UNIVERSITY OF NIGERIA, ENUGU

CAMPUS

PROJECT TITLE:

ASSESSMENT OF IMMUNE BIOMARKERS OF

SICKLE CELL DISEASE PATIENTS IN RELATION TO
DISEASE SEVERITY AND BODY MASS INDEX.

BY

OZOCHUKWU OGOAMAKA CHINYERE

Phd/MLS/19/90215

SUPERVISOR:

PROF. SILAS UFELLE

INTRODUCTION
Sickle cell disease (SCD) is a global genetic disorder caused by a mutation in the β
-globulin gene that result in the replacement of a single glutamic acid with valine.
As a consequence, an abnormal haemoglobin, Hbs is produced which polymerizes
under deoxygenation conditions. Hbs polymerization causes changes in the shape
and physical properties of erythrocytes, resulting in hemolytic anemia and the
occlusion of small blood vessels. The release of erythrocyte content into the
plasma by intravascular hemolysis interferes incisively on vasculopathy with the
involvement of coagulation and immune system component, such crises are
spontaneous with recurrent complications and microvascular infarction leading to
episodes of extreme pain, multiple organ dysfunction and infections. The vaso-
occlusion process that occurs in patients with SCD is a complex phenomenon that
involves the aggregation of erythrocytes, which interacts with the endothelium
and other blood cells, decrease nitric oxide (No) bioavailability, oxidative stress
and the release of inflammatory cytokines into the bloodstream such as TNF – α ,
IL-6 and IL-8. Repeated microvascular occlusion, tissue hypoxia (oxygenation-
deoxygenation), and cytokine production perpetuate the chronic inflammatory
state with a pivotal role in the vaso-occlusive crisis and painful episodes. Impaired
leucocyte function, defective humoral and cell mediated immunities underlie the
immuno compromised state of patients with SCD (Pinto et al, 2019)
Sickle cell disorder has been acknowledged to have a global impact by the WHO
with a remarkable public health implication for Africa. Available evidence suggests
4.4 million people have sickle cell disease. While about 43 million are living with
the sickle cell traits. About 80% of sickle cell disease causes occur in sub-saharan
African and the mortality rate for children <5 years of age ranges from 50% -80%
Evidence suggests that about 90% of the world’s sickle cell disease population
lives in Nigeria, India and the Democratic Republic of Congo, where the disease
affects up to 2% of the population.
Sickle Cell diseases is characterized by the presence of sickle-shaped RBCs with
altered biophysical and biochemical properties. Unlike normal RBCs, which have a
biconcave disc shape for flexibility and deformability, sickle shape RBCs lose their
ability to deform and navigate through small blood vessels efficiently, leading to
blockages and reduced blood flow. To compensate for the RBC loss, bone marrow
produces more immature levels of surface adhesive molecules such as
Lutheran/basa cell adhesion molecules (LU/BCAM) and alpha 4 beta 1 ( ∝4B1)
integrin………………. These adhesion molecules facilitate the binding of
reticulocyte to the endothelium lining the blood vessels causing activation of
endothelial cells and the release of inflammatory molecules ……………. Moreover,
reticulocytes in SCD interact with circulating leukocytes, including
polymophonuclear neutrophils and monocytes, leading to the formation of
aggregates and increasing the occurrence of vaso-occlusive crisis (VOC) (Ware et
al, 2017)……….. these interactions are primarily mediated by 4B1 intergrin and
LU/BCAM; reinforcing their adhesion to the endothelium………………. Invitro
studies have demonstrated that sickle-shaped RBC’s can also bind to peripheral
leukocytes, especially monocytes, via erythroid LW/ICAM. 4 and CD44 receptors
these RBC/reticulocyte-leukocyte aggregates interact with endothelia cells,
resulting in endothelial cell activation……………………. Thus, SCD is associated
with increased adhesion of RBC reticulocytes to leukocytes and ECs, which
contribute to the complex pathophysiology underlining SCD pain (Conran &
Belcher, 2018).The highly unstable sickle erythrocytes reduce life span to < 75%
which induces an increased response of bone marrow and therefore increased
erythropoiesis reflected by the circulation of reticulocytes which account for 20%
of the red blood cells in individuals with SCA.
Hemolysis is thought to occur principally via extra vascular process. Heme
and other danger -associated molecular pattern (DAMP) molecules released from
erythrocyte activate the innate immune system. Destruction of red cell membrane
cause exposure of membrane proteins, thereby inciting auto antibody formation.
These antibodies such as IgG anti-band 3 antibodies are believed to promote
erythrophagocytosis. Episodic microvascular occlusion in sickle cell disease even
in steady state results in ischemic cell reperfusion injury which leads to increased
inflammatory tone, elevation in total leucocyte count. It has been increasingly
suggested that innate immune cells including monocytes. Dendritic cells,
neutrophil, eosinophil’s, Basophils. Invariant natural killer (INKT) T cells, platelets
and mast cells, have a role in promoting inflammation, adhesion and pain in SCD,
multiple organ dysfunction and infection. Toll like receptor 4 (TLR- 4) and TLR-2
activte monocytes and macrophages to release inflammatory cytokines which
promote an inflammatory state and activation of endothelial cells. TLR-4
activation on platelets promotes their adhesion to Neutrophils which releaseDNA
to form neutrophil extracellular traps (NET). The circulating blood cells adhere to
the activated endothelium contributing and initiating vaso occlusion (Conran and
Belcher, 2018). The vaso-occlusion process that occurs in patients with SCD is a
complex phenomenon that involves the aggregation of erythrocytes, which
interacts with the endothelium and other blood cells, decreased nitric oxide (No)
bioavailability, oxidative stress and the release of inflammatory cytokines into the
blood stream such as TNFα ,IL6 and IL8. Episodic microvascular occlusion in sickle
cell disease even in steady state results in ischemic cell reperfusion injury which
leads to increased inflammatory tone and elevation in total leucocyte count.
(Idzko et al, 2014)
It is well documented that children with SCD were underweight, particularly those
with Hbss Poor growth in children with SCD is complex and multiple factors are
likely to contribute, including increased energy and nutrient requirements
resulting from increased hemolysis and erythropoiesis and elevated protein
turnover.
Reports in the USA found that 19% -22% of children with SCD were overweight or
obese. Nutritional status and growth may have improved in SCD due to the
increased use of SCD-directed therapies and lifestyle factors. Preventing obesity
in children with SCD is vital as obesity is associates with obstructive sleep apnea
which increase the risk of nocturnal hypoxia and vasocclusive episodes. Episodic
microvascular occlusion in sickle cell disease even in steady state results in
ischemic cell reperfusion injury which leads to increased which leads to increased
inflammatory tone, and elevation in total leucocyte count.
STATEMENT OF THE PROBLEM
It has been increasingly suggested that acquired immune cells including 1gM,
1gG, dendritic cells, have a role in promoting chronic inflammation, adhesion,
multiple organ dysfunction, increased susceptibility to infection, impaired vaccine
response and pain in SCD. Despite advancement in management, SCD remains a
significant public health burden. The complex interplay between immune
biomarkers, disease severity and treatment outcomes is poorly understood,
hindering the development of effective therapeutic strategies. In Nigeria, many
sickle cell disease patients die due to low immunity which could be as a result of
poor diet, environment and social economic status.
Evidence from various studies suggests that patient with sickle cell disease (SCD)
have altered components of hemostasis such as platelet function, pro-coagulant,
anti-coagulant, and fibrinolysis pathways and modulate inflammation such as
acute or chronic inflammatory state. These components may alter based on race,
regions or environment.
JUSTIFICATION
i. SCD patients exhibit altered immune responses, making them more
susceptible to infections and auto immune phenomena.
ii. Activated immune cells and coagulation cascade contribute to
endothelial damage and dysfunction.
Considering the divergent information about the complex immune response
on the progression of SCD, in this study we propose to assess some immune
biomarkers of SCD in relation to disease severity and body mass index (BMI)
which could help to uncover novel insights into disease mechanisms,
identify potential therapeutic targets, nutritional interventions and improve
patient outcomes.

