SICKLE CELL DISEASE

THE INDIAN PERSPECTIVE

From Lab. To Clinic, And Research To Policy

V L GUPTA

i
SICKLE CELL DISEASE
THE INDIAN PERSPECTIVE
From Lab.to Clinic, Research to Policy

V L GUPTA
MD, PhD, BA(SOCIO), MNAMS, MGSA,FICE, FICP, FISN, FASN
(NEPHROLOGY)
CONSULTANT NEPHROLOGIST,AVANTI INSTITUTE OF CARDIOLOGY
Ex.VICE CHAIRMAN ZTCC, NAGPUR
Ex. PROF. & HEAD, DEPT. OF NEPHROLOGY, GMC, SSH, Nagpu
Ex. DIRECTOR, CEU, GMC, Nagpur

Mailing Address
Dr. V.L.Gupta, 302, Giri Gaurav Apartments,
C-9 Ambazari Hill Top Lay Out
Nagpur, 44 00 33 Maharashtra
Contact: 09822228016 E-mail: vishwanathgupta14@gmail.com,
vlgupta14@rediffmail.com

ii
 CONTENTS

About Author & Book IV

Publications V
Original Contributions VII
Research Contributions IX
Sickler’s Lament XI

PAGE NO.
Chapter I Historical Aspects 1−11
Chapter II The Epidemiology of Sickle Cell Gene 12−34
Chapter III The Sickle Haemoglobin, HbS, 35−49
Process of Sickling, and Sickled Erythrocytes

Chapter IV Sickle Cell Crisis 50−73

Chapter V Systemic Manifestations of Sickle Cell Disease 74−109

Chapter VI Management of Sickle Disease 110−140

References 141-197

iii
ABOUT AUTHOR

DR. V. L. GUPTA, MD, Ph.D., BA (SOCIO), MNAMS, MGSA, FICP, FICE, FISN, FASN, is
consultant nephrologist at Ashwini Kidney Dialysis Center, and Avanti Institute of Cardiology,
Nagpur. He is Ex-Professor & Head dept. of nephrology, Super Speciality hospital & PG Institute
of Medical Sciences, Nagpur. He has also been, Director, Clinical Epidemiology Unit, GMC,
Nagpur. He has UG, & PG teaching experience of over thirty-two years in medicine and
nephrology. He has been UG & PG examiner across universities in India. He was a postgraduate
guide for MD, DNB, and Ph.D. for RSTM university Nagpur.

Winner of research awards & medals, He is a recipient of several oration and

lecturership awards at National level, including, “Searle Oration” API 2004. He was awarded,
“INCLEN, USAID, fellowship in Clinical Epidemiology, in 1987-88, during which he was part of
Master’s program and was trained in Clinical Epidemiology, Research Methodology, at UNC, NC,
USA, 87-88, besides he has received several other fellowships at national & international level.
Being member over dozen professional bodies, and scientific societies. He has been heading
several of them. His name has been included in “Asia’s Who’s Who” of Medicine, 2014. He has
authored, edited, and published four books, beside contributing to other published monograms
& Books, has published over 100 research papers. Last year, received award from Maharashtra
Medical Council for exmplery contribution to medical education.

He started working on sickle cell disease in year 1975. In last 45 years has worked on
several aspect of sickle cell disease. Has 29 publications on the subject, and has contributed to
couple of monograms on the subject. He was awarded PhD in this subject, and has submitted
dissertation for award of DSc in this subject of sickle cell disease. He received several awards
and orations at national and international level. Received grants from Indian Council of Medical
Research and USAID- INCLEN- ROCKFELLER foundation for research projects on the subject of
sickle cell disease. He has contributed in drafting. “Maharashtra Sickle Cell Control and
Prevention Program” for Govt. Of Maharashta.

This book is the compilation of important milestones, history landmarks, India specific
epidemiological data, clinical variations in presentation, research done and it’s impact, and
future directions, so that what can be done for these patients, which might help to formulate
policies and implement them for betterment of the suffering population.

iv
PUBLICATIONS ON SICKLE CELL ANAEMIA

1. Saoji AM, Raichur BS, Gupta VL, Dubey GK,: Fibrinogen and MISFI–An
Immunohaematological study in sickle cell disorder, Proceed. AI Immuno Con. Cuttuck,
1978
2. Dubey GK, Gupta VL and ChaubeyBS: Radiographic changes in osseous structures in
patients with sickle cell anaemia with crisis. JAPI:30:764;1980.
3. Gupta VL, Dube GK, and Choubey BS.: Spleenic vein thrombosis in homozygous sickle
cell disease (case report). Current Med. Pract. 24:419; 1980
4. Gupta VL., Dubey G.K.and Choubey B. S.:Clinical study of sickle cell crisis, Ind. Med. Gaz.
CXV, 145, 1981.
5. Gupta VL, Choubey BS, Dube GK, and Saoji AM.: Platelet functions, fibrinogen and MISFI
An immunohaematological study in sickle cell crisis. JAPI, 29, 183, 1981
6. Gupta VL,Borse SD, Dubey GK, and Chaubey BS.: RBC survival in sickle cell anaemia.
Proceed. XXXVI APICON, 1981
7. Gupta VL, Dube GK, and Choubey BS. :Sickle cell anemia clinical profile in Central
India.Jour. Acad. Med. Scienc. Nagpur. 1, 5, 1981
8. Saoji AM, . Gupta VL, Dubey GK, and Kelkar SS. : Hepatitis B surface antigen Hbs/Ag and
antiHbs/Ag in sickle cell haemoglobinopathy. Ind.Med.Gaz.XCV:158;1981
9. Gupta VL, Dube GK, Choubey BS : Two cases of salmonella oesteomylitis affecting spine.
CLINICIAN, 46:308.1982
10. Saoji AM, Gupta VL, Dube GK; Fibrinolysis in patients with sickle cell crisis, inhibition of
fibrinolytic activity in patients with vc with infection Ind. jour. path. micro;23:55,1982
11. Gupta VL, Dubey GK, Chaubey BS, and Tungar VS. Zinc deficiency in sickle cell anaemia,
a preliminary report. Jour. Academ. Med. Scienc. Ngp. 1:10;1982
12. Gupta VL, Dubey GK, Chaubey BS and Waghela JD. N-Phenylethylanthranilic acid in
patients with sickle cell crisis. JAPI. 30:764;1982
13. Waghmare BG, Gupta VL, Deshmukh PY, and Dubey GK, Iron metabolism in patients
with sickle cell anaemia. Proceed. XXXVII, APICON, 1982
14. Gupta VL, Chaubey BS. Zinc deficiency in relation with RBC survival and it’s effect on
sickling process and menifestations in sickle cell anaemia. Dr. SS Mishra memorial
award and medal 1983 NAMS, New Delhi 1983.
15. Attal HC, Gupta VL, and Salkar HR. Budd Chiari syndrome in patient with sickle cell
trait (case report) JAPI 32:526;1984
16. Jajodia PB, Gupta VL, and Waghmare BG, Left ventricular dysfunction in sickle cell
anaemia JAPI 33:40;1985
17. Gupta VL (1986) Zinc deficiency in sickle cell anemia it's effect on cell membrane, course
of disease and future place in management. A Thesis for PhD, medicine, Nagpur
University.
v
18. Gupta VL, and Chaubey BS. Efficacy of zinc therapy in management of sickle cell crisis
Ind. Jour. Med. Res. 86:803-807;1987
19. Gupta VL, Dubey GK, Chaubey BS. RBC survival, Zinc deficiency, and efficacy of zinc
therapy in sickle cell disease. Thalasaemia = pathophysiology and management Part A,
Alan R. Liss Inc. New York 1987
20. Gupta VL, Zinc in sickle cell anaemia, Bull Research Soc, GMC, 1989-90
21. Gupta VL, and Fletcher H. Management of sickle cell anaemia, A review. Ind. Jour.
Haemat. 7:1-5;1989
22. Gupta VL, efficacy of zinc therapy in prevention of crisis in sickle cell anaemia proceed.
INCLEN, Mexico, 1990
23. Gupta VL, Study of precipitating factors of sickle cell crisis, Proceed. INCLEN,
Mexico,1990
24. Gupta VL, Chaubey BS. Electrokinetic potential of RBCs which varies according to the
blood group as well as disease such as sickle cell anaemia. JAPI 38:191-2;1990
25. Gupta VL. Pathophysiology of sickle cell disease. Medicine update, 1995, APICON
1995
26. Gupta VL, Epidemiology of sickle cell and profile in central India, epidemiology of sickle
cell gene in India. Maharashtra sickle cell prevention and control program, DMER,
Mumbai, 2002
27. Gupta VL. A update in management of sickle cell disease, editorial, MGIMS, Jour. 2006
28. Gupta VL: Management of sickle cell anaemia, zinc therapy, and electrokinetic
potential of red blood cells . A treatise submitted for “D.Sc.” medicine, Nagpur
university, 2007

vi
Original Contributions
1. Precipitating factors are identified and studied in detail

Publications: Gupta VL., Dubey G.K. and Choubey B. S.:Clinical study of sickle cell crisis, Ind.
Med. Gaz. CXV 145, 1981.
Gupta VL, Dube GK, and Choubey BS. :Sickle cell anemia clinical profile in Central India. Jour.
Acad. Med. Scienc. Nagpur. 1, 5, 1981.
Gupta VL, Study of precipitating factors of sickle cell crisis, Proceed. INCLEN, Mexico, 1990.
Impact: Avoiding exposure to known precipitating factors will significantly reduce number of
crisis and thereby mortality and morbidity in sickle cell disease.
2. Predisposition to infections is major cause of morbidity as weii as mortality, apart from
precipitating crisis, it also inhibits fibrinolysis thereby adding to vaso-oclussive
phenomenon.
Publications: Gupta VL, Choubey BS, Dube GK, and Saoji AM.: Platelet functions, fibrinogen
and MISFI An immunohaematological study in sickle cell crisis. JAPI, 29, 183, 1981.
Saoji AM, Gupta VL, Dube GK; Fibrinolysis in patients with sickle cell crisis, inhibition of
fibrinolytic activity in patients with vc with infection ind. jour. path. micro;23:55,1982.
Gupta VL, Dube GK, Choubey BS : Two cases of salmonella oesteomylitis affecting spine.
CLINICIAN, 46:308.1982.
Impact: Infection prophylaxis will reduce number of crisis, morbidity and mortality in
patients with sickle cell disease.

3. Iron deficiency do exist in Indian subset of patients with sickle cell disease because of
various reasons, this is against the popular belief of “Iron overload” in these patient

Publication: Waghmare BG, Gupta VL, Deshmukh PY, and Dubey GK, Iron metabolism in
patients with sickle cell anaemia. Proceed. XXXVII, APICON, 1982.
Impact : If established, correcting iron deficiency will improve quality of life in these patients
apart from reducing morbidity
4. Zinc deficiency is common in patients with sickle cell disease. In zinc deficient patients,
zinc therapy is efficacious in prevention of sickle cell crisis.
Publications: Gupta VL, Dubey GK, Chaubey BS, and Tungar VS. Zinc deficiency in sickle cell
anaemia, a preliminary report. Jour. Academ. Med. Scienc. Ngp. 1:10;1982.
Gupta VL, Chaubey BS. Zinc deficiency in relation with RBC survival and it’s effect on sickling
process and menifestations in sickle cell anaemia. Dr. SS Mishra memorial award and medal
1983 NAMS, New Delhi 1983.
Gupta VL (1986) Zinc deficiency in sickle cell anemia it's effect on cell membrane, course of
disease anduture future place in management. A Thesis for PhD, medicine, Nagpur University.

vii
Gupta VL, and Chaubey BS. Efficacy of zinc therapy in management of sickle cell crisis Ind.
Jour. Med. Res. 86:803-807;1987.
Gupta VL, Dubey GK, Chaubey BS. RBC survival, Zinc deficiency, and efficacy of zinc therapy in
sickle cell disease. Thalasaemia = pathophysiology and management Part A, Alan R.Liss Inc.
New York 1987.
Gupta VL, Zinc in sickle cell anaemia, Bull Research Soc, GMC, 1989-
Gupta VL, and Fletcher H. Management of sickle cell anaemia, A review. Ind. Jour. Haemat.
7:1-5;1989.
Gupta VL, efficacy of zinc therapy in prevention of crisis in sickle cell anaemia proceed.
INCLEN, Mexico, 1990.
Gupta VL: Management of sickle cell anaemia, zinc therapy, and electrokinetic potential of
red blood cells . A treatise submitted for “D.Sc.” medicine, Nagpur university, 2007
Impact: Identifying and establishing and then treating zinc deficiency will reduce number of
crisis and morbidity in these patients with sickle cell anaemia.
5. Predictive modeling for vaso-oclussive episodes based on electrokinetic potential or
“Z” potential developed
Publication: Gupta VL, Chaubey BS. Electrokinetic potential of RBCs which varies according
to the blood group as well as disease such as sickle cell anaemia. JAPI 38:191-2;1990
Gupta VL: Management of sickle cell anaemia, zinc therapy, and electrokinetic potential of
red blood cells . A treatise submitted for “D.Sc.” medicine, Nagpur university, 2007
Impact: This will help in identifying high risk patients prone for crisis and give them complete
evidence based management.

viii
 Research Contribution

Zinc deficiency established in sickle cell disease

Co-relation of the zinc deficiency with severity parameters of sickle cell disease

Zinc effective in preventing sickling in peripheral blood in vitro

Favourable results on other disease parameters

Pilot study showing Encouraging results of zinc therapy in sickle cell disease

Clinical trial showing efficacy of zinc therapy in management

Randomized controlled clinical trial

“Zinc therapy is efficacious in prevention of crisis in sickle cell anaemia”

Research Spiral

ix
 Research Contribution

Blood group of patients EKP of red blood cells

Scoring system for prediction of vas-oclussive epispdes

Validation

Reduplication

Scoring system for vas-oclussive episodes

Predicting Vaso-oclussive Episodes

x
 “Why do I have to be in pain

every day of my life

I wake up each day, the “pain” appears

I go to sleep but, it does’nt disappear

You have got, “Sickle Cell”, the GPs say

There is nothing we can do for you today

I wish people can understand, just how I feel

The sudden pain, which makes me reel

You have got “Crisis” the houseman cry

But don’t you worry,

We won’t allow you to die !

*Courtsey sickle cell soc.UK

xi
xii
 CHAPTER I

HISTORICAL ASPECTS

1
 CHAPTER I

Prior to the first formal report on 1910, sickle cell disease must have been misdiagnosed
because it's cardinal manifestations were common to other tropical disorders.The disease had
probably been recognized for generations in West Africa, where 'cold season rheumatism'was
given the repetitive onomatopoeic names appropriate to a chronic recurrent condition
(Konotey-Ahulu 1968). This the Ga tribe referred to it as Chwecheechwe, the Twi as Ahututuo,
the Ewe as Nuidudui and the Fante as Nwiiwii.

Konotey-Ahulu (1968) also stated that ' some elderly men could trace back several
generations, relatives who had died young from the disease '. The first recorded description in
Africa has been attributed to Africanus Horton (1874) who described the fever of crises, the
shifting joint pains, the exacerbation during the rainy season, and the constant abnormality in
blood.

in North America, two early reports had features highly suggestive of sickle cell disease.
Lebby (1846) described a runaway slave upon whom he performed an autopsy after execution
for murder. The history detailed a full chest, narrow hips, very spare build and bilious
intermittent and remittent fevers. At autopsy the 'spleen (was) wanting'.

In 1898, Hodenpyl reported the case of a 32 year old black male who presented with
pains all over the body, pleuratic symptoms and jaundice and was noted to have scars on the
anterior surfaces of both legs. He died in hospital, and it was recorded that at autopsy, 'not
withstanding careful search no trace of spleen was discovered.'

The first generally accepted report of the disease in North America in the November
1910 edition of the Archives of Internal Medicine when Dr James Herrick of Chicago described a
young Negro student from Grenada in the West Indies in a paper entitled ' peculiar elongated
and sickle shaped red blood cells in a case of severe anaemia. The illustration of the blood film
left no doubt that this patient has a severe form of sickle cell disease probably homozygous
sickle cell disease.

Within 3 months of the original report, a second case was described in a 25 year old Black
woman who was under observation at the University of Virginia Hospital (Washburn 1911). The
third case was a 21 year old Black woman attending Washington University Hospital in St. Louis
who had similar features (Cook and Mayer 1915). The fourth case was a 21 year old Black male
at the Johns Hopkins Hospital and in that report, Mason (1922) summarized the first four cases.
He concluded that this was a new disease entity and was first to use the term 'sickle cell
anaemia’

2
 CHAPTER I

Fig.1.1 The peripheral blood from Herrick’s first case (From Herrick 1910)

Fig.1.2 Dr James B Herrick (1861-1954), taken in 1925 ( Photo courtesy of Dr.L.W.Diggs)

3
 CHAPTER I

Fig. 1.3 Scanning electron micrograph of sickled cells X19000

(Photo courtesy of Drs. Klug, Lessin, and Albert)

The haemolytic aspects of the disease were recognized in the first four reports all cases
having anaemia and jaundice. Sydenstricker (1924a) noted elevated reticulocyte counts, early
autopsies confirmed marked erthroid hyperplasia (Sydenstricker et al 1923). It was also noted
at this time that Sydenstricker (1924a) drawing on comparison of sickle cell disease and
hereditary spherocytosis, introduced the concept of haemolytic crisis. Although in hereditary
spherocytosis this term was limited to exacerbation of haemolysis and abdominal pain from
from acute spleenic enlargement. It was misused in sickle cell disease to describe the juxta-
articular pain in which there is usually no evidence of acutely exacerbated haemolysis and the
confusion still persists.
Gallstones associated with increased bilirubin excretion were described in the second
reported case (Washburn 1911) and in other early cases (Graham 1924, Hamilton, 1926,
Hein et al 1927, Matthews 1936, Mallory 1941, Schaefer 1942). The bone changes with
medullary expansion, cortical thinning, and sclerotic lessions were noted in early autopsy
(Graham 1924, Diggs et al 1937) and radiological reports (Vogi and Diamond 1930, LaWLd
4
 CHAPTER I

1932, Grinan 1935). The increased requirements for folic acid secondary to haemolysis were
not noted until much later (Zuelzer and Rutzky 1953, Jonsson et al 1959, Maclver and Went
1960).

