneumonic
complication had lower median IFN-γ value 7.96pg/mL (mean±SD-7.18±3.74 pg/mL) than
SCA patients without previous pneumonic complication 9.47 pg/mL (mean±SD-
10.31±8.30pg/mL) The relationship between pneumonia and IFN-γ level was not significant
(p=0.437).
Table 13: Median Comparison of IFN-γ level in SCA patients with and without
previous pneumonia
Pneumonia n(%) IFN-γ (pg/mL) z p
Yes 6(15) 7.96 -0.777 0.296
No 34(85) 9.47
Total 40(100) 8.61
60
4.17 Association between pneumonia infection and level of IL-4 in SCA
patients
Table 14 shows median comparison of IL-4 level in SCA patient with and without previous
pneumonic complication in the past 1 year. SCA patients with previous pneumonic
complication had higher median IL-4 value 7.99pg/mL (mean±SD-8.24±3.59 pg/mL) than
SCA patients without previous pneumonic complication (6.74pg/mL, mean±SD-6.61±3.55
pg/mL). The relationship between pneumonia and IL-4 level was not significant (p=0.296).
Table 14: Median Comparison of IL-4 level in SCA patient with previous pneumonia
Pneumonia n(%) IL-4 (pg/mL) z p
Yes 6(15) 7.99 -1.045 0.296
No 34(85) 6.74
Total 40(100) 6.74
61
CHAPTER 5
DISCUSSION
Sickle cell disease (SCD) is characterized by significant morbidity and mortality and studies
have shown that infection is the leading cause of death in Africa patients. Overwhelming
infections caused by encapsulated bacteria (such as salmonella spp.) and Plasmodium
falciparum are an important cause of morbidity and death in patients with sickle cell
disease37,38. The most important contributing factors to this increased susceptibility to
encapsulated bacteria are: a state of functional asplenia, an opsonophagocytic defect due to
abnormality in the alternative complement pathway, deficiency of specific circulating
antibodies, impaired leukocyte function and loss of both humoral and cell mediated
immunity.
This study assessed the cellular immunity using CD4+ T lymphocytes count, serum INF-γ
and IL-4 levels in patients with sickle cell anaemia. These values were correlated with spleen
sizes, in order to predict the susceptibility of HbS patients to infection.
The study population comprised 40 HbS patients in steady state and 40 HbA normal control
subjects. The median age for HbS patients was 25.5 years. There were more females subjects
with sickle cell anaemia than male. This was not unexpected as female patients have been
shown to live longer than their male counterpart47, and this has been reflected in the pattern
of clinic attendance of HbS patients. The levels of CD4 T lymphocytes, serum IFN-γ and IL-
4 were not affected by gender status both in patients and control population.
Eight (20%) of the HbS patients had autosplenectomy, which was confirmed by
ultrasonography as indicated by partial or no visualization of the spleen. Twenty-six (65%)
had normal splenic size on ultrasonography while splenomegaly was observed in 6 (15%) of
62
the patients. Studies have shown that most HbS patients do not have palpable spleen beyond
the eighth year of life29,34 when the spleen is expected to have undergone autosplenectomy,
from repeated vaso-occlusion and infarction. However, this is not always the case with some
HbS patients living in the tropics, where the spleen remains palpable in the older age group
(adolescent and adult)30. Factors such as high levels of foetal haemogobin, chronic malaria
infection and co-inheritance of alpha thalassaemia, have been associated with the persistence
of splenomegaly in older HbS patients32
In this study, the median CD4 T lymphocytes count in both control subjects (937 cells/µL)
or HbS patients (939 cells/µL) was lower than the mean CD4 T lymphocytes count reported
by Koffi et a l(2003)8 in Cote d’Ivoire in control (1215 cells/µL) and HbS (1656 cells/µL)
population but similar to the value reported in healthy Nigeria adults ( median 847 cells/µL)
by Oladepo et al(2008)48 . There was no significant difference between median values of
CD4 T-cell in patients with SCA and in the controls (p=0.704). This was in accordance with
previous report by Koffi et al(2003)8 but in contrast to the study by Kaaba et
al(1989)12,where decreased number of CD4 cells were reported in SCA patients compared
with HbA controls. Sixty-five percent of the HbS patients in this study had normal spleen
size which may explain why they have similar median CD4 T-cell value compared with
control. Koffi et al8 indicated a significant decrease in CD4 T-cell in sickle anaemia patients
with asplenia when compared with those with normal spleen size. This study showed lower
value of median CD4 T-cell in HbS patients with asplenia (923 cells/µL) compared with
HbS patients with normal spleen size (1030 cells/µL), though not significant (p=0.539). This
may be related to few number of asplenic patients. Persistence of the spleen in some HbS
patients has been attributed to chronic malaria infection in Nigeria 31. In this study 37.5% of
63
the patients had malaria in the preceding 1year of the study and 80% had normal or enlarged
spleen (6-16.4cm). The highest CD4 count value was recorded among HbS patients with
normal spleen size, however there was no significant difference in the median levels of CD4
count value among the 3 groups of HbS according to the spleen size (p=0.539). This is in
contrast to Koffi et al8 (2003) who observed significantly higher mean CD4+ T lymphocytes
count among HbS patients with normal spleen size compared with those with asplenia.