AIMS
SPECIFIC OBJECTIVE
- To assess the level of some immune biomarkers (IgG, IgA, IgM, dendritic
cell), protein C, protein S and correlate them with disease severity, clinical
outcome and treatment responses in SCD patients.
- To evaluate the relationship between BMI and immune biomarker levels in
SCD patients.
- To determine the predictive value of some immune biomarkers for disease
complications (Hbf, LDH, reticulocytes, acute chest syndrome, stroke) in
SCD patients.
RESEARCH QUESTION
- Are there differences in immune biomarker profiles between SCD patients
with different genotypes (HbSS, HSC)?
 - Does BMI influence the association between immune biomarkers, protein C
and S and disease severity in SCD patients?
- Do changes in BMI impact the effectiveness of hydroxyurea therapy on
immune biomarker levels in SCD patients?

LIMITATION OF THE STUDY

- Small sample size – Many studies have limited sample sizes, making it
challenging to draw conclusion and generalize findings.
- Variability in immune response – Immune responses can vary widely
between individuals making it challenging to identify consistent biomarker
patterns.
- Limited standardization – Different studies may use different methods for
measuring immune biomarkers making comparisons challenging.
- Need for longitudinal studies – Longitudinal studies are needed to
understand how immune biomarkers change over time and in response to
treatment.
- Integration with clinical data and outcome- other factors such as age, sex
and co-morbidities may influence immune biomarker levels and disease
severity.