Vaso-oclusion

The contribution of infarction to the pathology of sickle cell disease was perceived
more slowly. Wedge shaped areas of pulmonary consolidation were described by Graham
(1924), Wollste in and K reidel (1928), and Steinberg (1930) noted small and medium sized
pulmonary vessels contained fresh and organized blood thrombosis with consequences of fresh
and old infarcts. Multiple infarction of the kidneys and lungs was recognized by Yater and
Mollari (1931) and by Baird (1934). The contribution of repeated splenic infarction to
progressive splenic fibrosis was described by Diggs (1935).

Bone infarction was recognized to cause dactylitis (Danford et al 1941), cortical

infarction (Graham 1924, Kraft and Bertel 1947, Legent and Ball 1948), and femoral head
necrosis ( Bauer and Fisher 1943, Kraft and Bertel 1947, Legent and Ball 1948). Priapism was
first recognized as a complication in the 1930s ( Diggs and Ching 1934, Obendorf 1934), and
lessions in the central nervous system about the same time (Sydenstricker et al 1923,
Anderson and Were 1932). Serious retinal complications were not recognized until the last 20
years (Watch and Goldberg 1966).

Other clinical manifestation

A variety of features not directly attributable to either haemolysis or vaso- occlusion
were recognized. Leg ulceration was frequently reported in sickle cell disease in 1930s ( King
1936, Netherton 1936, Schwartz 1938, Cummer and LaRocco 1939), the later authors first
drawing attention to the association of the two conditions.( Cummer and LaRocco 1940).
Pregnancy was realized to be adversely affected by sickle cell disease ( Yater and Mollar I 1931,
Lash 1934, Richter et al 1934) Physical and sexual development of children was also noted to
be retarded (Winsor and Burch 1944a 1945a). and adults were considered to have a
characteristic habitus ( Winsor and Burch 1944a Sharp and Vonder Heide 1944).

An entertaining account of many personalities involved in the discoveries of sickle cell

disease has been presented by Conley (1980).

Second and third case of sickle cell anemia was reported by Washburn(1911) and Cook
Meyer(1915) respectively. In 1922, Mason first used the term 'sickle cell anemia '. It is generally
agreed upon that sickle cell anemia and trait are due to some inherent defect in red blood cells.
Material parasites were thought to be the cause. Beets (1947) proved that there is no
5
 CHAPTER I

appreciable difference in the incidence of malarial parasite infestation in sicklers and

nonsicklers. Inheritance of haemoglobin 'S' can be described as autosomal intermediate
(Wintrobe, 1967). Ingram showed that the chemical difference in the haemoglobin 'S' is the
substitution of valine for glutamic acid at the sixth position of beta chain.

Sickling can be demonstrated in peripheral blood by

1. Seriver and Waugh' s modified Emmel's method

2. Donald and Castle 's method

Sickle cell crisis

The word 'crisis ' has it's origin from Greek word , 'krisis' which means 'Turning Point '.
Dictionary meaning of the word crisis - (i) a striking change in the symptoms attended by
outword manifestation s, (ii) a painful paroxysmal disturbance of function accompanied by
pain

Sydenstricker (1924) used first time word "crisis" in association with sickle cell anemia.
Graham described pains in the same year. Penbetty and Cooley described haemolytic crisis
occurring in patients with sickle cell anemia in association with streptoccocal infection.

However accepted defination and classification of sickle cell crisis was by L.W. Diggs in
1965.

Greenburgh M.S. and Kass E. H. Postulated that metabolic acidosis is initiating factor.
Smith E. W. and Corley 1959) described common denominators.

Diggs L.W. and later Felix I.D. et al put schematic presentation of crisis with hypoxia
and reduced PH being initiative factors and vicious circle starts from lowered oxygen tension
which lead to Hb 'S' crystallization increased blood viscosity stasis and further hypoxia.

Many precipitating factors have been identified of sickle cell crisis e.g. Infection,
dehydration, stress ( Diggs LW, Smith EW, and Corley , Felix ID et al, Gupta VL). Infection is
most Important as sicklers are prone to infection by salmonella and gram negative organisms,
because of defective C3 essential opsonin ( WA Wilson et al).

Diggs LW, (1965), has reported vascular occlusive crisis as typical type of crisis that
occurs in most of the patients. G. Karayalcin et al (1975) in their series of 100 patients reported
highest number suffering from painful crisis.

In a series of 75 cases, Gupta V. L. et al reported incidences of various types of crisis,

and described clinical manifestation in detail.
6
 CHAPTER I

Cardiac manifestations in sickle cell anemia are very common indeed. Heart failure may
occur, secondary to multiple oclusions of small and medium sized pulmonary arteries by masses
of sickled erythrocytes. A systolic murmur of variable intensity is usually heard.

Pulmonary manifestation s are serious complications in patients with sickle cell disease.
Pulmonary infarction may result from primary occlusion of small vessels. Multiple pulmonary
thrombosis leads to increased pulmonary resistance -- pulmonary hypertension – Cor
pulmonale.

Jenkins M.E. and Scott R.B. first reported temporary splenomegaly ( Hb 'C' or Hb 'S'
thalassemia). in these cases spleen may be packed with sickle cells and may be grossly
enlarged in infants who die during acute episodes. Later there are siderofibrotic in spleen pulp
subsequently replaced by scar. There may be perispleenitis with multiple spleenic infarcts
which heal and may be followed by calcification and cystic degeneration in elder children and
adult spleen may be fibrotic and atrophic (autospleenectomy).

Liver may be enlarged during painful crisis. 10% cases may show obstructive jaundice
over and above haemolytic jaundice. Choubey BS et al. (1974) detailed histopathological
changes occurring in liver patients with sickle cell anemia.

Renal changes in sickle cell disease recognized long back by Herrick. Later Abel and
Brown, Calcagno et al., Sweeney et al., studied these changes in detail and summarised
manifestation as (a) hyposthenuria (b) low GFR (c) haematuria (d) nephrotic syndrome and (e)
renal failure.

Retinal changes were first described by Goodman G. Five stage classification of ocular
manifestations of sickle cell disease was given by Mansoor F. Firmaly.

The haemostatic balance is shifted in the direction of increased coagulability . Fenichel

el al reported raised levels of fibrinogen, specially with associated infections (Green et al).

Gupta VL et al reported unchanged platelet functions, raised fibrinogen level in vaso-

occlussive crisis and decreased fibrinolytic activity.

Molecular basis of sickling, it's patrheological rheological abnormalities, vaso-occlussive

phenomenon were explained by contributions from Sherman, John F. Bertles, James G White,
and Panpit P et al.

7
 CHAPTER I

Haemolysis in sickle cell disease

In sickle cell disease, RBC half life is decreased. Various attempts have been made to
find out method for determination of RBC half life span and also for determination of site of red
blood destruction. Jennifer and Mollison, Jandl et al, 1956, 1958, Bernini et. al. Sprague and
Peterson, Toghill, Hathorn, Adwani et. al.

Management Review
Precipitating causes of crises must be sought for and treatment of infection should be
started. Analgesics, and patient must be rehydrated. Acidosis should be treated.

Large doses of potent analgesics may be required. Dextran, Phenothiazines,

bicarbonates, and urea solutions have been suggested. Carbonic unhydrase inhibitors,
papaverine hydrochloride, vasodialators, antithyroid drugs, inhibitors of blood coagulation,
steroids, pyridoxine hydrochloride and fibrinolytic agents have been suggested but are not
widely used suggesting low efficacy. Partial exchange transfusion may have a role.( Fleming AF,
1982, Charche S, 1974, Co-operative urea trial group, 1974, May A, and Huehns B, 1974, Kark
JA, kale MP, 1978, Costa PF, Zago MA, and Bother a C, 1979, Franklin IM, 1980, Benett AJ and
Rosner F, 1979).

Carbon monoxide and agents, sodium cynate, mechlorethamine hydrochloride have

been thought off but given up because of toxicity or inadequate clinical benefits.

Phlebotomy, long term transfusion therapy, partial exchange transfusion too laborious,
and hazardous as well. (Charche S, 1974).

Zinc deficiency in sickle cell anemia is an established fact. (Serjeant GR, Galloway RB,
1970, Greaves MR, Skillen AR, 1970, Tellot TR Jr. and Ross JF, 1960, G. Karayalcin, Fred Rosnel
et al, 1974). There were encouraging results of oral zinc therapy in chronic nonhealing ulcers in
sickle cell anemia ( Hussain SL, 1969, Auckland G, 1970).The use of zinc as an antisickling agent
gained momentum because of it's ability to reduce ISC ( Irreversibly sickled cell). Use of zinc in
sickle cell anemia has sound laboratory rationale (Kruckberg WC, Delshel egal FJ, 1975,
Hellerstein S, Bunthrarungroj T, 1975, Prasad AS, ORtega J, et al, 1976, Brewer GJ, Oeshelgel TH
et al, 1975, Brewer GJ, 1976).

Indian milestones
Sickle cell trait was first reported in India by Lehman and Cutbash (1952), among the
aboriginal tribes of South India. Dunlop and Muzumdar (1952) reported presumptive cases of
sickle cell anemia from tea garden labourers of Asam. RN Shukla and BR Solanki (1958),
8
 CHAPTER I

examined 1010 adult labourers of different castes for the sickle cell trait. In Mahars, kunbis, and
Tells it's prevalence ranged from 9.4 to 22.2 percent. Subhedar BJ, Bhargava HS, and Choubey
BS (1961) reported sickle cell anemia in adolescents and adults.

Gupta VL (1981) reported prevalence of sickle cell in Nava Boudha and Kunbis as 60
percent and 13.33 percent respectively. It is worthwhile mentioning that the sickle cell gene in
Indian subcontinent is mostly confined to certain tribes and ethnic groups in some regions only.
(Mukharjee BN, 1985).

The Diagnosis
Sickling Test

The method of Doland and Castle (1948) has become the standard technique for
performing sickle test. One drop of 2% solution of sodium metebisulphite is mixed with one
drop of blood on a microscope slide covered with cover-slip and sealed with molten paraffin
wax to exclude air and to prevent drying . Sickling of cells is usually appearant under a
microscope within one hour, (Fig.1.4) (Early Sickling). Although the preparation should be read
after 24 hours, (Late Sickling).

Fig.1.4 A positive sickle test

9
 CHAPTER I

Solubility Test

The relative insolubility of deoxygenated HbS in solutions of high molarity (Goldberg

1958) has led to a profusion of tests employing concentrated phosphate buffers, lysing and
reducing agents. With these reagents haemolysates with HbS become cloudy and those without
HbS remain clear (Fig1.5). The solutions can be made cheap!y in any laboratory ( Huntsman et
am 1970, French 1971, Mustafa and Fielding 1971, Cook and Raper 1971, Greenberg st al 1972,
Naumann and Diggs 1974). Refinements involving different molarity buffers (Raper 1971),
centrifugatipn (Huntsmsn e t al, 1970, Sergeant and Serjeant 1972, Diggs et al 1975) or
separation on standing (Diggs and Walker 1973) have been used to differentiate between
heterozygous HbS genotype and SS disease. Auto mated versions of the test have been
described (Henry et al 1970) but have little applicability in most laboratories.

Fig.1.5 Solubility test

Haemoglobin Electrophoresis

The principles of haemoglobin electrophoresis at an alkaline pH are simple and are

based on the charge in a haemoglobin molecule which follow a certain amino acid substitution.
In conventional alkaline electrophoresis (pH 8.4-8.9) with haemoglobin movements towards
anode HbS moves more slowly than HbA.

10
 CHAPTER I

In practice the method requires a source of current , a buffer system of the media most
used paper is cheapest but gives poor defination while starch gel give good defination but is
time consuming to make. Cellulose acetate membranes give excellent defination and can be
purchased ready to use. Expensive but cellulose acetate is the most widely used medium at
present. Haemoglobin Electrophoresis on acid agar can provide valuable confirmatory and
additional evidence.

The electrophoretic patterns in four major genotypes of sickle cell disease are shown in
(Fig1.6.). It is apparent that SS disease and SBo thalassemia have closely similar electrophoretic
pattern and their differentiation requires further study (Sergeant GR, 1985).

Fig.1.6 Haemoglobin Electrophoresis

11
 CHAPTER II

CHAPTER II

THE EPIDEMIOLOGY OF
SICKLE CELL GENE

12
 CHAPTER II

Origin of the sickle cell gene

Controversy surrounds the origin of the sickle cell gene and the question of whether it
arose as a single mutation spreading to occupy it's present distribution, or originated from
identical mutations in different areas.

Single mutation theory

A single mutation occurring in Neolithic times in then fertile Arabian peninsula was
favoured by Lehman (1954a,b) who postulated that the changing climatic conditions and
conversion of this area to desert, caused migration of people to India, Eastern Saudi Arabia, and
down to Equatorial Africa. Lehman and Huntsman (1974), supported the hypothesis by citing
the distribution of certain agricultural practices and anthropological evidence as well as the
geographical distribution of the gene within Africa which manifested a decline in gene
frequency from East to West Africa together with higher levels on the North compared to the
south bank of the Zambesi river compatible with river having acted as a barrier to southern
migration.

A single mutation theory was favoured by Gelpi(1973) who considered that the
evidence from blood groups and other genetic markers was more compatible with an origin in
Equatorial Africa and subsequent diffusion of the gene to India, Arabia, and the Mediterranean
by the East African slave trade (Kamel and Awny 1965).

Fig. 2.1 Spread of Sickle Cell gene

Evidence from DNA polymorphisms , Multiple mutation theory

Recently the multiple mutation theory has received more support. The increasing use of
restriction endonuclease analysis has detected relatively common normal variations in DNA
structure (polymorphisms) which are inherited in Mandelian manner and may be used as
13
 CHAPTER II

genetic markers. The first such polymorphism to be reported was a variation in the recognition
site of the restriction endonuclease Hpa I to the 3' side of the beta globin gene (Kan and Dozy
1978a)

The variety of polymorphisms could have arisen on DNA bearing a beta s gene of single
ancient origin, or different polymorphisms may have become associated with beta s gene by
recurrent cross-border or gene conversion events. Current opinion favours the hypothesis that
the beta s mutation occurred independently several times on chromosomes manifesting a
variety of different haplotypes , probably between 3000-6000 generations (70,000-150000
years) ago (Kurnit 1979, Solomon and Bodmer, 1979).

Sickle hemoglobin (Hb-S) is so called because of the sickle shape it imparts to the
deoxygenated red cells. The highest prevalence of Hb-S is in tropical Africa and among blacks in
countries that participated in the slave trade. It occurs with lower frequency In the
Mediterranean region, Saudi Arabia, and parts of India. Result of studies of DNA polymorphism
linked to the beta-S gene suggests that it arose from three independent mutations in tropical
Africa. The most common beta-S chromosome is found in Benin and central West Africa. The
second haplotype is prevalent in Senegal and the Africa West Coast and third haplotype is seen
in Central Africa. The Hb-S gene in Eastern province of Saudi Africa and in Central India is
associated with different DNA structure not encountered in Africa and probably represents a
fourth independent occurrence of the sickle cell mutation. Only the Benin and Senegal
haplotype are prevalent among North Africans, Greeks and Italians, suggesting that this
mutation spread to the Mediterranean basin from West Africa. Sickle Cell Disease is highly
prevalent in certain ethnic groups in Central India like SC, ST & OBC.

Fig.2.2 Distribution of the sickle cell gene throughout the world with
arrows indicating spread of the gene to the Americas and latterly Europe (Serjeant GR, 1985)
14
 CHAPTER II

Balanced Polymorphism
Population survey among older children and adults of high beta-S gene in Equatorial
Africa noted a striking deficiency of homozygous (Allison 1956a, Lehman and Raper 1956, ) and
it was apparent that few homozygous lived long enough to reproduce. In the face of loss of
beta-S genes, other factors might have contributed to the maintenance of the high gene
frequency. A calculated mutation rate was far too low to balance the loss of beta-S genes
occurring in the African environment (Allison 1954a). The alternative explanation was that
persons heterozygous for the gene possessed some advantage in terms of survival or
reproduction over those with normal haemoglobin.

The concept of balanced polymorphism is the balance of deleterious and advantageous

effects of gene which allows it to remain at a relatively constant level in population.