Donadi and Falcao49 queried whether the changes of lymphocyte subsets in sickle cell
anaemia are due to the loss of splenic function. His study also showed that the number of
total lymphocytes, T-lymphocyte subsets and B-lymphocyte subsets were increased in both
splenectomised patients and sickle cell anaemia patients. It would be of interest if a subset of
splenectomised patients is compared with HbS patients in Nigeria.
By secreting cytokines, CD4+ T lymphocyte influence the function of virtually all other
cells of the immune system, including other T cells, B cells, macrophages, and natural killer
cells10,41,42.
Patterns of cytokine expression regulate lymphocyte effector mechanisms41,42. Human Th1
cells within the CD4 T cells population produce the following cytokine IFN-γ, TNF-β, and
IL-2, while the Th2 clones produce IL-4, IL-5, IL-6, IL-9, IL-10 and IL-13. Th1 cytokines
are associated with cell-mediated inflammatory responses, whereas Th2 cytokines are
associated with strong antibody and allergic responses. Cytokines from Th1 cells inhibits the
actions of Th2 cells and vice-versa, thus an immune response becomes polarized to a
predominant Th1 or Th2 type.
The prototype of human Th1 cytokines is IFN-γ, they promote the production of IgG2a,
opsonizing and compliment-fixing antibodies, macrophage activation, antibody –dependent
64
cell-mediated cytotoxity, and delayed type hypersensitivity. In this study, IFN-γ was
significantly higher among HbS patients (median 8.6pg/mL) than control (5.58pg/mL),
(p=0.04) which might be accounted for by subclinical chronic inflammatory state in HbS
patients. However, there was no significant difference in IFN-γ level in patients that have had
pneumonic infectious complication within a year preceding the study. The elevated value
obtained in these HbS patients might also be from other sources of interferon gamma such as
CD8 T lymphocytes as the study by Koffi et al (2003)8 showed that numbers of CD8 T
lymphocytes were significantly increased in HbS patients . This was in agreement with
Pathare et al (2004)50 but in contrast to report obtained by Raghupathy et al (2000)51 and
Taylor et al (1999)52 who found that there was no significant difference in the IFN-γ level
between HbS and HbA individuals.
IFN-γ level was found to be significantly higher in female HbS patients as compared to their
female HbA counterparts. However, IFN-γ value was not affected by gender status both in
HbS patients and control population. This was in agreement with Raghupathy et al (2000)51
who found that there was no gender bias on the IFN-γ level. Karpuzoglu-Sahin et al (2001)53
found that estrogen increases the production IFN-γ. The increase in IFN-γ level in female
HbS patients in this study may be due to the disease couple with hormonal factor. Study by
Miller et al (1990)54 found that IFN-γ significantly decreases synthesis of HbF. One of the
survival factors which the female HbS patients have over the male HbS patients has been
identified as increase in HbF level. It has been shown by miller et al (1990)54 that IFN-γ
suppresses the production of HbF. The rise in IFN-γ level and degree of negative influence
on the production of HbF has to be further evaluated in HbS patients. Marcal et al (2007)55
recorded significantly higher IFN-γ in HbS patients and implied that this may contribute to
65
inflammation and tissue damage in these patients. IFN-γ level was highest in HbS group with
asplenia compared with HbS group with normal spleen and splenomegaly who had similar
values. However this difference did not reach stastical significance, perhaps a larger
population of patients will resolve this issue. However, there was a significantly higher IFN-γ
in HbS with asplenia when compared with the control population (p=0.025). There was a
negative relationship (r=0.023) between spleen size and IFN-γ level though not statistically
significant (p=0.887).
The prototype of human Th2 cytokines is IL-4; they provide optimal help for humoral
immune responses, including IgG1 and IgE switching and mucosal immunity, stimulation of
mast cells, eosinophil growth and differentiation, and IgA synthesis. HbS patients
(6.74pg/mL) in this study had similar IL-4 values with the HbA individuals (7.67pg/mL).
This is in agreement with Pathare et al (2004)50 and Musa et al (2010)45 who found that
there was no significant difference in the levels of IL-4 level in HbS and HbA individuals in
Oman and Nigerian population respectively. Taylor et al (1997)56 found significantly higher
level of IL-4 in HbS patients than the HbA control subjects in America.
IL-4 is important in antibody production, in view of the fact that in this study there was no
difference in the levels of IL-4 between HbS patients and HbA controls, this could explain
the finding by Olaniyi et al57 on similar values of immunoglobulin classes in HbS patients
and HbA individuals. As regards influence of gender in this work there was no stastically
significant difference in both HbS patients and HbA controls which is in agreement with
Raghupathy et al (2000)51. There was a negative relationship (r=0.179) between splenic size
and IL-4 in HbS patients however this difference was not significant (p=0.269).
66
The commonest causes of infectious complication warranting hospital admission in the
University College Hospital Ibadan include malaria (37.5%) and Pneumonia (15%) in HbS
adult patients.
CONCLUSION
The results in this study showed that the median IFN-γ level in HbS patients was
significantly higher than in HbA individuals. This significantly higher value of IFN- γ may
contribute to inflammation and tissue damage in HbS patients, and this could worsen
morbidity and mortality. There was no significant difference in the median values of CD4 T
cells and IL-4 in HbS patients and HbA controls. There was no significant correlation
between splenic size and CD4 T cell, IFN-γ and IL-4 respectively.