LITERATURE REVIEW
Sickle cell disease is an inherit disorder of Hb that results in chronic hemolysis.
Affected individuals are prone to frequent and sometimes several infections.
Several factors predisposing sickle cell disease individuals to infection have been
reported and included abnormalities of opsonins and antibody production, defects
in the alternate compliment pathway, leucocyte functions and cell mediated
immunity. The spleen which is uniquely positioned to bring circulating antigen
with close contact with antigen presenting cells of the reticulo endothelia system
in the spleen is also lost early in life as a result of fibrosis resulting in recurrent
vaso occlusive crisis leading to defective function of the organ.. Chudwun et al
(2009) in their study from patients with sickle cell disease concluded that immune
dysfunction is evident in Nigerian SCD patients. Inflammatory or environmental
stimulus including infection, hypoxia, epidemiological evidence indicates that VOC
is frequently initiated by dehydration or acidosis.
The diverse clinical heterogeneity of SCD is related to two main pathogenetic
processes: Chronic hemolysis and high viscosity/vascular occlusion. Infarctile
events in SCD results from eythrostasis caused by rigid sickled cells in various
vascular beds especially organs with sluggish blood flow such as spleen and bone
marrow capillaries which are about 2-3 um in diameter. Sickle cells due to loss of
flexibility are unable to transmit the microvasculature, hence vessel occlusion.
Aside from these mechanical processes, sickle cells are also shown to exhibit
increased adhesiveness to vascular endothelium, leucocyte, platelet, and
themselves. Sickle reticulocyte are even more adhesive to the endothelium than
sickle discocytes. Molecular interactions between the red cells and the vascular
endothelium include CD 36 and thrombospondin, VLA 4 and VCAM-1 respectively.
Fibronectin & Von will brand factor are also involved in these interaction. It is
known that increased adhesiveness of different cellular surfaces with formation of
heterocellular aggregates is believed to propagate the phenomenon of vascular
occlusion, especially in the post capillary venules.
Innate immune cells possess a larges repertoire of receptors for cytokines and
chemokines, which enable them to respond to various inflammatory signal and
the activation of innate immune cells in SCD is probably promoted by both chronic
and acute inflammation (Keikhaei et al, 2013). Once activated, innate immune
cells release a wide range of cytokines and chemokines, thus promoting a vicious
cycle of immune cell recruitment and activation ( Lanaro et al,2009, Qauri et al,
2012). Pro-inflammatory cytokines, produced mainly by monocytes and
macrophages but also by platelets and other innate immune cells, can activatethe
endothelium along with heme resulting in increased expression of adhesion
molecules ( Pathare et al, 2004, ). Neutrophils are crucial factors in endothelial
adhesion but monocytes and platelets also contribute to decreased blood flow and
vaso-occlusion through the formation of red blood cell- monocyte, platelets-
monocytes and platelet- neutrophils aggregate (Zhang etal, 2016). Moreover,
patients with SCD are predisposed to infections mainly due to splenic dysfunction
which can favor deficit in innate, humoral and cellular immune function
(Ochocinski etal, 2020).
Patients with SCD are more susceptible to infection due to splenic hypofunction
and impared immune function. In addition to the deficiency involved in innate
immunity including insufficient opsonization and phagocytosis of bacteria,
evidence of changes in T and B lymphocyte dysfunction in SCD has been reported.
These changes include reduction in the proportion of circulating CD4 + and CD8+T
cells, defects in regulating T cells, the polarization of cells to a T helper profile and
the loss of Immunoglobin (Igm).
Immunoglobins are serum protein that help fight infections and also involve in
activation of complement system. Several investigators have reported varying
serum concentration for these immunoglobin in steady state asymptomatic sickle
cell patients. Dieye et al, natta and ourschoom reported significantly high serum
IgA and IgG, but reduce and normal serum IgM levels in their separate studies,
respectively. While Adeola et al reported significantly high serum IgA and IgG,
Mohamed et al did not find any significant difference in the complement and
immunoglobin levels in their SCD subject. Even in steady state, SCD is a chronic
inflammatory conditions, this induces oxidative stress which further contributes to
progressive tissue damage. The leucocytes in SCD also express high level of L.
selectin exposure on the red cells surface which further contributes to progressive
tissue damage.
Evidence from various studies showed that patients with sickle cell disease (SCD)