The deleterious effects are oblivious since the early death of homozygote leads to the
loss of two genes. The possible mechanism of an advantage for the sickle cell gene are
controversial. An increased fertility of heterozygotes has been proposed (Foy et al 1954, Allison
1956a, Ashcroft et al 1969) but never convincingly demonstrated . Raper (1949) was first to
suggest that the sickle cell trait may have a positive survival value against some adverse
condition in the tropics and Mackey and Vivarelli (1952) suggested that factor might be malaria.

The close geographical association between the distribution of malaria and of the sickle
gene supported the concept (Allison 1954b). and lead to the exciting period in the history of
research on sickle cell disease.

Malaria and the sickle cell trait

The first observations on malaria and the sickle cell trait were from Northern
Rhodesia where Beet (1946, 1947) noted in two communities that malarial parasites
occurred less frequently in blood films from individuals with sickle cell trait. Alison (1954c)
first drew attention to this association and concluded that persons wi th sickle cell trait
developed malaria less severely than those without the trait. The communication marked
the beginning of a considerable controversy.

Two studies were unable to document differences in parasite densities between

'sicklers' and 'non-sicklers ' ( Moore et al 1954, Archibald and Bruce -Chawtt 1955). Raper
(1955) supported this hypothesis by noting significantly lower parasite rates among the
sicklers. A protective effect was also noted in northern Greece (Deliyannis and Tavlarakis
1955) and in Nigeria death from cerebral malaria appeared uncommon in sickle cell trait
(Edington and Watson -Williams 1965).

15
 CHAPTER II

There appeared to be good evidence that the sickle cell trait conferred some
protection against falciparum malaria during the critical period in early childhood between
the loss of passively acquired maternal immunity and the development of active immunity
(Allison 1957a, 1964, Rucknagel and Neel 1961, Motulsky 1964, Livingstone 1971). Data on
the resistance of subjects with sickle cell trait to other types of malaria are sparse and
conflicting (Reper 1955, Colbourne and Edington 1956, Garlick 1960, Fle Ming et al 1979).

Fleming et al (1979), were able to show the apparent protective effect was limited to
30-60 week age group although in the dry season, lower parasite densities could be
demonstrated up to the age of 3 years. The higher prevalence of the trait in this population
(29%) compared to that in Ibadan (17%) where there is continuous malarial transmission, was
compatible with Raper's (1960) hypothesis.

Mackey and Vivarelli (1954) postulated that the malarial parasite metabolized HbS less
easily than HbA. Edozian et al (1960) proposed that a difference in immune response between
sicklers and non sicklers might account for the relative resistance of the former. The parasite
was thought to bring about it's own destruction by identifying it's host cell to the reticulo
endothelial tissue. This proposed mechanism depended on significant sick ing at physiological
oxygen tension in the sickle cell trait which seemed unlikely although it is now recognized that
in falciparum malaria parasitized cells are sequestered deep within the reticulum-endothelial
system and exposed to low oxygen tensions permitting potassium loss and low pH, factors
known to inhibit parasite growth in vitro (Pasvol et al 1978, Friedman 1978, Friedman et al
1979)

Fig. 2.3 Distribution of the sickle cell trait, shown in pink and
Purple Sickle cell disease From Wikipedia, the free encyclopedia

16
 CHAPTER II

Fig. 2.4 Historical distribution of malaria (no longer endemic

in Europe), shown in green Sickle cell disease From Wikipedia,
the free encyclopedia

Effects of control of Malaria

Arguing from the concept of balanced polymorphisms removal of the selective

advantage of the sickle cell trait by the control of malaria should be accompanied by a
decreasing prevalence of the gene. However control of malaria will also improve the prognosis
in patients with sickle cell SS disease.

These two effects are unlikely to be balanced and evidence on the effect on S gene
frequency of removal to a non malarious environment is available in two situations.

In Black Americans it is probable that the prevalence of the sickle cell trait has declined
from an original level exceeding 20 percent to the current frequency of 8 percent over a period
of approximately 300 years.

In North America there was a selective pressure from malaria which was eradicated over
3-5 generations and it was calculated that the fall from 15 percent to 8 percent was compatible
with the loss of the increased fitness of the heterozygote over the period of about 12
generations (Alison 1954a).

In Zambia the effects of transfer from malarial to non malarial environment could be
examined during the urbanization of the tribal population (Barclay and Splaine 1972).

17
 CHAPTER II

Fig. 2.5 Modern distribution of malaria Sickle cell disease

From Wikipedia, the free encyclopedia

The Epidemiology of Sickle Cell Disease

It is common misconception tthat HbS is limited to the Negro race whereas it is widely
distributed among non- Negro peoples throughout an area which includes southern Italy,
northern Greece, southern Turkey, the eastern province of Saudi Arabia, and India as well as
equatorial Africa. (Fig.2.5)

In Africa marked tribal

ibal variations occur between people living in the same environment
and in both Uganda and Tanzania, Bantu speaking tribes have higher and more consistent
frequencies than Hamitic speaking tribes ( Lehmann and Raper 1949, Lehmann 1954a, Allison
1954b). The most likely explanation for these variations is tribal migration in the recent past
and the historical and ethnological evidence for this is discussed by Allison (1954b), and by
Lehmann (1954a).

Other important foci of the sickle cell gene occur in the Q

Qatif
atif and Al Hasa Oases of the
Eastern Province of Saudi Arabia, central and southern India (Fig. (Fig.2.6)) and northern Greece
especially the Chalkidhiki peninsula. The gene is also distributed in Sicily, Turkey, Israel, and the
Americas. For the references are Livingstone (1967), and Serjeant (1974a).

The highest frequency of sickle cell disease is found in tropical regions, particularly sub
sub-
Saharan Africa, tribal regions of India and the Middle
MiddleEast. (Powars DR, ElliottMills DD, et. al
1991). Migration of substantial populations from these high prevalence areas to low prevalence
countries in Europe has dramatically increased in recent decades and in some European
countries sickle cell disease has now overtaken more familiar genetic conditions such
18
 CHAPTER II

as haemophilia and cystic fibrosis.( Weatherall DJ, Clegg JB (2001). In 2015, it resulted in about
114,800 deaths (GBD 2015 Mortality and Causes of Death, Collaborators 2016).
Sickle cell disease occurs more commonly among people whose ancestors lived
in tropical and sub-tropical sub-Saharan regions where malaria is or was common. Where
malaria is common, carrying a single sickle cell allele (trait) confers a heterozygote advantage:
humans with one of the two alleles of sickle cell disease show less severe symptoms when
infected with malaria. (Roberts I, de Montalembert M July 2007 ). This condition is inherited in
an autosomal recessive pattern, which means both copies of the gene in each cell have
mutations. The parents each carry one copy of the mutated gene, but they typically do not
show signs and symptoms of the condition ( Wellems TE, Hayton K, Fairhurst RM September
2009).
Africa
Three-quarters of sickle cell cases occur in Africa. A recent WHO report estimated that
around 2% of newborns in Nigeria were affected by sickle cell anaemia, giving a total of 150,000
affected children born every year in Nigeria alone. The carrier frequency ranges between 10%
and 40% across equatorial Africa, decreasing to 1–2% on the north African coast and <1% in
South Africa ( National Libray of Medicine. URL = https://ghr.nlm.nih.gov/condition/sickle-cell-
disease#statistics Archived 2016-05-15). There have been studies in Africa that show a
significant decrease in infant mortality rate, ages 2–16 months, because of the sickle cell trait.
This happened in predominant areas of malarial cases. (WHO. "Sickle-cell anaemia – Report by
the Secretariat" (PDF) Archived ( Retrieved 2010-11-27).
United States

The number of people with the disease in the United States is approximately 1 in 5,000,
mostly affecting Americans of sub-Saharan African descent, according to the National Institutes
of Health. (Aidoo M, Terlouw DJ, et. al. 2002). In the United States, about one out of 500
African-American children and one in every 36,000 Hispanic-American children have sickle cell
anaemia. (National Heart, Lung and Blood Institute. "Sickle cell anemia, key points". Archived
from the original on 2010-12-02. Retrieved 2010-11-27).

It is estimated that sickle cell disease affects 90,000 Americans. ("September is Sickle
Cell Awareness Month" CDC. Archived from the original on 27 September 2010. Retrieved 6
February 2011). Most infants with SCD born in the United States are now identified by routine
neonatal screening. As of 2016 all 50 states include screening for sickle cell disease as part of
their newborn screen (Centers for Disease Control and Prevention 16 September
2011. Archived from the original on 31 October 2011. Retrieved 8 November2011).

19
 CHAPTER II

France
As a result of population growth in African-Caribbean regions of overseas France and
immigration from North and sub-Saharan Africa to mainland France, sickle cell disease has
become a major health problem in France. ("Sickle Cell Patients Endure Discrimination, Poor
Care And Shortened Lives". NPR.org. Retrieved 12 November 2017). SCD has become the most
common genetic disease in the country, with an overall birth prevalence of 1/2, 415
in Metropolitan France, ahead of penylketonuria (1/10,862),
congenital hypothyroidism (1/3,132), congenital adrenal hyperplasia (1/19,008) and cystic
fibrosis (1/5,014) for the same reference period.
Since 2000, neonatal screening of SCD has been performed at national level for all
newborns defined as being "at risk" for SCD based on ethnic origin (defined as those born to
parents originating from sub-Saharan Africa, North Africa, the Mediterranean area ,South Italy,
Greece and Turkey), the Arabic peninsula, the French overseas islands and the Indian
subcontinent. (Bardakdjian J, Wajcman H (September 2004).
United Kingdom

In the United Kingdom, it is thought that between 12,000 and 15,000 people have sickle
cell disease (Thuret, I; Sarles, J; Merono,et.al.June 2010) with an estimate of 250,000 carriers of
the condition in England alone. As the number of carriers is only estimated, all newborn babies
in the UK receive a routine blood test to screen for the condition.("Inheriting sickle cell anaemia
– Live Well – NHS Choices". www.nhs.uk. Archived from the original on 2014-12-02).

Due to many adults in high-risk groups not knowing if they are carriers, pregnant
women and both partners in a couple are offered screening so they can get counselling if they
have the sickle cell trait. ("Sickle cell anaemia – NHS Choices". www.nhs.uk. Archived from the
original on 2011-12-13.)

In addition blood donors from those in high-risk groups are also screened to confirm
whether they are carriers and whether their blood filters properly.[("Who is offered screening
and when?". screening.nhs.uk. Archived from the original on 2014-12-31.) Donors who are
found to be carriers are then informed and their blood, while often used for those of the same
ethnic group, is not used for those with sickle cell disease who require a blood transfusion.
("Give Blood – Resources – Sickle Cell and Blood Donation". Give Blood. Archived from the
original on 2014-12-31)

20
 CHAPTER II

Middle East

In Saudi Arabia, about 4.2% of the population carry the sickle cell trait and 0.26% have
sickle cell disease. The highest prevalence is in the Eastern province where approximately 17%
of the population carry the gene and 1.2% have sickle cell disease. ("Why is Blood from Afro-
Caribbean Donors Special?". Sickle cell society. org. Archived from the original on 2014-12-30).
In 2005, Saudi Arabia introduced a mandatory pre-marital test including HB
electrophoresis which aimed to decrease the incidence of SCD and thalassemia. ( Jastaniah W
2011).
In Bahrain a study published in 1998 that covered about 56,000 people in hospitals in
Bahrain found that 2% of newborns have sickle cell disease, 18% of the surveyed people have
the sickle cell trait, and 24% were carriers of the gene mutation causing the disease. (Memish
ZA, Saeedi MY (2011). The country began screening of all pregnant women in 1992 and
newborns started being tested if the mother was a carrier. In 2004, a law was passed requiring
couples planning to get married to undergo free premarital counseling. These programs were
accompanied by public education campaigns ](Al Arrayed, Sheikha, 1995).

Caribbean Islands
In Jamaica, 10% of the population carries the sickle cell gene, making it the most
prevalent genetic disorder in the country (Asnani MR, McCaw-Binns AM, Reid ME 2011).

India and Nepal

Sickle cell disease is common in ethnic groups of central India who share a genetic
linkage with African communities, ("Sickle Cell Anemia". www.hematology.org. 2014-12-
16. Archived from the original on 2017-06-25. Retrieved 2017-05-01). where the prevalence has
ranged from 9.4 to 22.2% in endemic areas of Madhya Pradesh, Rajasthan and Chhattisgarh.
*Awasthy N, Aggarwal KC,et. al. 2008 ) It is also endemic among Tharu people of Nepal and
India; however, they have a sevenfold lower incidence of malaria despite living in a malaria
infested zone ("Life with sickle cell – Nation – Nepali Times". Archived from the original on
2015-06-24).

21
 CHAPTER II

Fig.2.6 Distribution and prevalence of the sickle cell trait in

India (Serjeant GR, 1985}

India
The first description of sickle haemoglobin in India was by Lehman and Cutbush in 1952
in the tribal populations in the Nilgiri hills in south India. In the same year, Dunlop and
Mazumder also reported the presence of sickle haemoglobin in the tea garden workers of
Upper Assam who were migrant labourers from tribal groups in Bihar and Odisha. Since then,
many population groups have been screened and the sickle cell gene has been shown to be
prevalent among three socio-economically disadvantaged ethnic groups, the scheduled tribes,
scheduled castes and other backward classes in India (Bhatia HM, Rao VR. 1987, Rao VR
1988. Kaur M, Das GP, et. al. 1997, Kate SL, Lingojwar DP. 2002, Patra PK, Chauhan VS, et. al.
2011, Urade BP. 2012, Kaur M, Dangi CBS, et. al. 2013, Colah R, Mukherjee M,et.al. 2014).

The prevalence of sickle cell carriers among different tribal groups varies from 1 to 40
per cent. Madhya Pradesh has the highest load with an estimated number of 9, 61,492 sickle
heterozygotes and 67,861 sickle homozygotes. Further, 27 of the 45 districts in Madhya
Pradesh fall under the sickle cell belt and the prevalence of HbS varies from 10 to 33 per cent. It
has also been estimated that 13,432 pregnancies would be at risk of having a child with sickle
cell disease in this State and the expected annual births of sickle homozygotes would be 3358
(Gupta RB. 2006). Gonds and Bhils constitute the largest tribal groups in central India.

The entire tribal population of 1,25,000 individuals in the Wayanad district of Kerala was
screened, followed by genetic counselling where carriers of HbS were advised not to marry

22
 CHAPTER II

carriers. A very high prevalence of HbS is seen in these tribes (18.2 to 34.1 %) (Feroze M,
Aravindan K. 2001)

L e t u s b r e a k t h e c h a i n o f S i c k l e C e l l

The Indian Perspective

North-East India 0 – 18 %

C A Central India 22 – 44 %
B

West India 0 – 33 %
D

South India 0 – 40 %

Fig. 2.7 Region wise prevalence of Sickle Cell, highest in Central India
The Indian Perspective

In Gujarat, the Dhodia, Dubla, Gamit, and Naika tribes have a high prevalence of HbS
(13-31 %). More recently very extensive population surveys have been done by the Indian Red
Cross Society, Gujarat State Branch where 1,68,498 tribals from 22 districts were screened and
the overall prevalence of sickle cell carriers was 11.37 per cent ( Patel AP, Naik MR, et.al. 2012).
Some tribal groups in south Gujarat like Chaudry, Gamit, Rohit, Vasava and Kukana have shown
both a high prevalence of HbS (6.3 to 22.7%) as well as β-thalassaemia trait (6.3 to 13.6 %)
( Patel AG, Shah AP, et.al. 2012) These tribal groups would have the likelihood of co-inheriting
both these genes.
As per the 1991 census, the total population of Gujarat state is 50, 671, 017. Gujarat
ranks 10th amongst the most populated states of India and is the 7th largest state, area-wise.
Scheduled Tribes, comprise of about 10%-15% of India’s population.5 The scheduled tribe
population of Gujarat is estimated to 7,481,160; accounting to 14.8 % of the total population.
Gujarat is the 4th most schedule tribe populated state of India after Madhya Pradesh,
Maharashtra and Orissa. The tribal community of Gujarat inhabitates in the geographically
difficult terrains of the Eastern belt, extending from Ambaji in the North to Dang in the South.
Sickle cell disorder is known to be prevalent in the descendents of Bhil community, namely

23
 CHAPTER II

Damor, Bhabhor, Hathila, Ninama, Nayala, Bariya, Bhuriya, Rathva, Vasava, Tadvi, Chaudhary,
Gamit,Ghodiya, Kukna, Dubada, Varali, Kodcha, Dangi and others.