67
Recommendations for further research
1. Future research on the relationship between IFN-γ and modifiers of SCD such as HbF
and clinical severity is hereby suggested.
2. Serial IFN-γ level in HbS with acute infectious complication may help predicts the
outcome of treatment of infection.
LIMITATION OF THE STUDY
1. In the retrospective review of the laboratory data generated before this study, it was
observed that some diagnostic investigation of infections looked at were incomplete probably
due to limited funds.
2. Small sample size due to limited funds
68
REFERRENCES
1. Fleming AF Historical introduction. Molecular biology and inheritance. In: Sickle
Cell Disease. A Handbook for the General Clinician. 1st ed. Churchhill
Livingstone Edinburgh.1982. p. 1-19.
2. Beutler E. The Sickle cell Disease and related Disorders. In: Williams’ Haematology.
6th ed. McGraw-Hill, Medical Publishing Division New York. p. 581-600.
3. Steinberg MH. Management of SCD. New England Journal of Medicine. 1999;
340(13):1021-30.
4. Lagundoye SB. Splenic calcification and Sickle Cell Disease in Nigerians.
Trop.Geog. Med.1975; 23:135-140.
5. Falcao RP, Donadi EA. Infection and immunity in sickle cell disease. AMB Rev
Assoc. Med Bras 1989 Mar-Apr; 35(2): 70-4.
6. Overturf G, Powars D. Infections in sickle cell anaemia: Pathogenesis and control.
Tex Rep Biol Med.1981; 40:283-92.
7. Ahmad AM, Mohammed MS. Pattern of bacterial infections in homozygous sickle
cell disease. A report from Saudi Arabia. Am J Dis child; 1985 Aug; 139(8):820-2.
8. Koffi KG, Sawadogo D, M Meite, et al. Reduced levels of T-cell subsets CD4+ and
CD8+ in homozygous sickle cell anaemia patients with splenic defects. The
Hematology Journal 2003; 4: 363–365.
69
9. Hoffbrand AV, D Catovsky, EGD Tuddenham. Sickle cell disease. In: Postgraduate
Haematology. 5th ed. Blackwell Publishing Limited, London. 2005. Chapter 7 p. 104-
106.
10. Cotran RS, Kumar V, Robbins SL. Robbins Pathologic Basis of Disease, 5th ed.
Chapters 13 and 14.p. 594-595,667-669.
11. Landesman SH, Rao SP, Ahokhai VI. Infections in children with Sickle Cell
Anaemia. Special reference to pneumococcal and salmonella infections. Am J pediatr
Hematol Oncol. 1982 Winter; 4(4); 407-18.
12. Kaaba SA, Al-harbi SA. Reduce level of CD2 +Cells and T- Cell subsets in patient
with sickle cell anaemia. Immunol Lett 1993 Jul, 37(1): 77-8113.
13. DV Desai and H Dhanani: Sickle cell Disease: History and Origin. The internet
Journal of Haematology. 2004 vol.1 Number 2.
14. Savitt TL, Goldberg MF. “Herrick’s 1910 case report of sickle cell anaemia. The rest
of the story”. Journal of the American Medical Association; 1989, 261(2): 266-71.
15. Sergeant GR. Distribution of sickle cell disease. In Sickle disease. 2nd ed. Oxford
University Press, Oxford. 1988; p.13-24.
16. Fleming AF. Sickle Cell Trait Genetic Counseling. Sickle cell Disease. In: A Hand
book for General Clinician. 1st ed. Churchill Livingstone Edinburgh.1982. p. 22-31
17. Serjeant GR. The Geography of Sickle Cell Disease. In: Opportunities for
Understanding its Diversity. Annals of Saudi Medicine.1994; 14(3): 237-246.
70
18. Aidoo M, Terlouw DJ, kolczak MS, McElroy PD, Kulie FO, Nahlen BL et al.
Protective effects of the sickle cell gene against malaria morbidity and mortality.
Lancet 2002; 359: 1311
19. Raper AB: Sickling in relation to morbidity from malaria and other disease. BMJ
1:963, 1956.
20. Burn HF, Epstein MD. Pathogenesis and treatment of SCD. New England Journal of
Medicine. 1997; B37 (11): 762-769.
21. Sodeinde O, Ambe JP and Fatunde OJ. Anaemic Crises in Patients with Sickle Cell
Anaemia. Nigerian Journal of Paediatrics. 1997; 24:55.20.
22. HR Hoffman, et al. Haematology basic principles and practice. 4th ed. Elsevier church
Livingstone. 2005. p.615.
23. Shokunbi WA, Kotila TR, Dare OA. Earliest presenting sign and morbidity profile of
sickle cell disease patients attending the adult SCD clinic at the UCH Ibadan, Nigeria.
Cent Afr J Med 2001; 47(7): 186-187.
24. Weiss L.: Red pulp of the spleen: Structural basis of blood flow. Clin. Hematol. 1983;
12:375.
25. Rosse WF. Spleen as filter. N. Engl. J Med. 1987; 317:704.
26. Hoffbrand AV, Moss PAH, Petit JE. Genetic disorder of haemoglobin. In: Essential
Haematology. 5th ed. Blackwell Publishing, Oxford UK. 2006. Chapter 6, p. 85-92.