have altered components of hemostasis such as platelets function, pro-coagulant,
anticoagulant and fibrinolysis pathways, that modulate inflammation such as
acute or chronic inflammatory state. These components may alter based on race,
regions or environment.
Protein C and protein S are glycoproteins, predominantly synthesized in the liver
and they are important competent natural anticoagulant system in the body. They
are vitamin K dependent and serve as essential components in maintaining
physiologic hemostasis. Protein C and S deficiencies can be secondary to inherited
gene mutations or due to acquired causes. In the healthy general population, the
incidence of asymptomatic protein C deficiency is I in 200 to 500 individual, while
clinically significant venous thrombo embolism is estimated to occur in 1 in
20,000 individuals. No clear racial or ethnic predispositions are known.
Approximately 160 mutations in the protein C gene (PROC) located on
chromosome 2q14.3 have been described. Protein C deficiency may also be
acquired by one of several mechanisms. Newborns may have physiological low
levels of protein C at birth. Levels have been reported to be as low as 35% in
otherwise healthy full-term infants. This is an age-related acquired form of protein
C deficiency: Protein C levels increase to the lower level of the adult referred
range by 6-12 months of age. Other causes of acquired protein C. deficiency
include inflammatory or infectious processes, liver disease, malignancies,
coagulopathy and vitamin K deficiency.
The exact prevalence of proteins S deficiency in the general population is
unknown. However, some studies have estimated a prevalence of 0.0 3% to
0.13% healthy individuals. Because of the alteration of nearly every component of
hemostasis, namely platelet function, the procoagulant, the anticoagulant and the
fibrinolytic systems in favor of the procoagulant phenotype, SCD is frequently
referred to as a hyper cooagulable state ………. Abnormal exposure of
phosphatide serine (ps) may occur due to repeated cycles of sickling and
unsickling linked to the polymerization and depolymerization of Hbss that results
in the production of terminal spicules or macrovesicles with exposed PS.
Moreover, external exposure of PS alters the adhesive properties of sickle blood
cell (RBC) and appears to be involved in the hemostatic changes observed in
SCD………. It has also been observed that plasma levels of prothrombin fragments
are associated with the number of circulating PS positive RBC………. Patients with
SCD have increased levels of markers of thrombin generation and thrombin-anti
thrombin complexes in the no crisis state……….. decreasing levels of natural anti-
coagulant proteins are also observed in SCD………. Therefore, the levels of protein
c and s are decreased in the no crisis steady state and perhaps even more so
during acute pain episodes………. There has been some studies with variant result
considering the use of anticoagulant drugs among sickle cell patients and the
positive effect of Heparin has been reported. (Bashanuri etal, 2007).
Protein c and its cofactor, proteins are the main anticoagulant proteins that inhibit
coagulation by inhibiting factors V and VIII (Guptal etal 2022). The anticoagulant
asbility of protein C deficiency has been reported from 0.2% in general population
to 2% in sickle cell patients and considering the proteins these members are from
0.7% in general population to 15% among patients with sickle cell anemia (Tupta
etal 2022).
According to Amiral etal (2019), patients with sickle cell disease had reduced
protein C and S levels and increased activity of factor V Leiden compared to
control which might cause hyper coagulation state among these patients.
Dendritic cells (DCS) are antigen presenting cells (also known as accessory cells)
of the mammalian immune system. Their main function is to process antigen
material and present it on the cell surface to the T- cells of the immune system.
They act as messenger between the innate and the adaptive immune system.
They are the main cells responsible for adaptive immune response activation,
polarization and regulation. They are activated by both the pathogen-and
damage- associated molecular patterns which can be released by tissue necrosis
or hemolysis which are frequent occurrences in patients with SCD. Change in DC
activation or function may, therefore be responsible for previously observed
dysfunction in the T lymphocyte response. DCs can produce pro- or anti-
inflammatory mediators depending on the tissues, the situation, and the cell
subpopulation, as DCs form a heteregenous population of cells with different
functions.
Patients with SCD demonstrate dysfunctional T and B lymphocyte responses and
they are more susceptible to infection. Dendritic cells are the main component
responsible for activating and polarizing lymphocyte function and are able to
produce pro-inflammatory cytokines found in the serum of patients with SCD.