Fig.2.8 Sickle Cell Disease in Tribal Population in India

Roshan B. Colah, Malay B. Mukherjee, et. al 2015

With a large population, burgeoning birth rate, and consanguineous marriage practices,
there is a dangerously high prevalence of genetic disorders among tribal populations. There
remains a conspicuous lack of maternal and child health services among the hilly tribal areas
and consequently, the tribal demographic scenario is one of high fertility, high maternal and
infant mortality rates. Epidemiological studies confirmed that sickle cell anemia is rampant in
the tribal populace. Genetic diseases have traditionally received little attention from urban
health services in India, and even less so in tribal areas. As a result, virtually all studies carried
out regarding tribal populations and sickle cell disease have strongly recommended that genetic
health services be integrated into existing primary health care and medical services to combat
the epidemic.The higher prevalence of the sickle cell trait may be a result of a higher frequency
of consanguineous marriages within the relatively small community. Association for Health
Welfare in the Nilgiris (ASHWINI), Tamil Nadu also reported prevalence of sickle cell trait in
24
 CHAPTER II

non-tribal Chetti community to be as high as 30%. 5 Studies by S. L. Kate, at B.J. Medical

College, Pune and other scientists from different institutions indicate that the overall
prevalence of sickle cell disorder in different tribal populations is 10% for carrier state and 0.5%
for the sufferer. In the present study, the prevalence of sickle cell trait was found to be 7.86 %.
The prevalence amongst the different communities in the decreasing order of frequency was
Rathod (71.4%), Vasava (21.4 %), Chavda (3.6 %) and Solanki (3.6 %). These observations
support the hypothesis that the sickle cell disorders are present in scheduled castes, tribals and
few communities of other backward classes (OBC), and not found in so called higher castes;
though the review of literature says it is present invariably in all castes. The prevalence of the
disorder was 50% in males and 50% in females. As regards to sex distribution of the disorder,
Wintrobe states that sickle cell trait is more common in females. But Samal et al, P. Deshmukh
et al did not find any such correlation. In the present study also, such a correlation could not be
found (1 pISSN: 0976 3325 eISSN: 2229 6816 National Journal of Community Medicine Vol 2
Issue 2 July-Sept 2011 Page 288). Although we carried out these activities for a limited period,
our experience suggests that we have definitely created awareness among people about sickle
cell disorders in the areas where the disease is prevalent. However, it is necessary to continue
with such activities and with our experience we are hopeful that with devotion from medical
scientists from multi-disciplinary fields, people will accept marriage-counseling program. With
advances of molecular genetics, it is possible to detect this defect at early stage (10 to 15
weeks) of pregnancy.The management cost of these patients shall be exorbitant and the
resources with the Government are limited. Hence, prevention appears to be the only solution
in present circumstances.
In a large multicentre study (Mohanty D, Mukherjee MB, et.al. 2015) where 15200
individuals from 14 primitive tribes from Maharashtra, Gujarat, Tamil Nadu and Odisha were
screened, the HbS allele frequency varied from 0.011 to 0.120 and β-thalassaemia allele
frequency varied from 0.005 to 0.024. Associated iron deficiency was seen in 26.2 per cent of
sickle heterozygotes as well as in 67.7 per cent of sickle homozygotes in this study ( Mohanty D,
Mukherjee MB, et. al. 2008) Kaur et al 2013 have summarized the distribution of HbS in
different tribal groups from individual States. Although a large number of tribal groups have
been screened for HbS, there are still many gaps in our knowledge about the distribution of the
HbS gene in tribal communities in India. The figure shows the district-wise distribution of sickle
gene among tribal populations in different States.

India has the largest concentration of tribal populations globally. They are believed to be
the early settlers in the country and are considered to be the original inhabitants. According to
the Census of India 2011, the tribal population of India is 8.6 per cent of the total population
which is about 67.8 million people. The States of Madhya Pradesh, Maharashtra, Odisha,
Gujarat, Rajasthan, Jharkhand, Chhattisgarh, Andhra Pradesh, West Bengal and Karnataka

25
 CHAPTER II

account for around 83 per cent of the total scheduled tribe population in the country and
majority of these tribal groups live in rural areas. In all, 461 scheduled tribes have been
listed6 and they have their own characteristic cultural patterns, languages and social systems,
by and large keeping to themselves. However, Reich et al concluded that “several thousand
years ago, the entire subcontinent underwent a period of massive intermarriage, shuffling its
population's genetic deck so thoroughly that it left clear traces even in the genomes of today's
most isolated tribes”(M. Kamble and P. Chatruvedi, 2000).

Table 2.1: Prevalence of Sickle Cell in Some States of India

State Prevalence of sickling (%)

Andhra Pradesh 0-34.6
Bihar 0-0.6
Gujarat 0-30
Karnataka 0-25
Kerala 0-29.7
Maharashtra 0-45.4
Madhya Pradesh 0-48.5
Orissa 0-12.4
Tamil Nadu 0-35.3
Utter Pradesh 0-32.6
West Bengal 0-1.1
Adapted from Rao 1991

More than 300 tribal groups have been screened to look for the presence of sickle gene (Bhatia
& Rao 1987, Balgir 1996, Mohanty & Mukherjee 2002).

The prevalence varies considerably among different tribal groups ranging from 0-35%. In
certain states like Madhya Pradesh, Orissa, Chattisgarh, Jharkhand, Gujrat and Maharashtra it
forms a major public health problem. It has also been detected among scheduled caste
populations (Table.2.1). Although it is considered as a ‘tribal gene’ it has been detected in
certain general caste groups also. Still certain population groups of various states remain
untouched as far as screening for sickle gene is concerned, and data on the prevalence of Hb S
is being published even today (Ramesh et al, 2006).

Table 2.2: Prevalence of HbS in different scheduled caste populations

Caste Place HbS (%) Reference

Mahars, Aurangabad 5.14 Lele et al 1962
Pana, Orissa 8.9 Ambekar et al 2001
Haddi, Orissa 6.8 Balgir 2005

26
 CHAPTER II

In Maharashtra, the sickle gene is widespread in all the eastern districts, also known as
the Vidarbha region, in the Satpura ranges in the north and in some parts of Marathawada. The
prevalence of sickle cell carriers in different tribes varies from 0 to 35 per cent. The tribal
groups with a high prevalence of HbS (20-35 %) include the Bhils, Madias, Pawaras, Pardhans
and Otkars. It has also been estimated that Gadchiroli, Chandrapur, Nagpur, Bhandara, Yoetmal
and Nandurbar districts would have more than 5000.
Epidemiology of sickle cell disorder: The urban scenario in Maharashtra, India

(A. U. Deore1 and S. B. Zade 2013) The present study was designed to determine the
current status of sickle cell disease (SCD) in urban population of eastern part (Vidarbha) of
Maharashtra State in India. A total of 3479 subjects belonging to 40 ethnic groups, including 29
non tribal, 8 tribal and three migratory groups were sampled. Blood samples were collected
aseptically from all individuals and positive samples were further subjected to cellulose acetate
hemoglobin electrophoresis for discriminatory confirmation. The overall prevalence of sickle
cell trait was found to be 4.94%, of which genotype HbS and HbSS were found to be
represented by 3.88% and 1.06% respectively. All tribal populations were found to be SCD
positive with moderate frequency (0-14%). Among non tribes, out of 32 ethnic groups, SCD was
diagnosed from 14 groups with frequency ranging between 0and 10%. The age group 0-30
years was found to be more prone to the disorder and the rate.

Table 2.3: Prevalence of Sickle Cell Disease in Selected Districts in Urban Maharashtra

District Screened population HbAS HbSS Total (AS+SS) Percentage

Amravati 696 19 07 26 3.73

Bhandara 388 29 06 35 9.02
Chandrapur 513 34 08 42 8.18
Nagpur 1125 33 12 45 4.00
Yeotmal 757 20 04 24 3.17
Total 3479 135 37 172 4.94

Table-2.4: Distribution of SCD gene in different ethnic groups of E. Maharashtra

Sr.No. Community Category Populatio screened AS SS Total (AS+SS) %

1 Bari OBC 92 - - - -------
2 Banjara NT 99 05 02 07 7.07
3 Bhangi SC 117 03 - 3 2.56
4 Bharadi OBC 13 - - - -------
5 Bhavsar OBC 08 - - - --------
6 Bhil ST 105 10 02 12 11.42
27
 CHAPTER II

7 Bhoi OBC 66 - - - ---------

8 Boudha SC 262 15 03 18 6.87
9 Charmkar SC 114 - - - ---------
10 Christian - 59 02 - 02 3.39
11 Daudi Bohra - 19 - - - ---------
12 Dhangar NT 53 - - - ----------
13 Dhanwar NT 21 - - - ----------
14 Gond ST 126 14 04 18 14.28
15 Gowari ST 102 04 02 06 5.88
16 Halba OBC 132 - - - ----------
17 Jain - 61 - - - ----------
18 Kalar OBC 141 04 - 04 3.83
19 Kolam ST 29 - 02 02 6.89
20 Koli Mahadeo ST 38 01 - 01 2.63
21 Komati OBC 77 - - - -----------
22 Korku ST 42 03 01 04 7.89
23 Kosti OBC 80 - - - -----------
24 Kumbhar OBC 12 - - - -----------
25 Kunbi OBC 275 11 02 13 4.72
26 Mali OBC 133 07 01 08 6.01
27 Maratha GC 89 02 01 03 3.37
28 Marwadi GC 92 - - - ----------------
29 Matang SC 217 15 04 19 8.75
30 Muslim - 69 01 - 01 1.44
31 Muslim Gawali - 40 - 01 01 2.5
32 Nhavi OBC 45 - - - ----------------
33 Parit OBC 22 - - - -------------
34 Pinjari OBC 08 - - - -------------
35 Powar OBC 123 05 01 06 4.87
36 Pradhan ST 87 08 02 10 11.49
37 Sonar OBC 84 04 - 04 4.76
38 Teli OBC 248 18 08 26 10.48
39 Wadhai OBC 28 - - - --------------
40 Warli ST 61 03 01 04 6.55

Table 2.5: Prevalence of sickle cell disorder (carrier) amongst OBC population groups
(State of Maharashtra)

Communities known as Scheduled Caste (SC), Scheduled Tribe (ST) and Other Backward
Communities (OBC) groups. It is rare in othercommunities.

District Prevalence Groups District Prevalence%

Chandrapur 24 Teli Gadchiroli 12

Gadchiroli 23 Teli Nagpur 10
Nagpur 22 Kunbi Gadchiroli 10
Bhandara 18 Kunbi Nagpur 04
28
 CHAPTER II

Gondia 15 Banjara Nanded 05

Thane 12
Wardha 07
Washim 07
Aurangabad 07
Nandurbar 05
Pune 04
*nEpidemiology of Sickle Cell Disorder in the State of Maharashtra © Kamla-Raj 2002 Int J Hum
Genet. 2(3): 161-167 ( 2002) S. L. Kate and D. P. Lingojwar

District showing High

prevalence of Sickle Cell
disorder in the State of
Maharashtra

Fig.2.9 Districts showing high prevalence of Sickle Cell

Disorder in the state of Maharashtra
Maharashtra Sickle Cell Prevention & Control program 2005-7

Studies are available giving the prevalence of Sickle Cell in Central India. Shukia and
Solanki in 1958 reported Sickle Cell gene frequency rate in various communities in Central India
as follows: Mahar (22.2%, Kunbi (9.4%), Teli (11.3%). Dr.S.L.Kate in 2000 reported Sickle Cell
gene frequency rate in various communities in Vidarbha Region and Rest of Maharashtra as
follows; SC (25%), Kunbi (9%), Teli (10%), ST(20%), Others (4%). In rest of Maharashtra, Sickle
Cell gene frequency in variour communities is SC(15%), ST (15% ).
RN Shukla and BR Solanki (1958), examined 1010 adult labourers of different castes
for sickle cell trait In Mahars , kunbis, and Telis, it's prevalence ranged from 9.4 to 22.2%.
Gupta V L (1981), reported prevalence of sickle cell in Nava Boudha, and Kunbis as 60% and
13.33% respectively. It is highly probable that this trait was brought to India by Negros from
Africa. More probably the present population acquired it from the primitive Veddoids and
29
 CHAPTER II

proto-Australoids. Highest prevalence of 55% has been reported in Parjah-kondas of South

India. Shukla and Solanki (1958), emphasized that the presence of sickle cell gene in this focus
near Nagpur has significance in that the cases reported above belong to the present day
scheduled castes and tribes and other communities and not to the aboriginal tribes. Nava
Boudha (Gupta V L, 1981) community includes mostly Mahars who en-mass embraced
Buddhism few years ago.

Population survey of Central India reveals that significant percentage of population

comprises of Scheduled castes and tribes and therefore high expected prevalence of sickle cell
gene.

Table. 2.6: Prevalance of Sickle Cell in Central India

R N Shukla B R Solanki 1958

Caste No.Examined No.Positive for Sickling Prevalance (%)

Mahar 450 100 22.2
Kunbi 116 011 09
Teli 080 09 11.3
Koshti 046 − −
Gond 053 − −
Mohemmdan 068 − −
Brahmin 026 − −
Mis celleneous 171 − −
Total 1010

Table.2.7: Caste wise distribution of 75 cases of Sickle Cell Crisis

Gupta VL, 1981

Caste No. of cases Percentage

Nava Buddha 45 60.0
Kunbi 10 13.33
Mahar 06 8.00
Koshti 02 2.66
Vanjari 02 2.66
Miscelleneous 10 13.33

A ongoing Pilot Project initiated by the Directorate of Medical Education & Research as
per G. R. No. IGM-2000/CR 368/2000/Prashasan-1, dated 10th July 2001 at GMC, Nagpur, IGMC
Nagpur & GMC, Dhule covered the following districts. Gadchiroli - Bhamragarh taluka, Aheri
taluka, District Yeotmal Pandharkawada taluka, Ghatanji taluka, District Nandurbar, District
Dhule, District Nagpur & District Wardha.
A total of 7534 people from general population screened in Nagpur showed a positivity

30
 CHAPTER II

of 18.48%. Of these positive cases 13.4% were SC, 1.3% were of ST, 0.46% of VJNT, 1.9% of OBC
& 0.63% others. Of the 21,051 hospital patients screened the percentage positivity was of
17.78%. Of these positive cases 11.3% were SC, 1.6% were of ST, 0.95% of VJNT, 2% of OBC &
1.75% others. Looking at the population size, percentage of SC, ST and the gene frequency in
these communities, a prevention and control program for Sickle Cell Disease is needed, which
will help in identifying & treating the patients, and also help in preventing occurrence of the
new cases by timely counseling & intervention.

Table.2.8: Demographic statistics of Maharashtra as per 2001 census:

Distribution of SC, ST communities district wise, which shows very high prevalence of Sickle Cell

Sr. Population Total SC ST

1 Maharashtra 96752247 8757842 7318281
2 Nandurbar 1062545 37298 661386
3 Dhule 1473170 97061 375105
4 Jalgaon 3187634 295047 313551
5 Buldhana 1886299 216687 95389
6 Akola 1351959 142485 91104
7 Wasim 862312 122069 64591
8 Amravati 2200057 384499 316448
9 Wardha 1067857 149975 166391
10 Nagpur 3287139 619226 457715
11 Bhandara 1021408 185176 81273
12 Gondia 1086221 107308 228549
13 Gadchiroli 787010 95996 304535
14 Chandrapur 1771994 299533 349169
15 Yewatmal 2077144 226820 445840
16 Nanded 2330374 422948 275972
17 Aurangabad 2213779 305246 83502
18 Nashik 3851352 326755 931069
19 Thane 5249126 271797 951205

31
 CHAPTER II

Prevalence Of Sickle Cell Disease In Certain

Communities
30

25

20
MAX
15 MIN

10

0
SC ST OBC

Fig.2.10 Prevalence of Sickle Cell gene in some communities,

Central India (Gupta VL, 1981)

With such a high prevalence in certain communities, (Fig.2.10) and distribution of

these communities (percentage of total population) across the districts in Maharashtra. (Table
2.8). A comphrenssive Sickle cell prevention and control program is needed, which in turn then
can be implemented at other parts of India where the prevalence of sickle cell gene is high

Genetics of sickling disorders

It was discovery by Emmel (1917), of the sickle cell phenomenon in the father of the
third described case of sickle cell anemia (Cook and Mayer 1915) that first reported a genetic
basis for sickling. Both Huck (1923) and Sydenstricker et al (1923), noted 'latent sicklers'
among relatives of patients with the disease and analysis of the pedigree of Huck's patient
(Taliaferro and Huck 1923) led to the conclusion that the sickle phenomenon was inherited as
Mendelian autosomal dominant characteristics. Diggs et al (1933) first clearly differentiated
symptomatic cases which they called sickle cell anemia, from latent asymptomatic cases which
they termed as sickle cell trait.

Although the great majority of cases fitted this pattern of homozygous inheritance,
apparent exceptions continued to occur. Cases with non-sickling parent of Mediterranean
origin were subsequently recognized to have sickle cell B thalassemia (Powell et al 1950,
Sturgeon et al 1952, Neel et al 1953a) and others sickle cell haemoglobin C disease ( Kaplan et
32
 CHAPTER II

al 1951, Neel et al 1953b) or sickle cell haemoglobin D Punjab disease ( Cooke and Mack 1934,
Sturgeon et al 1955).

Mendelian genetics
The inheritance of sickle cell disease obeys the principles of Mendelian inheritance.
When one parent is heterozygous for the sickle cell gene and other parent is normal, the
offspring would have an equal chance of having either sickle cell trait of a normal AA genotype.
If both parents have sickle cell trait , there is 1 in 2 chance of offspring having sickle cell trait
and 1 in 4 chance of the offspring being normal AA, being SS disease. This 1 in 4 chance of
offspring with SS disease remains for each pregnancy regardless of result of previous
pregnancies. (Fig. 2.11)

In applying Mendelian terminology to the sickle haemoglobin genotypes, the disease is

recessive In the sense it's characteristics are not manifest in heterozygous, the sickle cell gene is
dominant in the sense that it's presence is always expressed and codominant in the sense that
both normal and abnormal genes are expressed in heterozygote (Sergeant GR, 1985).