27. Rogers DW, Vaidja S and Serjeant GR. Early Splenomegaly in Homozygous Sickle
cell Disease: An Indicator of Susceptibility to Infection Lancet. 1978; 2:652-76.
71
28. Watson JR, Herbert C, lichtman H and Henry D. Splenomegaly in Sickle cell
Anaemia. Am. J. Med. 1956; 196-206.
29. Serjeant GR. The Spleen in Sickle cell Disease. 2nd ed. Oxford University Press,
Oxford, 1988. p.109-23.
30. Adekile AD, Adeodu OO, and Odesanmi WO. Persistent Gross Splenomegaly in
Nigerian Patients with Sickle Cell Anaemia Relationship to Malaria. Annals of
Tropical Paediatric, 1988; 8: 103-107.
31. Adekile AD, Mckie KM, Adeodu OO, et al. Spleen in sickle cell anaemia:
comparative studies of Nigerian and U.S. patients. Am J Hematol 1993; 42: 316-21
32. Olatunji AA, Olatunji OO. Splenic size determination in sickle cell anaemia: an
ultrasonographic study. East Afr Med J.2001 Jul; 78(7):366-9
33. Pearson HA, Mcintosh S., Ritchey A.K et al. Developmental Aspects of Splenic
Function in Sickle Cell Disease. Blood.1979; 53 (3):359-365.
34. Powers DR. Natural History of Sickle Cell Disease- The First Ten Years. Seminars in
Haematology. 1975; 12:3.
35. Ikemefuna AN, Emodi IJ. Hospital admission of patients with Sickle cell anaemia,
pattern and outcome in Enugu area of Nigeria. Nigerian J of Clinical Practice March
2007; 10(1): 24-29.
36. Platt OS, Branbilla DJ, Rosse WF. Mortality in sickle cell disease: life expectancy
and the risk factors for early death. N Engl J Med 1994; 330;1639-1644.
37. Robinson MG, Watson RJ. Pneumococcal meningitis in sickle cell anemia. N Eng J
Med 1966; 277: 1006–1008.
72
38. Wright JG, Hambleton IR, Thomas PW. Postsplenectomy disease course in
homozygous sickle cell disease. J Pediat 1999; 134: 304–309.
39. Onwubalili JK. Sickle Cell Disease and infection J Infect. 1983 Jul; 7(1): 2-20.
40. Pearson HA. Sickle cell Anaemia and severe infections due to encapsulated bacteria.
J Infect Dis, 1977 Aug; 136 Suppl. S35-20.
41. Rastogi SC. Elements of Immunology. The T- Lymphocytes 1st ed. CBS publishers
and distributors New Delhi India. 2002 p. 24-25, 84-94.
42. Abdul K Abass, Lichtman AH, Pober JS. Cellular and molecular immunology. 3rd ed.
Saunders text and the review series. 1997 p. 267-269
43. Federal republic of Nigeria official gazette, 2007; 24(94):B194-B195.
44. Kirkwood BR, Sterne JAC .Essential Medical Statistics. 2nd ed. 2003 p.142,
Blackwell Science Limited, Massachusetts, USA.
45. Musa BOP, Onyemelukwe GC, Hambolu JO, Mamman AI, Albarka HI. Pattern of
serum cytokine expression and T-cell subsets in Sickle Cell Disease patients in vaso-
occlusive crisis. Clinical and vaccine immunology, Apr. 2010, p. 602-608
46. Goldberg BB, MaGhan JP. Atlas of Ultrasound Measurement, 2nd ed.ch 88, p.440
48. Oladepo DK, Idigbe EO, Audu RA, Inyang US, Imade GE, Philip AO, et al.
Establishment of Reference Values of CD4 and CD8 Lymphocyte Subsets in Healthy
Nigerian Adults. Clinical and Vaccine Immunology, Sept 2009; 16(9): p. 1374-1377.
49. Donaldi EA, Falcao RP. Are the changes of the lymphocyte subsets in sickle cell
anaemia due to loss of splenic function? Acta Haematol. 1988; 80:91-94
73
50. Pathare A, Kindi SA, Alnaqdy AA, Daar S, Knox-Macaulay H and Dennison D.
Cytokine Profile of Sickle Cell Disease in Oman. Am. J. Hematol.2004; 77:323-328
51. Raghupathy R, Haider MZ, Azizieh F, Abdelsalam R, D’Souza TM, Adekile AD.
Th1 and Th2 Cytokine Profiles in Sickle Cell Disease. Acta Haematol 2000;
103:197–202
52. Taylor SC, Shacks SJ, QU Z. In vivo production of type-1 cytokines in healthy sickle
cell disease patients. J Natl Med Assoc. 1999; 91: 619-624
53 Karpuzoglu-Sahin E, Zhi-Jun Y, Lengi A, Sriranganathan N, Ahmed SA. Effect of long
term estrogen treatment on IFN-ᵧ, IL-2 and IL-4 gene expression and protein
synthesis in spleen and thymus of normal C57BL/6 MICE. Cytokine May 2001;
14(4): p.208-217
54. Miller BA, Olivieri N, Hope SM, Faller DV, Perrine SP. Interferon-gamma modulates
fetal hemoglobin synthesis in sickle cell anemia and thalassemia. J Interferon Res
1990 Aug; 10(4):357-66.
55. Marcal LE, Dias-da-Motta PM, Rehder J, Mamoni RL, Blotta MHSL, Whitney CB et
al. Up-regulation of NADPH oxidase components and increased production of
interferon-gamma by leukocytes from sickle cell disease patients. American Journal
of haematology 25July 2007; 83(1):p 41-45
56. Taylor SC, Shacks SJ, QU Z, Wiley P. Type-2 cytokine serum levels in healthy sickle
cell disease patients. J Natl Med Assoc. 1997; 89:753-757
74
57. Olaniyi JA and Arinola GO. Humoral immunity and haemoglobin F (HbF) status in
steady state adult Nigerian sickle cell disease patients with asymptomatic malaria.