Minimal studies have thus far been devoted to these cells. Renata sestic costa et
al 2021, identified the subpopulations of circulating DCs in patients with SCD, and
found that the blood stream of the patients showed higher numbers and
percentage of DC’s than that of healthy individuals.
Among all the main DC’s subsets, inflammatory DC’s (CD14+DCs) were
responsible for this rise and correlated with higher reticulocyte count. The patient
had more activated monocyte derived DC’s (Mo-DCs), which produced MCP-1, IL-6
and IL-8 in culture. They found that CD14+ MO-DC subset present in culture from
some of the patients was the more activated subset and was mainly responsible
for cytokine production and this subset was also responsible for IL-17 production
in co-culture with T-lymphocyte. They suggested an involvement of heme
oxygenase in the upregulation of CD14 in Mo-DCs from the patient, indicating a
potential mechanism for inducing inflammatory DC differentiation from circulating
monocytes in the patients which correlated with inflammatory ssscytokines
production, T-lymphocyte response skewing and reticulocyte count.
IMMUNE BIOMARKERS
Biomarkers are a broad category of medical indicators that can be measured
objectively to determine different medical states or responses to an intervention.
They can be used for basic diagnosis, therapeutic prognosis or as end points in
clinical studies. Biomarkers may consist of unclerical acids, proteins, metabolites
and even images as long as they can be recorded and measured consistently and
objectively.
1. The majority of those genetic marks are single nucleotide polymorphisms
(SNPS) although more recently next generation sequencing techniques are
being successfully applied in particular for cancer mutation detection
(Edcefsenetal 2014).
In contract to the use of genetic markers an estimated 325 proteins can be
reliably measured in human plasma by conventional chnical cherusly or Enzyme
linked immunoserbent assay (Elisa) with concentian ranging between 10 -3m and
10-12m of these protains an estimated 175 are currently approved by the FDA as
clinical biomarkers highlighting the advanced maturity of these technological and
also potentically their greater chinical relevance as compared to genetic
biomarkers (French etal 2015).
2. Immunological biomarkers such as serum cytokines chernoknes, adipocy to
kines, soluble forms of cell receptons and immune activation and play and
important role in the function of the immune system. Complex interaction
between immune cells of imale and adaptive immune systems are modified
by the release of a variety of cell mediators that trigger inflammatory
responses which help to illuminate and destroy foreign antigens. They serve
numerous functions within our immune system and interact with
specific cell types that corresponds with different stapes of
disease. Changes in the levels of these biomarkers along with
changes in I ymphocytes value by reflecting under lying disease
conditions French enal (2015). Thus, it is essiesitial to be able to
detect laboratory and clinical relevant changes by accurately
measuring those blood biomarkers, making them potentially
beneficial for the monitoring, diagnosis and follow up of various
diseases. Clinical usefulness of these biomarkers depends upon,
the ability to account for a significant portion of the disease
being evaluated the ability to be accurately, reproducibility of the
assey for widespread application 2003. Since blood level of
immune digitalbiomarkers differ widely between individual based
on their gender, age and other factors baseline concentration of
biomarkers should be established for healthy individuals. In
addition, the suitable biomarker should have a temporal intra
individual variability in healthy subjects otherwise any significant
changes might met necessary reflect disease process.
3. (Fraser C.G, 2001)
Immune globules as immune response biomarkers
Immune globules also know as anti-bodies are proteins that can
be used as biomarkers to monitor the immune response. They
are produced by plasma cells in response to foreign substances,
like viruses or bacteria. They bind to these harmful substances;
called antigen and mark them for destruction.
The immerge or antigen reacts with a B-cell receptor (BCR) on
the cell surface of B lynphaseayt. A siginal is produced that
directs the activation of transcription factors to strucmlate the
synthesis of antibodies, which are highly specific for the
immunogenic that stimulate the synthesis of antibodies, which
are highly specific for the immugen that stimulate the B cell.
Furthermore, one done of a B cell makes an immune globules
(specificity).
Besides the immune system remembers the antigen that caused
a previous reaction (memory) due to the development of memory
B cells. These are intermediate, differentiated B cells that can
quickly become plasma cells.