Population genetics
When studying genes in populations rather than individuals the behavior of gene
frequency is determined by the Hardy- Weinberg principle, which states that with certain other
assumptions, genotype frequencies remain constant from generation to generation, and
provides a formula for the 'equilibrium ' frequencies of the genotypes and their constituent
genes. For two alleles (alternative genes at the same site on each of a pair of chromosomes),
the frequencies which are represented by p and q respectively, the expected proportion of the
respective genotype s in the population may be represented by the formula (Serjeant GR,
1985).
 + 2 +  = 1

33
 CHAPTER II

Male/Female Male/Female Male/Female Male/Female

Homozygus Homozygus Heterozygus Heterozygus
“SS” “SS” “SA” “SA”

Homozygus Homozygus Homozygus Homozygus Homozygus Heterozygus Heterozygus Normal

“SS” “SS” “SS” “SS” “SS” “SA” “SA” “AA”
Sufferer Sufferer Sufferer Sufferer Sufferer Carrier Carrier Normal

Fig.2.11 The Inheritance of Sickle Cell Disease

34
 CHAPTER III

CHAPTER III
THE SICKLE HAEMOGLOBIN,HBS,
PROCESS OF SICKLING, AND
SICKLED ERYTHROCYTES

35
 CHAPTER III

Haemoglobin S

Sickle haemoglobin (HbS) is an example of a single point mutation. Nucleotide

sequencing of beta globin mRNA from sickle beta globin genes has revealed that the normal
codon GAG at position 'beta-6' has been replaced by GUG (Marotta et al, 1977). This change
determines the insertion of valine at this position instead of glutamic acid which occurs in
HbA, and in some way causes tendency of deoxy HbS molecules to form polymers.

Haemoglobin C (HbC) results from an amino acid substitution at the same site in beta
chain as HbS, the first nucleotide in the codon determining the amino acid at beta-6 being
changed from G to A (GAG to AAG) with resultant insertion of lysine .

In HbD Punjab ( or D Los Angeles), glutamine (CAG) replaces glutamic acid (GAG) and
in HbO Arab, lysine (AAG) replaces glutamic acid at the same site. Haemoglobin E results from
the substitution of lysine for glutamic acid at position Beta-26.These abnormal haemoglobins
as well as Hb Lepore Boston when inherited along with the gene for HbS result in clinically
significant sickle cell disease.

Molecular basis of sickling

Basic defect in haemoglobin S is substitution valine in place of glutamic acid at 6 th

position in beta chain. Difference being glutamic acid hydrophilic while valine is hydrophobic,
with acidosis and hypoxia decreasing the distance between two HbS molecules. There is
hydrophobic bonding, crystallization, parallel alignment, which results in formation of sickled
RBCs.

Sherman's observation of birefrigence in sickled RBCs proposed that certain surface

regions of deoxygenated molecules bear sufficient configurational similarity to each other so
that one molecule can fit closely against another molecule like fitting of antigen to antibody.
Deoxygenated molecules of HbS according to this concept would aggregate to form aligned
structures of sufficient rigidity to distort RBC membranes into sickled shape.

Some other observations suggested loss of oxygen lowered the solubility of sickle haemoglobin
and that the insoluble polymers taking needle like configurations formed bundles of sufficient
rigidity to sickle the cells.

So in summary the fibres responsible for sickling are long tubular structures each made
up of six filaments wound around a hollow core with pitch of about 3000A ( approximately 45
rings of haemoglobin molecule). Each of these six filaments are in register so that the fibre in

36
 CHAPTER III

effect a stack of discs each disc being six HbS molecules in a planer hexagon (John F Bertles,
1974).
N Hydrophobic N

6 th position beta chain

Hydrophilic, “Glutamic acid” is replaced
By Hydrophobic “Valine”
144 AA Additional Hydrophobic

Hydrophilic

N Hydrophobic N

Beta Chain Beta Chain

Hb A Hb S
Fig. 3.1 Beta Chain of Hb A is made of 144 hydrophilic amino acids with 2 hydrophobic at “N”
terminals
Beta Chain of Hb S is made of 144 hydrophilic amino acids with 2 hydrophobic at “N” terminals and
additional hydrophobic valine at 6 th position in beta chain

Hb SI beta Hb S II beta

Hydrophobic Bonding

Crystalliazation of HbS

Crystalliazed, ansiomorphic
HbS molecule

Hb Polymerization
Parallel alinement

Hypoxia
Acidosis
Fig.3.2 Hypoxia and Acidosis, brings together two HbS molecules nearer which leads to hydrophobic
bonding and HbS crystalliazation

37
 CHAPTER III

Ultrastructural features of erythrocytes and haemoglobin Sickling

Haemoglobin polymerization from process of condensati on, interaction, aggregation,
and assembly. Molecular organization of structures formed during polymerization depends on
type of haemoglobin, it's specific defect and it's of oxygenation. Sickling exemplifies
haemoglobin polymerization in the reduced state. Sickle haemoglobin (HbS) alone or in
combination with other haemoglobin undergoes codensation, aggregation and assembly into
long rod like structures. Oxygenated or reduced normal and abnormal haemoglobin and oxy
HbS also polymerize, they form aggregates, microtubules or rods but molecular reorganization
of earlier structures into a new lattice is rewuired . This sickling is due to sol-gel transformation
rather than crystallization and polymers of HbS are unique because they differ in molecular
organization from haemoglobin aggregates, microtubules and crystals. (James G White, 1974).

Polymer is readily visible on electron microscopy, but it's appearance in reversibly and
irreversibly sickled cells, ( Dobler and Bertles 1968, Bertles and Dobler 1969) resembling the
patterns of polymer formation in vitro in HbS solutions of low and high concentrations
respectively. On oxygenation the haemoglobin of both cells presents a granular amorphous
appearance with no evidence of polymer but the deformity of the membrane in ISC's persists.
The nature of these membrane changes is unclear but the abnormal shape is maintained by
exhaustively washed red cell ghosts and even by Triton X extracted spectrin/actin shells (Lux et
al 1976). HbS appears to bind more readily to the membrane than HbA (Shaklai et al 1981) as
do separated betaS chains (Bank et al 1974). There are also changes in lipid bilayer, the
normal inner leaflet phospholipids being found in abnormal amounts in the outer leaflet of
irreversibly sickled cells (Lubin et al, 1981). The significance of these changes is currently
unclear.

Functional characteristics of sickled cells

Deoxygenated sickled cells have long been known to leak potassium and allow sodium
to enter (Tosteson et. al. 1952, 1955, Tosteson 1955) this abnormality being attributed to a
defective Na+, K+- ATPase pump ( Clark et al 1978). In reversibily sickled cells, potassium loss
and sodium gain are approximately balanced so ther e is no change in intracellular hydration
(Glader and Nathan 1978), but in ISCs potassium loss exceeds sodium gain with resultant
cellular dehydration ( Glader et al, 1978, Eaton et al 1979).

Sickled cells also accumulate calcium although the mechanism is controversial. Calcium
ions enter SS red cells more rapidly than normal (Eaton et al, 1973), although the experimental
evidence on Ca++ extrusion from the cell is conflicting being considered normal (Palek 1977a) or
defective (Litosch and Lee 1980, Bookchin and Lew 1980), depending on the control
observations. Activity of the calmodulin stimulated ATPase pump in SS red cells had been

38
 CHAPTER III

reported to be reduced, ( Gopinath and Vincenzi 1979, Dixon and Winslow 1981), although this
was considered normal in what was judged to be more appropriate assay system for(Luthra and
Sears 1982). The current evidence suggests that the most likely cause of raised calcium level s
is an increased membrane permeability . It is unclear whether the elevated calcium levels in the
cells of SS disease have pathological significance (Eaton et al, 1978, Steinberg et al, 1978).
Potassium loss, sodium and calcium retention, total action depletion and dehydration are
characteristics of normal red cells up on ATP depletion, but the interpretation of ATP Levels in
SS disease is difficult ( Weed and Bessis 1975, Clark at el 1978, Luthra and Sears 1982).

A variety of other abnormalities have been described. Sickled cells have decreased
polyunsaturated fatty acids, increased levels of malonyidialdehyde (MDA) (Das and Nair 1980),
and increased formation of MDA upon incubation (Chiu et al, 1979). Catalase activity is
decreaed, superoxide dismutase increased ( Das and Nair, 1980), and free oxygen radicals
generated in increased amounts. (Hebbel et al, 1982). Furthermore an unusual phospholipid
linked to MDA has been isolated from the membranes of irreversibly sickled cells (Jain and
Shohet, 1984). These abnormalities raise the possibility of increased lipid peroxidation but it's
contribution to membrane damage in SS disease (Palek 1977b) is currently unclear.

Defective Na+ K+ Potassium loss - - -

ATPase Pump Sodium gain +
Calcium gain ++
Potassium loss -- Zinc loss - -

Sodium gain +
Cellular dehydration
No cellular dehydration Rigid distorded

Fig. 3.3 Sickle Cell Fig 3.4 Irreversibly Sickled Cell (ISC)

Endothelial adherence
Oxygenated HbS, containing cells adhere abnormally to the plates of cultured human or
bovine endothelium (Hoover et al 1979, Hebbel et al 1980a) suggesting a further factor in the
pathophysiology of sickle cell disease. The finding has been demonstrated in both states.
(Hoover et al 1979, Hebbel et al 1980a) and flowing (Hebbel et al 1980a, kucukcelebi et al
1980) experimental models and has been attributed to an altered pattern of red cell surface
negative charge ( Hebbel et al 1980a). The degree of adherence varies between individuals and
has been claimed to correlate with indices of vaso-oclusion both in SS disease (Hebbel et al
1980b) and in other sickling disorders (Hebbel et al 1981).
39
 CHAPTER III

Sickled erytrocytes, surface, and membrane changes and it's effects on circulation
Riddick, 1968, has put forward the photochemical mechanism of blood coagulation
which implies the presence of substantial amount of electrical repulsive forces for maintaining
the discreteness of blood cells without any coagulation. It is well known that electrokinetic
potential (EKP), which is a measure of diffuse double layer charge density is mainly responsible
for such repulsion (Kitchnel JA, and Musselwhite PR, 1968). EKP of erythrocytes has been
studied under varying conditions ( Furchgott RF, and Pond er E. 1941) of PH, ionic strength and
agents modifying their characteristics ( Heard DH, and Seaman GVF 1960, Svennerholm L 1956,
Beleman JB, and Zellner A 1956, Cook GMV, 1962, Seaman GVF, and Heard DH, 1960). As per
the above physiochemical approach to the mechanism of blood coagulation, it appears that
EKP could be a substantially contributing factor.

So long as the EKP of system remains constant the fluidity of system will remain
constant. But the EKP is progressively lowered by the introduction of cationic electrolytes or
polyelectrolytes then the stability of system will undergo progressive changes from simple
agglomeration to fluid gel formation and finally to a rigid gel (Chapman D, Kamat VB, et al,
1968, Riddick T M, 1968).

Significance of EKP on erythrocytes

Difference in electrophoretic properties were first noted in case of sickle cell anemia
and depending on electrophoretic mobility (EM) ten different types of haemoglobin in human
subjects have been ascribed (Pauling L, Itoni HA, et al. 1949).

EKP of sickled erythrocytes

There are several factors which will interfere with EKP of sickled erythrocytes. In fact
reduced electrophoretic mobility was first identified with haemoglobin 'S' because of it's
distorted shape and change in polyelectrolyte atmosphere around, there is change in electrical
double layer on erythrocyte which in turn leads to change in electrokinetic potential.
Electrokinetic potential (EKP), was studied in patients with sickle cell disease, and it's possible
application in course of disease was analyzed (Gupta VL, and Choubey BS, 1982, 1984).

40
 CHAPTER III

Table-3.1: Electrokinetic Potential (EKP) of erythrocytes in Normal controls and Sickle cell disease

Bl. Gr*. Study Gr. Mean EKP. (mmV) 'P' value

O Control 34.2
SCD 22.8 <0.01
B Control 22.6
SCD 16.6 <0.01
A Control 26.6
SCD 20.3 <0.01
AB Control 19.3
SCD 13.5 <0.01
* Blood groups and EKP: Number of factors influence electrical double layer of erythrocytes , further rheological
atmosphere, cationic electrolyte input, polyelectrolyte balance all can affect electrokinetic potential of
erythrocytes. There is wide variation in readings. Electrokinetic potential also varies according to ABO blood
groups possibily because of change in one of above factors., or possibly because of presence of surface antigen
which decides blood group, contributing for resultant electrokinetic potential (Riddick TM, 1968, Panpaliya GM,
Dorle AR, 1980, Panpaliya GM, 1977).

Measurement of Electrokinetic Potential (EKP), or Zeta Potential of erythrocytes

Zetameter
The microelectrophoresis assembly designed and developed for electrophoretic mobility
and electrokinetic potential determination is shown in the figure. 3.5

The Zetameter essentially consists of power supply, multimeter, micrometer,

potentiometer, reversing key, microelectrophoresis cell. The source of potential is a power
supply unit which can supply the potential in two ranges i.e. 0-30 volts and 0-300 volts. The
terminals of power supply providing the high voltage are taken into circuit. The voltage
operating on the system can be varied by the use of potentiometer. The applied voltage can be
read on multimeter. The reversing key reverses the polarity of electrophoresis cell but does not
affect ammeter, which can be left in circuit. A circuit selector changes the range of micrometer.
The operator's left hand holds the stop watch, reverses the current and manipulates the
potentiometer to keep the current constant (Panpaliya GM, 1977).

41
 CHAPTER III

TO MAINS
MULTIMETER

PLATINUM
ELECTRODES HIGH VOLTAGE POWER SUPPLY

MICRO ELECTROPHORESIS CELL

ZETAMETER ASSEMBLY

Fig.3.5 Zeta Meter Assembly for measurement of EKP

Procedure: The erythrocyte suspension which is to be evaluated for electrophoretic

mobility and EKP is introduced into electrophoresis cell, care being taken to avoid air bubbles.
The cell is clamped in position under the microscope with magnification of 400. The
microscope is adjusted with fine focussing adjustment so that particles which are at stationary
level are in focus. Since the stationary level in cylindrical chamber is in horizontal cylinder, the
particles in the same microscope are at different levels unless the focus is directed against
exactly at high point of focus (Briton CA and Lauffer MA, 1959). However this difficulty is
overcome by counting only those particles that are in sharp focus (Hydon DA, 1961). The circuit
is closed and field strength is made just high enough to produce turbulence. The time
required for a specific particle in a sharp focus to migrate over given number of divisions of
chess board graticule, which is is calibrated with a standard stage micrometer for the
particular optical system is determined . A length of scale is selected for which the tracking
time is between 4 and 10 seconds. At times of less than 4 seconds errors in starting and
stopping the watch begins to be significant, whilst at times over 10 seconds there is a greater
chance of the particle moving out of the stationary level or drifts arising. A stop watch
reading to 0.1 second is used. The direction of current flow is reversed with the help of
reversing switch after every 3 to 4 measurements so as to minimise electrode effects. These
measurements should agree, and any divergence can usually be traced to air bubbles or
leak in the cell ( Northrop JH, 1922). When there is no potential across the c ell, the particles
should remain stationary. The voltage across platinum-iridium electrodes in the electrophoresis
cell is kept constant throughout measurement and is recorded in multimeter.
42
 CHAPTER III

The number of particles timed depends on the homogeneity of the suspension. A

minimum of ten particles should be timed, but it may be necessary in some instances to choose
greater number depending upon the degree of heterogenisity of a sample.The experimental
results are expressed in terms of u/sec/volt/cm and electrokinetic potential (Z) is calculated by
the use of the Helmholtz- Smoluchowski equation (Smoluchowski M, 1969).

Simplified formula to calculate EKP (Z)

Constants arrived and included

150 ∗  ∗ 1000

=
 ∗ 
Where. 150 = Constant
d = distance in cms, micrometer scale
1000 = Constant
 
PG = Potential. Gradient  =   !" "  #  $

Sec. = Time required in sec, stop watch,

Example:

Average time = 15 seconds

%&'
Voltage Applied = 150 , %' = 15
Ocular. 30 divisions. = 0.0357 cms

150 ∗ 0.0357 ∗ 1000 357

= = = 23 ++,
15 ∗ 15 15

43
 CHAPTER III

ELECTROKINETIC POTENTIAL, “Z” POTENTIAL OF RBCs

_ _ _ _
_ _ _ _ _
_ _ _ _ _
_ _ _ _

NORMAL “Z” POTENTIAL REDUCED “Z” POTENTIAL

REPULSE CONGLOMERATE

Fig.3.6 EKP in maintaining fluidity of blood

Irreversibly Sickled Cells

Irreversibly sickled cells (ISCs) have deformed membranes that have lost the capacity to
return to a normal shape even on exposure to oxygen.

It was these cells which led to Herrick's first description of the disease and Diggs and
Bibb (1939) first noted that the number of these cells remained relatively constant within
individuals but might vary markedly between different patients. The role of prolonged hypoxia
in ISC formation was suggested by Diggs and Bibb (1939) and by Shen et al (1949). The
number of ISCs obtained by direct splenic puncture was 4-10 times greater than that observed
in peripheral blood of the same patient (Watson et al 1955), suggesting that these cells had
either formed or been sequestered at this site ( Lichtman et al, 1953). The role of repeated
cycles sickling and unsickling in the genesis of ISCs was suggested by Padila e t al, 1973). who
noted with cycle in single preparation , the cell appeared to become less pliable, had greater
difficulty in unsickling and was finally fixed in the Sickl ed form . This implication.that the cells
more prone to the. Sickling readily may become ISCs was. consistent with observation of that
ISCs tended to have lower levels of HbF (Bertles and Miner 1968) and with the overall
relationship between high ISCs and low HbF levels (Bertles and Miner 1968, Serjeant 1970)

Early assumptions that ISCs were old were incorrect and probably based on the rarity
of reticulated ISCs and the relactant sickling of normoblasts . Using a cohort label incorporated

44
 CHAPTER III

only by reticulocytes and earlier precursors. Bertles and Milner (1968) were able to show
some cells became ISCs within 5 days of labeling.