Turk J Med Sci 2009; 39(6): 953-957
58. CD4 easy count kit (package insert). Otto-Hahn-Str.32, Germany: Partech:2008.
75
APPENDIX I
INFORMED CONSENT
TITLE:
CD4+ T-LYMPHOCYTE, GAMMA-INTERFERON AND INTERLEUKIN-4 AND
ULTRASONOGRAPHIC SPLENIC SIZE IN HOMOZYGOUS SICKLE CELL ANAEMIA
PATIENTS
NAME OF APPLICANT:
This study is being conducted by Dr Omotola T Ojo
Department of Haematology, University College Hospital, Ibadan.
SPONSOR OF RESEARCH: Self.
PURPOSE OF RESEARCH:
T o assess cellular immunity using CD4 T lymphocyte count, serum IFN-γ and IL-4 levels in
patients with sickle cell anaemia. These levels will be correlated with spleen sizes.
PROCEDURE OF THE RESEARCH: A total of 90 consenting people, comprising of 45
patients with sickle cell anaemia (cases) who present at haematology clinic and 45 people
whose haemoglobin electrophoresis is A(control) would be selected by consecutive sampling
method respectively. The participants’ will be the researching physician using an interviewer
administered questionnaire. The physical examinations, and systemic examinations, will be
carried out by the physician. Voluntary pre-testing counseling will be done for HIV screening
before the blood sample will be obtained for CD4, interferon gamma and interleukin-4
analysis; this is to prevent bias on the results. Abdominal ultrasound will be done thereafter.
EXPECTED DURATION OF RESEARCH AND OF PARTICIPANTS’
INVOLVEMENT: The study is expected to be over a period of three months, however you
will only be interviewed and examined once during this period. The questionnaire is expected
to be completed in 10-15 minutes and you will be examined for about 10 minutes.
RISK(S): You will not be at any risk by participating in this study. The routine examination
will be carried out by a female physician with a male chaperon as need arise and your blood
sample will be collected after which abdominal ultrasound will be done
COST(S) TO THE PARTICIPANTS, IF ANY, OF JOINING THE RESEARCH: Your
participation in this research will not cost you anything.
BENEFIT(S): The goal of this study is to assess an aspect of immune function in patients
with sickle cell anaemia; the result might be useful in the course of the disease for effective
management of patient with sickle cell anaemia.
CONFIDENTIALITY: All the information collected from this study will be coded and you
are not required to write your names. This cannot be linked to you in any way and your name
or identifier will not be in any publication or reports from this study.
VOLUNTARINESS: Your participation on this study is entirely voluntary.
ALTERNATIVES TO PARTICIPATION: If you choose not to participate, this will not
affect your treatment in this hospital in any way.
DUE INDUCEMENT(S): You will not be paid any fees for participating in this research.
You will be adequately informed of any abnormality detected and measures will be taken to
ensure that appropriate management is instituted.
CONSEQUENCES OF PARTICIPANTS’ DECISION TO WITHDRAW FROM
RESEARCH AND PROCEDURE FOR ORDERLY TERMINATION OF
PARTICIPATION: You can also choose to withdraw from the research at anytime. WHAT
HAPPENS TO RESEARCH PARTICIPANTS AND THE COMMUNITIES WHEN
76
THE RESEARCH IS OVER: You will be informed on the outcome of the research through
a subsequent follow-up clinic visits. During the course of this research, you will be informed
about any information that may affect your health.
STATEMENT OF PERSON OBTAINING INFORMED CONSENT: I have fully
explained this research to ______________________________________________ and have
given sufficient information, including about risk and benefits, to make an informed decision.
DATE:__________________________ SIGNATURE:______________________________
NAME:___________________ ___________
STATEMENT OF PERSON GIVING CONSENT: I have read the description of the
research or have had it translated into the language I understand. I have also talked it over
with the doctor to my satisfaction. I understand that my participation is voluntary. I know
enough about the purpose, method, risks and benefits of the research study to judge that I
want to take part in it. I understand that I may freely stop being part of this study at any time.
I have received a copy of this consent form to keep for myself.
DATE______________SIGNATURE/THUMBPRINT______________________________
NAME_________________________________
WITNESS’ SIGNATURE (if applicable)____________________________________
WITNESS’ NAME (if applicable)______________________________________
77
APPENDIX II
PARTICIPANT QUESTIONNAIRE
CD4+ T lymphocyte, INF-γ, IL-4 and ultrasonographic splenic size in
homozygous Sickle cell anaemia patients.
This questionnaire is part of a study on some aspects of the immune status of patients with
sickle cell anaemia. The information obtained could become useful for better monitoring and
management of patients with sickle cell anaemia. The information obtained from you will be
treated with utmost confidentiality.