In addition, circulatory anti-bodies recognized antigens in path

physiology of immune globulin. All antibodies exhibit one or more
function binational including activation of the complement
system, opsonisation of microbes to be easily phagocytosed,
prevention of attachment of the microbes to muciosal surfaces
and neutralization of texnis and viruses……………… (1)
There are five types of 1gsin humimian 1gm, 1ga, 1ge,1gd
immune globulin M (1gm).

1gm has a molcula weight of 970kd and an average surum

concetration of 1.5 mglml.

It is mainly produced in the primary immune respond to

inflections agents or antigens. It is a pentamer and activates the
classical pathway of the complement system. 1gm a regarded as
a potent agglutina. It is a pentamer and activates the classical
pathway of the complement system. 1gm a regarded as a potent
agglutinina leg auti A blood, respectively and a monomer of 1gm
is used as a B cell receptor (BCR) ……………. (3).

Immune globin G
IgGo a monomer with an approximately molecule weight of
146kd and a sermon cencen of a, ough 1gG is said to be divalent,
i.e it has two identical antiganbinding sites that comprises 2l
chains and 2H chains landed by divnefide bords. IgG is synthesis
mostly in the secondary immune response to pathogens.

1gG can activate the classical pathway of the complement

system and it also is highly protective. The four subcleases of
1gG include lgG1, lgG2, lgG3 and lgG4, lgg1 is around 65% of the
total lgG. lgG2 forms an important host defense against bacteria
that are encapsulated. lgG is the only immunoglobulin that
crosses the placats as its Fc mortion binds to the receptors
present on the surface of placents.
Protecting the neonate from infectious diseases lgG is thus the
most abundant anybody present in newborns
………………………… (4)

Immunoglobulin A
lgA appears in 2 different molecular structures: monomeric
(serun) and dimeric structure (secretory). The serum lgA has a
molecular weight of 385kd and a mean serum concen tration of
0.05 mylaul. lgA is the major antibody in secretive found
insalivia, tears, colostrum, intestinal, genital tract and respiratory
secretions. It appears in mucosa memberaous as a dimer (with a
J chain when secretal) and protcult the epithecial surface of the
gestive, repiratory and genitourinary systems. lgA possesses a
secretory component that prevents its enzymatic digestion. It
activates the alternaure pathway of activation of the complement
system (5). The lgA immune Globus are further subdivided into 2
subclasses lgA1 and lgA2.

Role of Biomarkers in the major complications SCD

Pain crisis or VOCs are the most common clinical manifestation of
SCD VOC is also the most common cause of hospitalization,
posing a major health burden in this population – in addition
pregnant painful episodes and chronic pain significantly
decreased the quality of life (QOL) in patience, with SCD.
Historically, it has been shown that advancing age, high
hemoglobin, low fatal honeybun (HB) and coinheritance of
thalassemia are associated with increased number VOCs an
children and adults with SCD (Oherie etat, 1998) among the
experimental biomarkers, elevated plasma thrombosporichi 1,
secretory product of activated platelets that promotes sickle
vascular adhesion, plasma levels and in balance of aphelion
(vasodilator) and endothelin 1 (vasoconstrictor) are associated
with a vaso-occusue phenotype (smith etal, 2015).