Estimating cell age by glycosylation In red cell fractions separated by stractan gradient
also suggested that the densest cells which included the ISCs were relatively young (Noguchi et
al 1985). Once formed ISCs appear to have.a very short survival ( Bertles and Milner 1968).

Clinical Relevance of ISCs

It is likely that ISC represents a cell at the end of it's functional life as such, it may have
limited relevance to clinical manifestation s . However if the ISCs reflect the ease of sickling
within the red cell population, they may be useful tool for investigating the relevance of in
vitro sickling to complications of the disease.

The relationship between ISC count and pathological processes in SS disease has been
explored in the conjunctival vessel anomalies ( Serjeant et al 1972, Nagpal et al, 1977a),
haemolytic rate (Serjeant et al, 1969, McCurdy and Sherman 1978), splenomegaly ( Serjeant
1970), Priapism (Emond et al, 1980), angoid steaks (Hamilton et al 1980), macular vasculature
( Marsh et al 1982), proliferative retinopathy ( Hayes et al 1981), epiphysial fusion (Serjeant
and Ashcroft 1973), and avascular necrosis head of femoral head (Hawker et al 1982). The only
significant effects were with barometric rate (fig.) and the density of conjunctival vessel
abnormalities. The later relationship may be explained by the highly atypical nature of the
conjunctival capillary bed.exposed on one side to the high oxygen tensions in the air. Under
these conditions potentially reversible sickled cells revert to no rmal and ISC may represent the
only cell capable of capillary obstruction.

45
 CHAPTER III

Fig. 3.7 Relatioship between 51Cr and red cell survival, expressed as Slope of the
survival curve (k), and the ISC count in 28 patients with SS disease (After Serjeant et al. 1989)

Red cell survival

Measurements of red cell survival vary markedly in SS disease and are difficult to
evaluate because of the variable clinical indications for performing the investigation.
Hypersplenism is a common indication and measurement of 51 Cr half.life in affected
patients may be as short as 1 day (Rossi et al 1964) Estimates in Jamaican patients
uncomplicated by hype rsplenism range from 2 to 21 days with mean of 10 days.

An importan t determinant of the haemolytic rate is the proportion of the red cell
population with low HbF levels and therefore more prone to sickling . The ISC count is related
to the haemolytic rate, and the ISC itself is a short lived cell (Bertles and Mi knee 1968)
although premature destruction must also affect cells that are not irreversibly sickled .

Hypersplemons also increases haemolysis although it's effects are complex involving
both red cell sequestration and influencing the plasma volume. An increased sensitivity to
shear stress in SS eruthrocytes has been demonstrated in vitro experiments and is occasionally
manifest as haemoglobinuria on vigorous exertion (Platt 1982).

We estimated red blood cell half life using radioisotope technique using Cr51 tagging.
Cr51 labeling and determination of RB C survival was performed under aseptic precaution By
the method described by Jandl et al. 1956
46
 CHAPTER III

The radioactivity of all blood was determined after hemolysis by freezing and
thawing . The activity of whole blood samples was determined in a well type scintillation
counter. The percentage of radioactivity was p lotted on semilog paper (Fig.) The RB C half
life is the number of days required for the radioactivity in the bl ood to reach 50% of the
initial level.

Ten normal individuals were studied as controls. Out of 20 patients studied 10 had
homozygous disease (SS), while 10 patients were of heterozygous type (SA) i. e. Sickle cell
trait.
Table.3.2: Red Blood Cell half life in controls and Sickle cell
Type of Hb No. of Cases Mean RBC half life
AA 10 25.25 days
SA 10 17.86+2.18
SS 10 13.8+0.95
* Gupta VL, and Chaubey BS 1988

RBC half life is less in sickle cell trait (SA) and is further reduced and is least in sickle
cell disease (SS).

The mean RB C survival in normal individuals was 25.25 + 2.88 days. Toghill PJ, 1964,
reported. similar RBC survival in normal individuals. The mean RBC survival in sickle cell anemia
was 13.80 + 0.95 days . Jandl et al, 1964, reported RBC survival in sickle cell anemia to be 11. 2
days. In Sprague and Peterson series (1958), it was 2 to 15 days, while in the Hathrom series
(1967) it ranged from 3.4 to 9.4 days . Values reported here are consistent with those reported
by Jandl et al (1964).

Fig.3.8 RBC half life

47
 CHAPTER III

The basic defect in HbS is substitution of hydrophobic valine in place of hydrophilic

glutamic acid at 6 th position in beta chain. Acidosis and hypoxia which are triggers for process
of sickling brings two HbS molecules nearer leading to hydrophobic bonding, resulting in HbS
crystallization. Two HbS molecules after crystallization forms HbS polymer which gets parallel
alignment along the cell wall, making cell membrane weak, which in turn forms tactoid process
and later assumes the shape of sickle. Thus sickle cell is formed.Fig.3.9.
Because of it's abnormal and distorted shape, there is shear mechanical stress while
flowing through capillaries, and small blood vessels. There is mechanical entanglement leading
to sluggish blood flow. Because of changes in polyelectroly lite balance and subsequently in
double electric layer at cell membrane, the negative charge on surface is reduced leading to
reduction in repulsive forces or electrokinetic potential (Zeta Potential). The RBCs therefore
tend to conglomerate leading to vaso-oclussion. Lot of other factors come into play to
accelerate and enhance vaso-oclussive phenomenon leading to Ischaemia and infarction
which are predominantly responsible for most of the clinical features Fig.3.9.
Because of shear mechanical stress, weakness of cell membrane, reduced RBC survival ,
and also because spleen picks them up as abnormal cells, get them lysed and removed from
circulation, the process of haemolysis is continuous at times get accelerated leading to
hyperhaemolytic state or crisis, another important presentation of sickle cell disease.
Most of the sickle cell do revert to normal shape, but quite few of them develop permanent
changes, like change in polyelectrolyte balance ,Importantly influx of calcium.in the.cell at
cost of exit of zinc. Hb S in the cell binds with calcium, (HbS+ Ca). The cell gets dehydrated
becomes rigid and further distorted and irreversibly sickled Fig.3.9.

48
 CHAPTER III

Hydrophobic Bonding
Hb Crystalliazation
HbS Poymer Parallel Alignment along cell membrane

Change in Electrical Double Layer

Change in Polyelectrolye Balance
Slow mobility of HbS Cell membrane Weakness

Tactoid Formation, Sickling

Reduced “Zeta Potential”
Electrokinetic Potential
Sickle Cell Sickle Cell

Conglomeration Sluggish blood flow

Mechanical entanglement

Vaso-oclussion Haemolysis

Change in Polyelectrolyte Balance

Cellular Dehydration
Entry of Calcium in the cell
HbS +Ca formation
Distorted, rigid, sickle cell

Irreversibly Sickled Cell, (ISC)

Markers of Organ Damage

Fig. 3.9: Flow Chart showing pathophysiology in Sickle Cell Disease

These irreversibly sickled cells (ISCs) are markers of organ damage in sickle cell
disease. There are some associated factors like predisposition for infections which also add
and modify the clinical course of the disease.
49
 CHAPTER IV

CHAPTER IV

THE SICKLE CELL CRISIS

50
 CHAPTER IV

The Sickle Cell Crisis

The word 'crisis' has it's origin from Greek word 'krisis' which means turning point.
Dictionary defination of the word crisis, (i) a striking change in the symptoms attended by
outward manifestations. (ii) a painful paroxysmal disturbance of function accompanied by pain.

Sydenstricker, 1924, used first time word crisis in association with sickle cell anemia.
Graham described pains in same year. Penbetty and Cooley described haemolytic crisis
occurring in patients with sickle cell anemia in association with streptococcal infections.

However accepted defination and classification of sickle cell crisis was by L W Diggs in
1965. The word crisis is defined as a sharp turn or definate change in the course of disease
with development of new symptoms and signs.

TABLE. 4.1: Types of sickle cell crisis

Vascular oclussive crisis Haematological crisis

A. Capillary Stasis A. Aplastic crisis
(i) Focal B. Haemolytic crisis
(ii) Systemic
B. Venous thrombosis
C. Arterial Emboli

(i) Pulmonary
(ii) Fat
*LW Diggs 1965

According to severity, crisis has been classified as (i) mild and (ii) severe. Felix ID,
Konotey Ahulu 1974 are also in agreement.with defination and classification given by Diggs LW
1965, in their opinion there is often a combination of two or more of these crisis types in a
single clinical picture - "Mixed Crisis"

Diggs LW 1965 has reported vascular oclussive crisis as typical type of crisis that occurs
in most of the patients. He has described following variants of oclussive crisis and studied them
in detail, (a) hand and foot syndrome (b) recurrent painful crisis (c) hepatic crisis with regur
gitant jaundice (d) Priaprism ( e) spleenic infarcts (f) aseptic necrosis of head of femur (g)
cerebral cortical infarction with hemiplegia (h) encephalopathies (i) papillary renal necrosis.

51
 CHAPTER IV

Table. 4.2: Recognized types of sickle cell crisis

1. Vaso-oclussive
2. Haemolytic or Hyperhaemolytic
3. Mixed (Vaso-oclussive + Haemolytic)
4. Aplastic
5. Sequestration
6. Megaloblastic

With (j) haematuria (k) infarcts of.other viscera ( l) Sudden and unexpected death (m) venous
thrombi (n) pulmonary artery emboli (o) fat emboli.(Table-4.1)

Till now, clinically recognized types of “crisis” are Vaso-ocussive (VOC),

Hyperhaemolytic, as is increasingly recognized that haemolysis is continuous phenomenon in
sickle cell disease, and whenever it is increases to crisis proportion, it should be lebelled as,
“hyperhaemolytic”crisis. More than often, there is combination of vaso-oclussive and
haemolytic crisis, third common situation, and is lebelled as, “mixed crisis”. Aplastic crisis
though rare is well recognized. Last two types i.e. sequestration and megaloblastic are rarest
types but are commonly reported in literature.( Table-4.2).
Table. 4.3: Frequency of various types of sickle cell crisis
Crisis Type. No. Percentage. No.of Episodes
Painful. 73. 73. 328

Sequestration 08. 08. 008

Haemolytic 13. 13. 013

Aplastic 06. 06. −

G. Karayalcin et. al. 1975

G. Karayalcin et. al. 1975, in their series of 100 patients reported highest number
suffering from painful crisis (73%), followed by haemolytic (13%), aplastic, and sequestration
crisis (08%), (Table-4.3).

In a series of 75 cases, Gupta VL et al 1981, reported incidences of various types of

crisis as follows: most common being vaso-oclussive (64%), followed by haemolytic (22.6%),
mixed, i.e voc+ haemolytic (10.6%) and aplastic (2.6%) (Table. 4.4).

52
 CHAPTER IV

Table. 4.4: Frequency of various types of sickle cell crisis, N = 75

Type of Crisis No. of Pts. Percentage
Vaso-oclussive 48 64.00

Haematological

a) haemolytic 17 22.66

b) aplastic 02 02.66

Mixed* 08 10.66

* Vaso-oclussive + Haemo

A
22
8 =8
MIXED

VOC
HAEMOLYTIC
17
HAEMO =17 VOC
48 = 48 MIXED
APLASTIC

Fig. 4.1 Pie diagram showing frequency of

Various types of sickle cell crisis, N= 75 *Gupta VL et al 1981

53
 CHAPTER IV

Table- 4.5: No.and type of crisis, days spent in hospital, and total working days lost in 25 patients of
sickle cell disease, in one year follow up

SNo. Crisis N/T. H- days. W- days

1. 3VO, 1H. 17. 26
2. 4VO, 1H, 1M 22. 35
3. 4VO, 2H. 16. 30
4. 5VO, 20. −
5. 3VO, 1H. 15. 21
6. 4VO, 2M 24. 32
7. 2VO, 3S. 30. 42
8. 2VO, 2M, 1S. 25. −
9. 3VO, 1H. 14. 16
10. 3VO,2H, 1M. 15. 46
11. 5VO, 1M. 25. 46
12. 4VO, 1H, 1S. 24. 41
13. 2VO, 1M 10. −
14. 5CO. 20. 25
15. 5VO, 3M. 17. 27
16. 4VO, 1A. 29. 38
17. 4VO, 2H. 21. −
18. 1VO, 3M, 1H. 22. −
19. 3VO, 2H. 16. 30
20. 3VO, 3M 20. 29
21. 2VO, 1H, 1M. 15. −
22. 3VO, 2M. 18. 19
23. 4VO 26. 38
24. 2VO, 2M. 24. 40
25. 3VO, 1H,1M. 17. 49

Abrev. SN = Seial Number, Crisis N/T = Crisis number and type, H-days = No. of days spent in hospital,
W-days= Number of total working days lost, VO= Vaso-oclussive, H= Haemolytic M= Mixed (Vaso-
oclussive + Haemolytic), S =Sequestration, A = Aplastic
*Gupta VL et al 1989

25 patients of homozygous sickle cell (SS), who had predisposition and propensity for
developing crisis were followed for 1 year at sickle cell clinic, Govt. Medical College

54
 CHAPTER IV

Nagpur,number of crisis, including mild and severe types were recorded. Severe crisis
required admission in hospital. Morbidity parameters in the form of days spent in hospital,
and total working days lost were also recorded. (Gupta VL et al 1989).

These 25 patients, over 1 year follow up, had total 128 episodes of crisis, (including
mild crisis, managed on outdoor basis). Commenest type was vaso-oclussive crisis, 84,
followed by mixed type(vaso-ocussive + haemolytic) 23, hyperhaemolytic 15, sequestration 5
and aplastic 1. So the typical painful crisis i.e. vaso-oclussive crisis was the commonest and
dominated the clinical course of the disease.(Table- 4.5)

Table-4.6: Morbidity parameters, in 25 patients of sickle cell disease over 1 year follow up
Parameter. Total no. Mean/Patient
Crisis. 128. 5.08
Days spent 502. 20
In hospital.

Working 635. 33.4

Days lost
*Gupta VL et al 1989

As far the morbidity parameters, average number of crisis in 1 year per patient was
5.08. On an average, they spent 20 days in hospital yearly and had loss of 33.4 working days
in a year. So in severe cases the disease has significant morbidity associated with it. (Table-
4.6)
Clinical menifestations of sickle cell crisis
The hand and foot syndrome , a common initial manifestation of sickle cell disease
during infancy refers to dactylitis resulting from symmetrical involvement of.metacarpel and
metatarsal bones. It is manifested as painful swelling of dorsum of hands and feet .
Symptomatic painful crisis involving.the joints and the extrimities usually begins in
early.childhood. Pain may be due to infarction of long bones and bone marrow or involvement
of periarticular tissues. Painful abdominal crisis are due to infarctions of areas of liver spleen
and lymph nodes . Pain is usually attributed to stretching of the capsules of these structures.

The other type of crisis in sickle cell disease is the hyperhaemolytic crisis , in which
there is increased destruction of red blood cells associated with erythroid hyperplasia in bone
marrow. Hyperhaemolytic crisis may be accompanied by pain, fever, anaemia,
normoblastemia, reticulocytosis, and increased serum bilirubin levels.

55
 CHAPTER IV

Aplastic crisis is a misnomer, since there is only an acute erytroblastopenia, ( Miosch

DC, Baxter MR), Leukopenia is quite uncommon and thrombocytopenia extremely rare. The
most common symptoms in association with fever, lethargy, pain annorexia, weakness,
nausea, vomiting and headache,. Most prominent physical findings include pallor,
hepatosplenomegaly, lymphadenopathy, and signs of upper respiratory infections, (Kim KY,
Karayalcin.G)

Table-4.7: Symptoms at the time of crisis, N=33

Symptoms No. of Patients

Fever 23
Abdominal pain 17

Pain in extremities 10

Back pain. 07

Chest pain 03

Aseptic necrosis 01

Rt hemiparesis, head ache 01

Lt hemuparesis , head ache. 01

Parasthesias arm,head ache. 01

* Karayalcin G, et al, 1975

“Pains and Aches” dominates the clinical picture of typical painful crisis or vaso-
oclussive crisis of sickle cell disease. Abdominal pain, pain in extremities, joints, back, chest are
basically ischaemic pains because of vaso-oclussive phenomenon, and therefore at times
excruciating and intolerable. If it produces infarction, then menifestations like aseptic
necrosis, neurological deficits like hemiplegia etc. occurs. It will depend on area of vaso-
oclussion and further infarction. Sicklers are predisposed to infections, it can precipitate crisis
or can be superadded complicating the crisis menifestations, therefore can be a common
sympotom (Table-4.7).