Kindly answer the questions truthfully.
Fill the appropriate or the appropriate number into the box.
Section A: Socio-demographic data
1. Serial number _______________________
2. Hospital number _________________________________________
3. SEX: Male Female
4. AGE:
5. Marital Status: 1.Married 2. Single 3.Widowed
4. Divorced 5. Separated
78
6. Level of Education
Primary Secondary Tertiary
Father
Mother
Patient
7. OCCUPATION
Employed Unemployed Unskilled Semi Skilled Professional
Father
Mother
Patient
8. Average monthly personal Income------------------
Section B: Past Medical History
9. Age at first diagnosis __________________
10. Presentation at diagnosis _______________
11. Approximate number of crises per year _____________
12. How long ago was your last crisis _______________
79
13. Approximate number of admissions in a year __________
14. Total number of blood transfusion till date _____________
15. Approximate number of blood transfusion per year
16. Clinic attendance: Regular Irregular
17. Regular medication: Malaria prophylaxis Folic acid
Herbal products Others e.g. Niprisan (specify)_____________
18. Have you had any of the following infectious complication/Number of times?
Pneumonia Osteomyelitis Malaria Urinary tract infection
Others (Specify) ___________________
19. Response to antimicrobial therapy Yes NO
80
Section C: Physical Findings
21. JAUNDICE
20. SPLEEN Yes No Scan Mild Mod Severe
Clinically Palpable
22. HEPATOMEGALY Yes No
23. LYMPHADENOPATHY Yes No
Section D:
24. Result: CD4 cells/µL INF-γ pg/mL
IL-4 pg/mL
81
82
APPENDIX IV
Flow Cytometer Principle for CD4/CD45 cell count
Flow cytometer is equipment designed for blood cells (particles) e.g. white blood cells. The
counting process involves passing the individual cells through a light source during which
the light scatter and fluorescence parameters are measured. Thus, a flow cytometer combines
the principle of a fluorescence microscope and an automated haematology analyzer.
Principle of CD4 cell count by flow cytometry
It is based on detection of CD4 cell by optical detectors. Cells in suspension injected into a
flow cytometer, are ordered into a stream of single particles by the fluidic system which
consists of a central channel/core through which the sample is injected, enclosed by an outer
sheath that contains faster flowing fluid. As the sheath fluid moves, it creates a massive drag
effect on the narrowing central chamber. The effect creates a single file of particles and is
called hydrodynamic focusing. Each particle passes through one or more beams of light
where they scatter light and emit fluorescence that is collected, filtered and converted to
digital values that are stored on a computer. Light scattering or fluorescence emission (if the
particle is labelled with a fluorochrome) provides information about the particle’s properties.
Light that is scattered in the forward direction, typically up to 20o offset from the laser beams
axis, is collected by a lens known as the forward scatter channel (FSC). The FSC intensity
roughly equates to the particle’s size. The light measured approximately at a 90o angle to the
excitation line is called side scatter. The side scatter channel (SSC) provides information
about the content within a particle.
83
PARTEC TECHNIQUE58
Flow cytometer with an excitation light source of 488 nm blue or green solid state laser. To
count CD4/CD45 cells, transfer the test tube with 840ul of the ready prepared blood sample
(see method) to the Partec flow chart. The CD4/CD45 cells receptor are passed through a
light source via the flow cuvette, the fluorescence helps to detect the CD4/CD45 cells stained
by the monoclonal Antibody-PE /monoclonal antibody PE-Dy647 and signal is sent through
photomultiplier tube and seen on the monitor and recorded by the software 3 decade log.
Reagents and materials
1. CD4 mAb PE (MEM-241, PE-conjugated monoclonal antibody to human CD4)
2. CD45 mAb PE (MEM-28, PE-Dy647-conjugated monoclonal antibody to human CD45)
3. Partec Flow Cytometry instrument (CyFlow®SL_3)
4. Partec test tubes
4. Micropipettes and pipette tips
5. Venous blood collection system with EDTA anticoagulant
6. Powder-free latex gloves
84
Method
1. 20ul whole blood was added to a Partec test tube
2. 10ul of CD4 mAb PE and 10ul CD45 mAb PE-Dy647 was added, mixed gently and
incubated for 15 minutes at room temperature protected from light
3. 400ul of Buffer 1 was added and shook gently
4. Directly prior to the measurement 400ul of Buffer 2 was added and analysed immediately
(within 10 minutes)
5. Analysis was done and results were recorded.
85
APPENDIX V
Principle of Enzyme linked immunosorbent assay IFN-
This assay employs the quantitative sandwich enzyme immunoassay technique. A
Polyclonal antibody specific for IFN- has been pre-coated onto a microplate. Standards and
samples are pipetted into the wells and any IFN- present is bound by the immobilized
antibody. After washing away any unbound substances, an enzyme-linked polyclonal
antibody specific for IFN- is added to the wells. Following a wash to remove an unbound
antibody-enzyme reagent, a substrate solution is added to the wells and color develops in
proportion to the amount of IFN- bound in the initial step. The color development is stopped
and the intensity of the color is measured.
Reagents and materials
IFN- Microplate- polystyrene microplate coated with a polyclonal antibody against IFN-
IFN- Conjugate--- polyclonal antibody against IFN- conjugated to horseradish peroxidase.