Flotel Hemoglobin in sickle cell Anemia

Hbf is the most powerful modulator of the clinical and
hermatologic features of sickle cell anemia (defined as
homozygosity for glu6val in the B globin gene or HBB). To protect
against various complication of disease, different cercentration of
Hbf were postuated to be required, although any increment in
TBF had a beneficial effect on mortality (Paikarieta 2018) higher
Hbf levels were associated with a reduced rate of acute painful
episodes, fever leg ulcer, less oseonegosis, less frequent acute
chest syndrome and reduced disease sevenly. However, Hbf level
had a weak or no clear association with priapis in, urimie
albumim excretion, strokes and silent cereblal infarction systemic
blood pressing and perhaps sickle vasculopathy (stariberg etal,
…), the failure of HBf to modulate unformly all complication of
sickle cell diseas might be releted to the pathophysiodge events
that impact the likelihood of developing these complication.
Many epidemiologic studies suggested that diease complication
most closely linked to sickle vaso occlusion and blood viscosity
were robustly related to Hbf concentration whereas
complications associated with the intensity of hemolysis were
less affected (kato et al) although HBF is protective for leg ulcers,
one complication closely associated with hyperhemolysis ............
After defining hyper hemolysis by higher aquatic of serum lactic
dehychogenes and controlling liver disease by examining only
patients with normal serum alanine aminotransferase, HBF levels
were lower in patients with the highest quartile compared with
the lowest quartile of hemolysis (Soloveige et al 2010).
Even when total Hbf levels are high perhaps intravascular
hermolysis of crythrocte containing little or no Bhf leads to
sufficient nitric oxide scaverigeing by plasma hermoglobin to
provoke hemolysis related complication fetal heamoglobin (BhF:
X2X2) encoded by two nearly identifical R-globingenes (HBG 2
HBG1) that are part of the Bglobrigene (HBB) cluster comprises
70 to 90% of the hemolysatein newborns talking to 1% after 12
months (Bauer e t al 2021) the switch from fetal to adult.
Hemoglobin synthesis in sickle cell. Anemia 5 to 10 years and is
rarely complete. The sickle hemoglobin gene is formed on fire
common genetic background or haplotypes: patients of African
descent have Bantu, Benin, Cameroon or Senegal haplotypes: In
most of these patients a stable Hbf level of -4 to 10 % is reached
at approx 5yrs of age. In individuals of middle eastern or Indian
extractive the Arab-Indian haplotype
9. Stable Hbf level of approx. 17% are achieved at approx. 10
years of age...... secretive in treatment-naïve adult of African
descent HBF levels between 15 and 30% are present; This can be
accompanied by disease (Steinberg, et al, 2013)

LACTATE DEHYDROGENSES (LDH) in sickle cell disease.

Lactate dehychegenase is a marker of hemolysis and endothelial
activation in sickle cell disease SCD.LDH levels can be used to
diagnose and predict complication in SCD. Its serum levels are
mildly elevated in extravascular hemolysis such as immune
hemolysticanemia, but are substantially elevated with
intravascular haemolysis such as thrombotic thrombocytopenic
Purpra and paroxysmal noctumial hemoglobinuria (Tabara 1992).
Although in sickle cell disease two thirds of hydrolysis occurs
extravasculary, the reviewing one third of red cells hemolyze
intravasculary potentially releasing as much as 10g hemoglobin
per day into blood plasma.
This robust hermolytic rate increase even further during
vasoclusive pain crisis (VOC). Elegant biochemical studies
performed 35years ago have demonstrated significant increases
in serum LDH and Plasma heameglobin level, the gold standard
marker of intravascular hemolysis during VOC ............(13) this
hyper-hemolysis recently has been formed by labelled red blood
cell studies that reveal further decreases in red blood cell
survival during VOC in patients with sickle cell disease.
Lactate dehychrogenase is one of the enzymes of the glycotytic
pathway that cataluzes the conversion of pyruvate to lactate with
concurrent C conversion of NADH to NAD +. It is a ubiquitous
enzyme found in all tissues. Serum LDH exists in 5 separable
Isoenzymes numbered 1-5 according to their electrophoretic
mobility (Neely et al 1969) the distribution of the 5 Iso enzymes
is not uniform across body tissues. LDH1 and LDH 2 are found
primary RBC’s and heart muscle, LDH3 is highest in skeletal
Muscle and Liver (Pinaus et al 2011) the routine determination of
serum LDH includes all of its Isoenzymes. Srum LDH is usually
elevated in sickle cell anaemia in the steady state Najim &
Hassan 2011 during painful vasooclusive crises (VDCs), the LDH
may increase further in some patients because of
hyperhemolysis as shown by RBC survival studies. However, the
increase in LDH during VOCs is not always because of hemolysis
Ballas & Marcohna 2006), Neely et al found that the increase in
serum LDH was not correlated with Plasma Hb level indicating
that the sources of LDH is not secondary to hemolysis but reather
to tissue damage most likely PSM infarction tosanovic et at 2012
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MATERIALS AND METHOD
3.1. Area of Study
3.2. Subjects
The study shall include 150 subjects categorized into: the sickle cell disease
patient group with 50 subjects in steady state, consisting of patients who are not
in crises and had not received blood transfusions in the preceding 3 months, and
apparently healthy individuals group with 50 (HbAA & HbAs) subjects without any
apparent diseases. The SCD subject group will be composed of both genders with
age range 18-65yrs. The apparently healthy group will be composed of both
genders with age range 18-65yrs.
For inclusion, patients will be required to be in steady state, that is, none had
received a blood transfusion 3 month prior to inclusion and no acute events, pain,
hospitalization, infectious or acute clinical symptoms or crises at least 3 months
established by a careful history and complete physical examination by a
physician.
Exclusion criteria for these participants were recent viral infection, diabetes, auto
immune disease, immune deficiency, pregnancy, obesity, presence of neoplastic
disease, steroid treatment, acute pain/bone crisis, displayed ulcers and
hydroxyurea use.
Subjects with other haematological diseases (leukemia and other anaemias),
subjects undergoing or have been treated for cancers, subjects with inflammatory
and infectious diseases such as malaria, pneumonia, pharyngitis, tonsillitis,
bronchitis etc will not be included.
3.3 STUDY DESIGN
All protocols and consent forms shall be approved by the ethical committee on
research from the UNTH and ESUTH teaching hospital in the Enugu metropolis.
The subjects involved in the study will sign the informed consent with absolute
confidentiality.
Social Demographic Characteristics
The patients will be interviewed and questionnaire given to collect such data as
last episode of crisis, frequency of crisis, previous hospitalization/ complications,
drug history, family history of haemoglobinopathies and social history. Some
demographic data (body mass index, Age, gender, weight, height, sex) of both the
subjects and controls will be documented.
Sample Collection
3.4 About 10ml of blood sample will be drawn from both patient and controls.
3mls will be mixed with 0.2ml sodium citrate for coagulation studies (protein c &
s), while 4mls will be put in a plain tube for serological studies and 3mls put in
EDTA container for hematological parameters.
3.5 Enzyme linked immunosorbent assay (ELIZA) will be used to assay the
immunoglobins, protein c and s while full blood count auto analyzer (mythic 22)
will be used to assay the hematological parameters, Reticulocytes will be done
with brilliant cresyl blue solution. The Hb electrophoresis will be done with high
performance liquid chromatography (HPLC) to assay Hbf and other sickle cell
disease type.
3.6 Statistical Analysis will be done using two-way analysis of variance (ANOVA).