Table- 4.8: Common symptoms encountered in 75 cases of sickle cell crisis

Vaso-oclussive crisis
Joint pain
Abdominal pain
Generalized “pains & aches”
Chest pain
Fever
56
 CHAPTER IV

Haemolytic and Mixed type of crisis

Fever
Yellowness of eyes
Extreme weakness
Bleeding diathesis
* Gupta VL, et al, 1981
Again generalized “pains and aches” dominates the the clinical picture of typical painful
vaso-oclussive crisis of sickle cell anaemia. Fever may be a symptom of associated infection
both in vaso-oclussive, and haemolytic crisis or mixed type. Hyperhaemolytic crisis may
manifest as jaundice mild though. Advanced liver dysfunction, or bleeding diathesis rare, is
usually because of associated sickle cell hepatopathy. (Table-4.8)

The typical painful crisis, vaso-oclussive crisis is commonest and representative of sickle
cell disease, which dominates the clinical manifestation and course of the disease. (Diggs
LW,1965, Karayalcin G, et al, 1975, Gupta VL, et al 1981).

The pathophysiology of sickle cell crisis

The elongated, multipointed, and rigid sickled erythrocytes cause an increase in
viscosity and obstruct the flow of blood in capillaries, terminal arterioles and veins. There is
also mechanical entanglement which hampers the smooth flow adding to obstruction. (Fig. 4.2
A).Hypoxia due to stasis of blood, is associated with cellular injury, vasospasm, leakage of fluids,
and cells into perivascular spaces, haemoconcentration and fibrin formation. Localized sickling
temporary or partial vascular occlusion and perivascular occlusion - oedema cause pain and
swelling in involved organs. Generalized sickling is associated with symptoms or sudden and
unexpected death. Chronic anaemia, stasis, hypoxia lead to endothelial proliferation, tissue
injury, and replacement of parachymal cells by fibrous tissue. If ishaemia is severe and
prolonged in a given anatomic site, there is infarction and necrosis.

57
 CHAPTER IV

Normal RBCs, Smooth Flow Sickled RBCs, Mechanical

Entanglement, Sluggish Flow
Fig.4.2 A Fig.4.2 B

In general, the degree of anaemia, and jaundice, and frequency and severity of the
vascular oclussive process are proportional to the percentage of sickle haemoglobin that is
present.

The fundamental abnormality resides in sickle cell. haemoglobin which is gene

determined type of abnormal haemoglobin. In the deoxygenated state Hbs is relatively
insoluble and forms needle like tactoids which distort shape of erythrocytes producing the
characteristic sickle cell deformity. Sickled erythrocytes are mechanically more fragile,
phagocytised readily and destroyed more rapidly in the circulation than are the nonsickled
cells. The increased blood destruction is associated with jaundice of hemolytic type,
hyperplasia of bone marrow, reticulo cytosis and immature cells in peripheral blood. As a
result of imbalance between. the rates of blood formation and destruction , there are
varying degree of anaemia which.f or given individual is fairly constant. Clinical presentations
depend on type of crisis, the patient has, most common being the vascular oclussive or
painful crisis , what is described as typical crisis in sickle cell disease.

58
 CHAPTER IV

Fig.4.3 Normal red blood cell and capillary flow, Sickled RBCs and capillary flow

The pain of the painful crisis , when it involves areas of the long bones, appears to be
due to the inflammatory response to bone marrow necrosis. This was initially demonstrated by
the aspiration of the.neurotic bone.marrow from the site of bone pain ( Charche and Page
1967, Hammel et al 1973, Hutchinson et al 1973, Alvi et al 1974, Majd and Frankel 1976).
but has since been amply confirmed by scintigraphy. Bone marrow scans with 99m Tc sulphur
colloid demonstrate reduced uptake and defective function of teriyaki-endothelial cells at the
site of pain (Hammel et al 1973, Hutchinson.et al .1973, Alavi.et al 1974, 1976, Majd and
Fran Kel 1976). and 52 Fe positron.scans which.are cons istent with.absent erytrhopoiesis in
affected areas (Hammel et al 1973). Bone marrow scans with 85 Sr show increased activity at
the site of pain compatible with accelerated calcium turnover and 18 F bone scans are normal
indicating normal. Bone blood flow (Hammel et al 1973) in contrast to the deceased
marrow.blood flow. Resolution of the painful crisis is accompanied by a gradual repopulation
of the affected areas with reticulo-endothelial cells (Alavi et al 1974).

Bone marrow scanning does not confirm marrow infarction in all painful crisis, normal
scan occurring in 4/10 in one series (Alavi et al 1974), and 3/12 in another (Lutzker and Alavi
1976), perhaps because the necrotic areas were too small. Decreased marrow.uptake,
consequent on presumed previous ingarction , occurred in 8/17 patients in the steady state
suggesting.that recolonization of infected marrow may not always occur (Alavi et .al 1974)
and permanent marrow fibrosis may develop (Diggs 1967).

59
 CHAPTER IV

Fig.4.4 3D representation of initiation of vaso-oclussive phenomenon In sickle cell anaemia

Bone marrow embolism

During some painful crisis, necrotic bone marrow, fat, and even sp icules of bone may
occur as emboli in the small vessels of brain, lungs, or kidneys. The first report described a
young woman with cerebral fat embolism ( Brittingham and Phinizy 1931) and many further
reports followed (Diggs et al 1937, Evans and Symmes 1957, Ober et al 1959, Chmel and
Bertles 1975), patients appearing to be at special risk in late pregnancy (Wyatt and Orrahood
1952, Ranney et. a l 1953, Hendrickse and Watson -Williams 1966. Diggs (1967) proposed that
the increased marrow pressure caused by infarction and the associated inflammatory response
drives particules into the sinusoids from where they pass to the lungs and via intrapulmonary
haunts to the arterial. circulation.

Clinically patient may commence with a typical crisis but then deteriorate with a stormy
course severe back pa in, chest tightness, dyspnoea , falling arterial.oxygen tension, and signs of
systemic embolism with respiratory, renal, central nervous system involvement, and
occasionally skin petechiae. Laboratory evidence of disseminated intravascular coagulation is
common. The diagnosis is based on the severe clinical course, and may occasionally be
confirmed by the findings of fat droplets in the peripheral blood (Smith and Krevans 1959),
sputum ( Hutchinson et al 1973), or urine (Newman 1948). Treatment is by supportive therapy
with blood transfusion, oxygen, and heparin.

60
 CHAPTER IV

Haematological findings in the painful crisis

Haemolysis
The term 'haemolytic crisis ' is frequently but incorrectly used interchangeably with the
painful crisis. Sickle cell disease is characterized by chronic haemolysis in the steady state and
there is no evidence in the great majority of cases of a further increase in haemolysis during
painful crisis (Diggs 1956). Serum benzidene positive pigments were raised in the steady state
(Lathum and Jens en 1962, Upshaw at al 1963) and did not change significantly in the painful
crisis ( Iuchi et al 1964, Stutman and Shinowara 1965). Plasma haemoglobin increased early. in
the course of painful crisis ( Neely et al 1969, Naumann e t al 1971), but.the magnitude of
change was considered consistent with.minor in the vessels in the area of necrosis and not
indicative of a more generalized increase in haemolysis.

Lactic dehydrogenase (isoenzymes 1 and 2) levels were elevated in steady state SS

disease and increased further during painful crisis (Neely et al 1969). Later observations.have
confirmed that this enzyme ( also known as alpha- hydroxy butyrare dehydrogenase or alpha-
H.B.D.) rises during painful crisis (White et al 1978, Karayacin et al 1981) and.that this rise may
precede symptoms. In the absence ofcardiac, skeletal muscle or liver disease, it is presumed
that this enzyme is released by necrotic bone marrow, and may therefore be a useful objective
indicator of the painful crisis.

Haematological indices
Interpretation of haematological events during the painful crisis is complicated by the
effects of associated infection, dehydration, and therapy. Bone marrow necrosis would not be
expected to affect haematological indices unless it was so extensive.that the total erytropoietic
capacity of marrow was compromised (Pardoll et al 1982). Infection associated with some
painful crisis could be also cause a general.suppression.of marrow activity.

Serial observations on red cell morphology during the painful crisis performed using
image processing analysis (Wasterman and Bacus 1983) reported a consistent macrocytosis
which they attributed to the accelerated release of young red cells. Barreras and Diggs 1964,
found no difference in sickle cell counts between groups of patients in the steady state and in
painful crisis, yet in individual patient’s counts were reported to fall with.recovery from pains.
No consistent changes in ISC count were observed by some workers (Diggs 1956, Richardson.et
al 1979,' Wasterman and Bacus 1983) al though the consisten t fall in ISC count was reported
by Rieber et al 1977. Recently the haemoglobin level has been reported to fall early in the
course of most painful crisis (Fabry et al 1984b) and there was striking decline in the dense
cell fraction during the painful crisis ( Fabry et al 1984b , Warth and Rucknagel 1984).

61
 CHAPTER IV

Rheology
Erythrocyte deformability, measured by filtration of washed erythrocytes through 5
micron pore polycarbonate filters, was decreased during the painful crisis ( Kenny et al 1981)
and improved with recovery from the painful crisis ( Rieber et al 1977).

Aerial observations in one patient indicated that decreased deformability preceded the
onset of pain by 24 hours ( Kenny et al 1981) raising the possibility that change in deformability
may be a etiologically related to the painful crisis.

Plasma volume
The plasma volume is increased in sickle cell disease in the steady state but has been
reported to fall during the painful crisis (Jenkins et al 1956, Erlandson et al, 1960, Barreras et
al 1966). Negative fluid balance !(Hatch and Diggs 1965) and weight loss ( Wilson and Alleyne
1978) have been observed early in the painful crisis and contraction of the plasma volume
below steady state values reported.

These results were at variance with observations of the painful crisis in Jamaica where
Wilson and Alleyne (1978) were unable to detect any significant change in plasma volume.
they found increased total body water, extracellular fluid volume and interstitial fluid volume
and also decreased intracellular fluid volume during the painful crisis , but no significant change
in plasma volume.

These conflicting observations on plasma volume in painful crisis may also reflect
differences in the duration of severity of the painful crisis at the time of estimation of plasma
volume. A constant reduction in plasma volume with consequent increase in haemoglobin
would conflict with.relatively stable haemoglobin levels observed by Diggs (1956).

White cells
The leucocyte count increases further during the painful crisis , occasionally simulating
a leukaemoid reaction ( Diggs 1965, Buchanan and Glader 1978). In the absence of overt
infection, the leucocytosis is presumed to reflect an inflammatory response to necrotic bone
marrow. In the presence.of infection the leucocytosis is more marked and associated with an
increase in band neutrophils. Leucocyte alkaline phosphatase may aid detection of infection
since levels which are normally low in steady state SS disease (Wajima and Kraus 1968, 1975)
increase in infection but remain unchanged in the presence of uncomplicated painful crisis.
(Rosner and Karayalcin 1974).

Platelet count and function

62
 CHAPTER IV

The platelet count appears to fall early in the painful crisis (Gor don et al 1974,
Freedman and Karpatkin 1975a, Alkjaersig et al, 1976), rising in the second week (Van der Sar
1973). Studies of platelet aggregation during the painful crisis have yielded conflicting results (
Haul et al 1973), Stuart et al 1974, Gruppo et al 1977) perhaps because of the large
functionally active megathrombocytes which.decrease early in the painful crisis and increase
above steady state levels during recovery ( Freedman and Karp takin 1975a).Gupta VL et 1981)
reported unchanged platelet functions i.e. Counts and aggregation during the period of crisis.

In a study of 50 cases of sickle cell crisis, which included , 40 vaso-oclussive crisis, 5

cases of haemolytic crisis, 3 cases of mixed crisis and 5 cases of aplastic crisis, platelet
count and platelet adhesiveness was studied. Platelet count was done by the method of
Kristension, later modified by Lampert, and platelet adhesiveness was studied by slight
modification of Wright’s method. Platelet count was normal in all types of crisis , except in
aplastic crisis, where it was low. Platelet adhesiveness remained unaltered during crisis.
(Gupta VL et al, 1981)

Fibrinogen and fibrinolysis

Fibrinogen levels have been reported to be unchanged (Fenichel et al 1950, Mahmood
1969a, Green et al, 1970) or elevated during the painful crisis compared to steady state ( Diggs
1965, Gordon et al 1974, Gupta et al. 1984). However serial observations reveal consistent
increases in fibrinogen levels during painful crisis (Alkjaersig et. al. 1976, Richardson et. al.
1979, Gupta et, al. 1984) reaching a peak approximately one week after onset, and increase
occasionally preceding.the onset of pain. These serial changes in fibrinogen level may be
attributable.to infection and an acute- phase reaction ( Richardson et,al. 1979, Gupta et, al.
1984), although.in some cases the fibrinogen increase appears to be independent of
demonstrable infection (Gordon et al. 1974).

Fibrinogen level was estimated and studied in 50 cases of sickle cell crisis. The
sample included proportionate number of cases of vaso-oclussive crisis, haemolytic crisis,
mixed type of crisis and aplastic crisis. The controls were studied to determine the normal
rane and mean value for comparison.

The method used for estimating the fibrinogen level was, “Single Radial
Immunodiffusion technique”. The antisera was raised in the laboratory and single radial
immunodiffusion was done for known quantity of fibrinogen levels to standardize the “ ring
diameter” which than as reference range will give fibrinogen levels in an unknown
sample. The procedure was standardize so as to give the accurate values.

63
 CHAPTER IV

Table-4.9: Fibrinogen levels in sickle cell crisis, n=50

Crisis type Fibrinogen mgm%

Range Mean
VC 450 – 800 526.26
HC 250 – 400 350.00
MC 200 – 300 250.00
AC 80 - 200 143.37
CONTROLS 221 – 330 275.00
Abbrev. VC= Vaso-oclussive crisis, HC= Haemolytic crisis, MC = Mixed crisis, AC = Aplastic crisis

Hyperfibrinogenimia Normal levels

Diameter of Inner ring gives levels of fibrinogen
Fig.4.5 Fibrinogen estimation was done by Standard Radial
Immuno-diffusion technique of Mancini et al

Fibrinogen levels were significantly raised in cases of vaso-oclussive crisis, were also
elevated in haemolytic crisis, marginally reduced in mixed type of crisis, and markedly
depleted in aplastic crisis ( Table-4.9).

In further analysis, 40 cases of vaso-oclussive crisis were grouped according to

presence or absecence of infection. 24 cases had established evidence of infection while 16
cases had no evidence of infection. Fibrinogen level was estimated in both groups. Fibrinogen
levels were raised in both groups. Vaso-oclussive crisis with infection group had more
remarkably elevated levels of fibrinogen as compared to vaso-oclussive crisis without infection.
(600 mgm% Vs 480 mgm%) (Table. 4.10).

64
 CHAPTER IV

Table-4.10: Fibrinogen levels in Vaso-oclussive crisis with and without infection

N = 40
VC with infection VC without infection

N = 24 N = 16

Range Mean Range Mean

550 – 800 600 mgm% 450 – 575 480 mgm%

Fibrinogen levels are still significantly higher in VC with infection

To further explore marked hyperfibrinogenaemia in patients of vaso-oclussive crisis with

infection, we tried to study, whether there was inhibition of fibrinolysis in these patients.
This has been proposed by various workers, that in patients with serious infections, there is
release of substances, which are the potent inhibitors of fibrinolysis which results in high or
very high levels of fibrinogen, adding to vaso-oclussive phenomenon, and to morbidity
and possibly mortality in these patients.

Test for fibrinolysis, (9a)

1. Euglobin Clot Lysis Time
2. Fibrin Plate Lysis
3. Demonstration of “MISFI”
In our study, we used third method to demonstrate decreased fibrinolysis. “MISFI”
stands for, “Molecules Immunologically Similar to Fibrinogen”. “MISFI”, molecules
immunologically similar to firinogen, gives direct evidence of fibrinogen disintegration
products. “MISFI” were demonstrated by Countercurrent Immunoelectrophoresis. (Saoji
AM, Gupta VL et al. 1982)

SPUR

Fig.4.6, Countercurrent Immuno- Electrophoresis showing IEP, spur

formation, indicating presence of “MISFI”

65
 CHAPTER IV

Out of 24 patients of vaso-oclussive crisis, MISFI were demonstrated in only 2

patients on countercurrent immune-electrophoresis, suggesting that there is marked
inhibition of fibrinolysis in patients of vaso-occlusive crisis with infections (Fig. 4.6) (Saoji
AM, Gupta VL et al. 1982). Fibrinolysis assessed by two assay systems is decreased in the
painful crisis ( Mehmood et, al. 1967, Green e t, al. 1970, Gordon.et, al. 1974, Stathakis et, al.
1975, Gupta et, al. 1984) returning to normal following the.crisis. (Mehmood 1969a). Inhibitors
of fibrinolysis have been demonstrated ( Kwaan and Green 1973, Gupta et, al . 1984). The
evidence appears to conflict with.reports of.increased fibrin degradation products during the
painful crisis ( Alkaersig et, al. 1976, Leishtman and Brewer 1978, Richardson et, al. 1979) but
may be compatible with limited thromboplastic activity and low grade consumption of
fibrinogen and platelets. Disseminated intravascular coagulation indicated by excessive platelet
consumption. and throbocytopenia, increased prothrombin, and partial thromboplastin.times,
together with.evidence of fibrin degradation products is uncommon.but may.occur in fat
embolism , overwhelming septicaemiss (McDonod and Eichner 1978), and occasionally in
uncomplicated sickle cell crisis (Corvelli et, al. 1979).
Other clotting factor do not appear to be changed in the painful crisis (Abildgaard e t, al. 1967,
Green et, al. 1970), although altered Factor VIII complexes have been described in some
patients (Mackie et, al. 1980).