IFN- Standard
Assay Diluent
Calibrator Diluent
Wash Buffer Concentrate
Color Reagent A- stabilized hydrogen peroxide.
Color Reagent B -stabilized chromogen (tetramethylbenzidine).
Stop Solution -2 N sulfuric acid.
Plate Covers - Adhesive strips.
86
Microplate reader capable of measuring absorbance at 450 nm.
Pipettes and pipette tips.
Graduated cylinder.
Distilled water.
Automated microplate washer.
Dilution tubes.
Sample collection and storage
Samples were allowed to clot for 30 minutes before centrifugation for 15 minutes at
approximately 1000 x g. Serum was removed and aliquot samples were stored at -20° C until
assay.
Reagent preparation
All reagents were brought to room temperature before use.
Wash Buffer - 20 mL of Wash Buffer Concentrate was diluted with distilled water to prepare 500
mL of Wash Buffer.
Substrate Solution - Color Reagents A and B were mixed together in equal volumes within 15
minutes of use. The Solution was Protected from light. 200 µL of the resultant mixture was
required per well.
Calibrator Diluent - 20 mL of Calibrator Diluent Concentrate was diluted with distilled water to
yield 100 mL of Calibrator Diluent.
87
IFN- Standard was reconstituted with 1.0mL of Calibrator Diluent. This reconstitution produces
a stock solution of 1000 pg/mL. The standard was allowed to sit for a minimum of 15 minutes
with gentle agitation prior to making dilutions. 500 µL of the appropriate Calibrator Diluent was
pipetted into each tube. The stock solution was used to produce a dilution series (below). Each
tube was thoroughly mixed before the next transfer. The undiluted standard served as the high
standard (1000 pg/mL). The appropriate Calibrator Diluent served as the zero standard (0
pg/mL).
Assay procedure
All reagents and samples were brought to room temperature before use.
1. All reagents, working standards, and samples were prepared as directed in the previous
sections
3. 100 µL of Assay Diluent was added to each well.
4. 100 µL of Standard or sample was added per well and covered with the adhesive strip.
Incubated for 2 hours at room temperature.
88
5. Each well was aspirated and wash, the process was repeated twice for a total of three washes.
Washing was done by filling each well with Wash Buffer (400 µL) using an autowasher. After
the last wash, remaining Wash Buffer was removed by decanting; the plate was inverted and
blotted against clean paper towels.
6. 200 µL of IFN-γ Conjugate was added to each well and covered with a new adhesive strip.
Incubated for 2 hours at room temperature.
7. The aspiration/wash was repeated as in “Step 5”.
8. 200 µL of Substrate Solution was added to each well. Incubated for 20 minutes at room
temperature. This was protected from light.
9. 50 µL of Stop Solution was added to each well. The color in the wells changed from blue to
yellow.
10. The optical density of each well was determined within 30 minutes, using a microplate reader
set to 450 nm wavelength.
CALCULATION OF RESULTS
A standard curve was Created by reducing the data using computer software capable of
generating a four parameter logistic (4-PL) curve-fit.
89
APPENDIX VI
Principle of Enzyme linked immunosorbent assay for IL-4
This assay employs the quantitative sandwich enzyme immunoassay technique. A monoclonal
antibody specific for IL-4 has been pre-coated onto a microplate. Standards and samples are
pipetted into the wells and any IL-4 present is bound by the immobilized antibody. An enzyme-
linked monoclonal antibody specific for IL-4 is added to the wells. Following a wash to remove
any unbound antibody-enzyme reagent, a substrate solution is added to the wells and color
develops in proportion to the amount of IL-4 bound in the initial step. The color development is
stopped and the intensity of the color is measured.
Reagent and Materials
IL-4 Microplate –polystyrene microplate coated with mouse mAb against IL-4
IL-4 Conjugate –polyclonal antibody against IL-4,conjugated to Alkaline phosphatase with
preservatives
IL-4 Standard-recombinant human IL-4 in a buffered protein base with preservatives, lyophilized
Assay Diluent
Calibrator Diluent
Wash Buffer Concentrate
Color Reagent A- stabilized hydrogen peroxide.
90
Sample collection and storage
As for IFN-
Reagent preparation
As for IFN-
IL-4 Standard - The IL-4 Standard was reconstituted with 1.0 mL of Calibrator Diluent. This
reconstitution produced a stock solution of 2000 pg/mL. The standard was allowed to sit for a
minimum of 15 minutes with gentle agitation prior to making dilutions. 500 µL of the
appropriate Calibrator Diluent was pipetted into each tube. The stock solution was used to
produce a dilution series (below). Each tube was thoroughly mixed before the next transfer.
The undiluted standard served as the high standard (2000 pg/mL). The appropriate Calibrator
Diluent served as the zero standard (0 pg/mL).
Color Reagent B -stabilized chromogen (tetramethylbenzidine).
Stop Solution -2 N sulfuric acid.
Plate Covers - Adhesive strips.
Microplate reader capable of measuring absorbance at 450 nm.
Pipettes and pipette tips.
Graduated cylinder.
Distilled water.
Automated microplate washer.
Dilution tubes.
91
Assay procedure
All reagents and samples were brought to room temperature before use.
1. All reagents, working standards, and samples were prepared as directed in the previous
sections
3. 100 µL of Assay Diluent was added to each well.