1.1 Role of Biomarkers in Major Complications of SCD

Biomarkers provide insights into the progression and management of SCD by helping predict complications and
monitor treatment efficacy. Key biomarkers include:
- Lactate Dehydrogenase (LDH): A marker of hemolysis and tissue damage, elevated LDH levels are strongly
associated with disease severity, VOCs, and endothelial dysfunction.
- Fetal Hemoglobin (HbF): High HbF levels inhibit HbS polymerization, reducing VOCs, chronic pain, and other
complications.
- Inflammatory Cytokines: Elevated levels of TNF-α, IL-6, and IL-8 exacerbate chronic inflammation and
endothelial cell activation, perpetuating disease severity.

1.2 Role of Immunoglobulins in Immune Response

Immunoglobulins (Igs) play a critical role in immune defense mechanisms:

1. IgG: The most abundant antibody, involved in pathogen neutralization, opsonization, and complement
activation. It crosses the placenta, providing passive immunity to neonates.
2. IgA: Protects mucosal surfaces by neutralizing pathogens and preventing their adherence to epithelial cells.
3. IgM: Acts as the first responder during infection, efficiently activating the complement system.

1.3 Genetics of Immunoglobulins

Immunoglobulins are produced through a process of V(D)J recombination in B cells, which generates a diverse
antibody repertoire. This process, along with somatic hypermutation and class-switch recombination, allows
immunoglobulins to adapt and target a wide variety of pathogens effectively.

1.4 Relationship Between Immune Biomarkers and Disease Severity

Immune biomarkers are closely linked to the pathophysiology of SCD:

- Cytokines: Elevated pro-inflammatory cytokines are associated with VOC frequency and increased risk of
organ damage.
- Immunoglobulins: Altered Ig levels reflect the chronic inflammatory state and compromised immunity seen in
SCD patients. Low IgM levels and elevated IgG are commonly observed.

1.5 References

1. Azan, T., et al. (2023). Emerging methods in biosensing immunoglobulin G. Sensors (Basel), PMID: 36679468.
2. Najib Aziz, R. D., Anthony, W. (2019). Biological variation of immunological biomarkers in healthy individuals.
BMC Immunology, 20(33).