4. 50 µL of Standard or sample was added per well and covered with the adhesive strip.
Incubated for 2 hours at room temperature.
5. Each well was aspirated and wash, the process was repeated twice for a total of three
washes. Washing was done by filling each well with Wash Buffer (400 µL) using an
autowasher. After the last wash, remaining Wash Buffer was removed by decanting; the
plate was inverted and blotted against clean paper towels.
6. 200 µL of IFN-γ Conjugate was added to each well and covered with a new adhesive
strip. Incubated for 2 hours at room temperature.
92
7. The aspiration/wash was repeated as in “Step 5”.
8. 200 µL of Substrate Solution was added to each well. Incubated for 20minutes at room
temperature. This was protected from light.
9. 50 µL of Stop Solution was added to each well. The color in the wells changed from blue
to yellow.
10. The optical density of each well was determined within 30 minutes, using a microplate
reader set to 450 nm wavelength.
CALCULATION OF RESULTS
A standard curve was Created by reducing the data using computer software capable of
generating a four parameter logistic (4-PL) curve-fit.
93
94
INTERFERON GAMMA(OPTICAL DENSITY)
1.521 0.056 0.057 0.049 0.054 0.06 0.056 0.061 0.056 0.065 0.081 0.031
1.022 0.062 0.062 0.062 0.056 0.078 0.061 0.058 0.061 0.067 0.065 0.062
0.519 0.088 0.069 0.8 0.105 0.113 0.06 0.051 0.086 0.087 0.079 0.065
0.331 0.054 0.052 0.046 0.048 0.044 0.046 0.067 0.061 0.5 0.047 0.053
0.187 0.056 0.048 0.049 0.046 0.049 0.042 0.068 0.043 0.048 0.047 0.059
0.121 0.054 0.063 0.054 0.053 0.063 0.078 0.115 0.125 0.057 0.052 0.075
0.088 0.049 0.054 0.043 0.045 0.05 0.05 0.064 0.083 0.079 0.058 0.064
0.043 0.047 0.043 0.038 0.035 0.04 0.042 0.051 0.037 0.04 0.039 0.047
INTERFERON GAMMA(CONCENTRATION) pg/ml
993.1 6.44 6.88 3.42 5.58 8.18 6.44 8.61 6.44 10.35 17.35 -4.34
518.2 9.04 9.04 9.04 6.44 16 8.61 7.31 8.61 11.21 10.35 9.04
225.4 20.37 12.08 16.87 27.82 31.34 8.18 4.28 19.49 19.93 16.44 10.35
131.1 5.58 4.71 2.12 2.99 1.26 2.1 11.21 8.61 3.85 2.55 5.15
64.37 6.44 2.99 3.42 2.12 3.42 0.4 11.65 0.83 2.99 2.55 7.74
34.87 5.58 9.48 5.58 3.42 9.48 16 32.22 35 6.88 4.71 14.69
15.59 3.42 5.58 0.83 5.58 3.85 3.85 9.91 18.18 16.44 7.31 9.91
0 2.55 3.42 -1.33 0.83 -0.46 0.4 4.28 -1.76 -0.46 -0.9 2.55
95
96
INTERLEUKIN - 4 (OPTICAL DENSITY)
1.392 0.077 0.064 0.07 0.069 0.069 0.067 0.07 0.071 0.069 0.064 0.06
0.941 0.081 0.075 0.081 0.071 0.069 0.071 0.066 0.073 0.07 0.07 0.066
0.608 0.065 0.065 0.066 0.065 0.068 0.065 0.06 0.062 0.062 0.063 0.065
0.347 0.065 0.061 0.06 0.063 0.057 0.062 0.061 0.062 0.062 0.063 0.066
0.219 0.068 0.061 0.065 0.062 0.058 0.059 0.06 0.062 0.062 0.066 0.075
0.155 0.068 0.061 0.061 0.058 0.062 0.064 0.064 0.064 0.067 0.059 0.06
0.107 0.054 0.057 0.054 0.059 0.056 0.056 0.055 0.055 0.055 0.051 0.057
0.052 0.053 0.053 0.063 0.055 0.048 0.056 0.058 0.058 0.053 0.052 0.059
s
INTERLEUKIN - 4(C0NCENTRATION) pg/ml.
2000.1 16.35 7.98 11.79 11.15 11.15 9.88 11.79 12.44 11.15 7.98 5.51
993.9 19.01 15.04 19 12.44 11.15 12.44 9.24 13.73 11.79 11.79 9.24
495.65 8.61 8.61 9.24 8.61 10.51 8.61 5.51 6.74 6.74 7.36 8.61
257.5 8.61 6.12 5.51 7.36 3.7 6.74 6.12 6.74 6.74 7.36 9.24
122.82 10.15 6.12 8.6 6.74 4.3 4.9 5.51 6.74 6.74 9.24 15.01
64.47 10.15 6.12 6.12 4.3 6.74 7.98 7.98 7.98 9.88 4.9 5.51
34.7 1.95 3.7 1.95 4.7 3.11 3.11 1.95 2.53 2.52 0.31 3.7
0 1.38 1.38 7.36 2.53 <0.31 3.11 8.61 4.3 1.38 0.83 4.9
97
Interferon-Gamma 4PL curve, plotting concentration against optical density
98
Interleukin-4 4PL curve, plotting concentration against optical density.
99
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