Aplastic crisis
Aplastic crises are acute worsenings of the patient's baseline anaemia, producing pale
appearance, fast heart rate, and fatigue. This crisis is normally triggered by parvovirus B19,
which directly affects production of red blood cells by invading the red cell precursors and
multiplying in and destroying them. Parvovirus infection almost completely prevents red blood
cell production for two to three days. In normal individuals, this is of little consequence, but the
shortened red cell life of SCD patients results in an abrupt, life-threatening
situation. Reticulocyte counts drop dramatically during the disease (causing reticulocytopenia),
and the rapid turnover of red cells leads to the drop in haemoglobin. This crisis takes 4 days to
one week to disappear. Most patients can be managed supportively; some need blood
transfusion.

Sequestration crisis
Sequestration crises results from trapping of large amounts of red cells in the spleen and
sometimes in the liver. Acute splenic sequestration is one of the important causes of early
mortality in children with SCD . In homozygos (HbSS) patients the life time prevalence of acute
splenic sequestration is reported to be between 7%-30%.

66
 CHAPTER IV

• it may be seen as early as 8 weeks of age although usually an initial event is seen in
toddlers
• in HbSC and HbS/thalassaemia patients the initial event tends to occur later in life (even
into adulthood)

During a crisis the sequestered sickle cells cause the spleen to become grossly enlarged. This
is more common in infancy as repeated sequestration and infarction of the spleen during
childhood gradually results in an auto- splenectomy. There is marked pallor with cardiovascular
collapse due to loss of effective circulating volume . A patient with a sequestration crisis is
usually severely anaemic (2-3 g/100 ml), but the reticulocyte count will be high, in contrast to
the aplastic crisis where the reticulocyte count is low or zero. Sequestration crises also occur in
pregnant women.

Immediate management of sequestration crises is aimed at correcting hypovolaemia with

emergency blood transfusion. The subsequent management depends on the recurrence rate of
splenic sequestration and may include observation, chronic transfusion and splenectomy (3).

The important features are:

• sequestration crises recur
• splenectomy is curative - and is recommended after two episodes
• educating the parents about the proper management is vital, particularly in the
developing world

Similar sequestration episodes may occur in the liver in adults.

Megaloblastic Crisis
Sickle cell disease usually presents with vaso-occlusive, aplastic, hyperhemolytic, splenic
sequestration, and acute chest syndrome as predominant crisis. Though megalo-blastic crisis is a
known phenomenon in thalassemia and hereditary spherocytosis. SCD with megaloblastic crisis is
rare. The possible explanation may be the nutritional deficiency of folate and vitamin B12.

67
 CHAPTER IV

Sickled Erythrocytes Sluggish flow in capillaries, small vessels

Mechanical Entangement
Endothelial reaction/ damage
Reduced Zeta Potential

Precipitating Factors Vaso-oclussive phenomenon

Infection

Decreased Fibrinolysis

Increased Fibrinogen levels Wide spread, increased vaso-oclussion, Crisis

Fig.4.7 Vaso-oclussive phenomenon and crisis

As shown in fig.4.7, vaso-oclussive phenomenon is initiated by slow, sluggish flow

because of sickled erythrocytes, which because of their peculiar shape gets mechanically
entangled, inviting endothelial reaction and later damage, which adds to vas-oclussion. Due
to reduced electrokinetic potential (Zeta potential), sickled erythrocytes tend to conglomerate
which also adds to severity of obstruction. If the situation is complicated by presence of
infection, it often lead to inhibition of fibrinolysis and increased levels of fibrinogen which
ultimately makes it worse.

This vaso-oclussive phenomenon may be local or fairly wide spread, accordingly it will
manifest in the form of ischaemia or infarction in the area involved. There are some
preferential sites, where it occurs commonly, but theoretically can occur anywhere in the
body. (Fig. 4.8)

As shown in fig.4.8, vaso-oclussive manifestations include, cerebral infarcts- stroke,

pulmonary infarcts, splenic infarcts- fibrosis-atrophy, renal infarcts, infarcts in extremities,
aseptic bone necrosis, peripheral vaso-oclussion. Other common findings are retinopathy,
cardiomegaly, bone marrow hyperplasia. Cholelithiasis. Predisposion to infection leads to
pneumonia, osteomyelitis.

68
 CHAPTER IV

Fig. 4.8 Manifestations of sickle cell disease

Precipitating Factors of Sickle Cell Crisis

Greenburgh MS, and Kass SH, postulated that metabolic acidosis is initiative factor.
Smith.EW, and Conley 1959 described common denominators as (i) sickled cells (ii) increased
viscosity (iii) circulatory stasis (iv) hypoxia (v) vasospasm (vi) dehydration (vii)
haemoconcentration and (viii) acidosis.

Diggs LW, and later Felix ID et al put schematic presentation of crisis with hypoxia
and reduced PH being initiative factors and vicious circle starts from lowered oxygen tension
which lead to 'S' crystallization increased blood viscosity stasis and further hypoxia.

Many precipitating factors have been identified of sickle cell crisis e.g. Infection,
dehydration, stress, (Diggs LW, Smith EW, and Conley, Felix ID, et al, Gupta VL. ) Infection for
say is more important as sicklers are prone to infection by salmonella group of organisms and
gram negative organisms, this is thought to be because of factor 'B' which in turn results in
defective C3, essential opsonin against gram negative organisms and pneumococci (WA
Wilson et al).
69
 CHAPTER IV

Infection
The importance of malaria was suggested by the observation that painful crisis
occurred more frequently during periods of high malarial transmission in Ghana (Edington
1953) and by the report that malaria accounted for 16 percent of painful crisis requiring
hospital admission ( Konotey Ahulu 1971c). The frequency of painful crisis has been reduced by
prophylactic therapy employing antimalarial drugs alone (Nwokolo 1960, Hendrickse 1965) or in
combination with.antibiotics (Lewthwaite 1962, Warley et al .1965) or with symphonies
(Hendrickse and Barnes 1966).
The role of other infections in the genesis of the painful crisis is less well documented
although repeatedly observed ( Margolies 1951, Peterson and Sprague 1959, Wright and
Gardner 1960). Infections may precede painful crisis more frequently in the African
environment (Konotey Ahulu 1971b) than in North America (Diggs 1965). The mechanism
whereby infection precipitates the painful crisis is unclear a although it is likely to be
multifactorial with contributions from fever, dehydration and acidosis. There is some
evidence.that.pyrexia alone may precipitate painful crisis. (Paterson and Sprague 1959, Lecocq
and Harper 1963).

Sickle cell patients are predisposed to infections,specially by gram negative organisms,

and organisms opsonised by C-3 compliment. this is thought to be because of factor 'B' which
in turn results in defective C3, essential opsonin against gram negative organisms and
pneumococci (WA Wilson et al). C-3, is essential opsonin, needs two factors, namely factor A
and B, each one has proactivator, proactivator of factor B is defective, in turn factor B which
leads to defective C-3, which renders these patients vulnerable against infections. Spleen
which tends to get multiple infarcts, and following fibrosis, gradually leads to loss of functions,
which again makes this patients more likely to have infective episodes, Associated co-morbid
conditions and compromised cell mediated immunity adds to the fire and virtually makes
them immune-compromised host. (Fig.4.9). During 18 months prospective follow up of 65 pts.
204 infective episodes were recorded. (Gupta VL, and Chaubey BS, 1995). Gupta VL, Dube GK,
et al. 1982, reported two cases of salmonella oesteomylitis affecting spine, where
aspirations were positive for salmonella cultures Infection by itself causing morbidity, also
triggers crisis, this combination then increases morbidity as well as mortality in patients with
sickle cell disease.

70
 CHAPTER IV

DEFECTIVE FACTOR B SPLEENIC HYPERTROPHY ASSO.CO-MORBID COMPROMOISED

DEFECTIVE C-3, SPLEENIC INFARCTS = FIBROSIS CONDITIONS CELL MEDIATED

AUTO SPLENECTOMY IMMUNITY

LOSS OF SPLEENIC FUNCTIONS

PREDISPOSITION FOR INFECTIONS

Gram negative organisms, Pneumococci, H.influenze, Strept, Parvo virus

Fig.4.9 Predisposition for infections

Cold
Diggs (1965) reported the impression that painful crisis were more common during cold
and dump periods in Memphis but was unable to document this in a 2 year prospective study of
37 children monitored at home ( Diggs and Flowers 1971). In Chicago, Sealer (1973a) was
unable to show any seasonal pattern in hospital admission s of children with sickle cell crisis. In
West Africa peak frequencies of painful crisis occurred just before the two rainy seasons (
Lehman 1968, Addae 1971a) and a 5 year retrospective review in Buffalo ( A mjad et al 1971a)
indicated admissions were more frequent in winter. In Jamaica hospital admission s for painful
crisis correlated closely with mean monthly minimum temperatures ( Fig) ( Redwood et al
1976).

It might be argued that the effect of cold was mediated through cold related viruses or
infection rather than direct temperature effect but the evidence is against this. Ghanaian adults
developed symptoms of the painful crisis on acute exposure to.20 C. ( Addae 1971b), and a
Ugandan infant with SS disease developed pain and swelling simulating dactylitis after
immersion of the hand in ice cold water. (Marsden and Shah 1964). Jamaican.patients report
the temporal relationship of the bone pain and rain and many recognize that bathing in cold
water or the sea may precipitate painful crisis.

The mechanism of such an effect is obscure but must in some way relate skin cooling to
a slowing of bone marrow blood flow sufficient to cause the marrow necrosis which is the
pathological basis of the painful crisis in most patients. In normal people skin cooling causes
cutaneous vaso- constriction with haunting of blood to deeper venous system (Glase r et al
71
 CHAPTER IV

1950) but in AS disease there is some evidence that reflex is diminished, since the skin cooling
produced only a negligible fall in skin blood flow compared with controls ( Ward 1975). During
the painful crisis the oxygen tensions may rise in the superficial veins of the forearm (
Manfredi et al 1960) Renal factors that may contribute include an increased distress
on.exposure to cold temperatures. The role of cold aglutinins and cryoglobulins ( Young 1946,
Diggs 1965, Sathiapalan 1965) in.temperature related painful crisis is difficult to assess at
present.

Acidosis
As acid PH increases sickling in vitro and acidosis in vivo may be associated with painful
crisis. Metabolic acidosis has precipitate d painful crisis when induced by the administration of
ammonium chloride (Barreras and Diggs 1964), acetazolamide (Dos Santos and Lehmann
1959), or both (Greenberg and kass 1958). The frequency of metabolic acidosis during the
painful crisis is controversial since it was constantly observed in a report from Memphis
(Berreras and Diggs 1964) but was not seen in studies in Jamaica (Ho Ping Kong and Alleyne
1969) or Ghana (Ringelhann and Konotey - Ahulu 1971).

Pregnancy
Painful crisis commonly occur during the last trimester and post-partum periods of
pregnancy. In some patients pregnancy precipitates the painful crisis for the first time (Smith
and Conley 1954, Anderson et al 1960) and pregnancy becomes an increasingly common cause
of the painful crisis over the age of 25 years when painful crisis precipitated by other causes
become less common.

Other factors
Less well documented precipitating factors of the painful crisis include emotional stress
hypoxia, dehydration, and increased viscosity. Emotional stress is difficult to quantitate
objectively but pains in association with school.or college examination and adverse home or
work situations are not uncommon.( Nadel and Portadin.1977). Painful crisis may occur in
patients traveling at high attitudes. One report documenting painful crisis in 8/39 (21%)
patients with.SS disease travelling above 2000 m, ( Mahoney and Githens 1979) and another
estimating that approximately two-thirds of SS patients normally resident at.sea level ,
developed painful crisis on travelling to 2000m (Claster et al 1981). The role of.dehydration
has been postulated in painful crisis induced by alcoholic intoxication and after.excessive
sweating or exercise (Diggs 1965). or following diuresis whether spontaneous or medically
induced in a patient with SS disease and the nephrotic syndrome (Sweeney et al 1962).
Frequent.severe painful crisis attended the marked increase in haematocrit.following successful
renal transplant in a patient with SS disease (Spector et al 1978).
72
 CHAPTER IV

In a study of 75 cases of sickle cell crisis, Gupta VL et al. 1981, could not detect any
precipitating factor in maximum number of cases of sickle cell crisis, (38, 50.66%),most
common precipitating factor was infection, (24,32%), next common was stress, (5,6.66%),
followed by exposure to cold, (4, 5.33%) hypoxia (3, 4%) and dehydration in one case (1.33%)
Fig.4.10.

PRECIPITATING FACTORS OF SICKLE CELL CRISIS, N=75

40
35
30
25
20
15
10
5
0
N

IA

.
.

SS

R
T
W

L
C

D
X
E
O

O
FE

Y
PO
R
N

H
ST
IN
K

E
D
H
T
O
N

Fig. 4.10 Precipitating factors of sickle cell crisis (N=75)

Recognising these precipitating factors, and avoiding them, e.g. stress, exposure to cold,
hypoxia, dehydration, or preventing or treating them promptly, like infection prophylaxis and
prompt treatment may prevent crisis and thereby morbidity and mortality.

73
 CHAPTER V

CHAPTER V

SYSTEMIC MANIFESTATIONS
OF
SICKLE CELL DISEASE

74
 CHAPTER V

The Liver
The Liver is frequently affected in SS disease. Red cells traversing the low oxygen
tension areas of the sinusoids are prone to sickli ng and the sinusoidal flow may be blocked by
clumps of sickled cells or by Kupffer cells grossly swollen by erythrophagocytosis. High levels of
bilirubin excretion lead to an increased prevalence of cholelithia sis and of exyrahepatic
obstruction . Blood transfusion therapy has in the past been associated wit h viral hepatitis and
it's sequelae and with haemosiderosis (Serjeant GR, 1985). Hepatomegaly occurred in 40-80%
of patients in early clinical reports. (McGavack and Nussbaum, 1942, Grover 1947). and in 80-
100% cases at autopsy (Green et. al.1953, Song 1957).

The anatomical basis of the hepatomegaly.appears to be related, at least in part to

distention of the sinusoids by sickled cells (Fig. 5.1) and possibly an element of sinusoidal
obstruction by Kupffer cells distended by phagocytosed erythrocytes.

Other common histological findings have included increased haemosiderin deposition

(Diggs and Ching 1934, Green et. al. 1953). extramedullary erythropoiesis, and a delicate fibrin
like network within the sinusoids (Green et. al. 1953).

Fig.5.1 Hepatic histology in sickle cell disease

75
 CHAPTER V

Chaubey BS et. al. 1974 proposed an arbitrary histological grading of liver in sickle cell
disease.
Grade I. Dilatation.of sinusoids with.or without sickled erythrocytes, Kuffer's cell
hyperplasia with.or without phagocytosis, parachymal.damage.
Grade II: Sinusoidal changes,, paranormal change (a,b). Minimal necrosis (c)
Grade III: Paranxhymal changes (b,c) Early regeneration of paranchymal.cells, minimal
collagen- perportal around necrosed cells, sinusoidal area and around central vein.
Grade: IV: Paranchymal damage (c), reticulin collapse, pseudolobule formation.fibrosis-
around groups of cells (monolobular) periportal (multilobular). Paranchymal damage.
(a) Moderate variations in the size of cells, and nuclei, cytoplasm uniform with minimal degree
of clumping
(b) Variation in nuclear cellular size, Nuclear change consist of pyknosis, karolysis, and
ballooning.
(c) Necrosis of liver cells and nuclear cell fragments.These
Vascular occlusion
The hepatic complications attributed to vascular occlusion encompasses a variety of
clinical syndromes for which our understandings of the relationships between clinical
presentation, biochemical findings and histologic features remain unclear. The liver is generally
enlarged throughout life, especially when adjusted for body size. The hepatic blood volume and
blood flow are increased and, presumably, contribute to hepatomegaly. Liver histology
invariably shows Kupffer cell hyperplasia with erythrophagocytosis, sinusoidal distension with
aggregates of sickled erythrocytes and fine fibrosis in the space of Disse (Johnson et al, 1985
and Omata et al, 1986). The histologic features correlate poorly with the clinical status and
amino-transferase level. The Kupffer cell erythrophagocytosis and sinusoidal aggregates likely
reflect the site of hemolysis as evidenced by relatively low ISC counts in the hepatic vein and by
the hepatic accumulation of red cell label in studies of RBC survival. However, massive
distension of sinusoids has been described in syndromes of hepatic failure, implying an etiologic
role for the severe degree of such findings.

Hepatic sequestration
Acute hepatic sequestration is a rarely recognized complication of VOC; in one elegant
study, there was one case in 161 hospital admissions (Davies and Brozovic, 1987). This
syndrome is characterized by a rapidly enlarging liver accompanied by a decrease in
hemoglobin/hematocrit and a rise in reticulocyte count. The liver is smooth and variably
tender. The bilirubin may be as high as 24mg/dl with a predominance of the conjugated
fraction. The alkaline phosphatase can be as high as 650 IU/L but can be normal. The
transaminases are only minimally elevated and often are normal. Recurrence is common.

76
 CHAPTER V

Ultrasonography and CT scanning show only diffuse hepatomegaly. Liver biopsy shows
massively dilated sinusoids with sickle erythrocytes and Kupffer cell erythrophagocytosis.
Intrahepatic cholestasis with bile plugs in canaliculi can occur. Hepatocyte necrosis is unusual.
The pathophysiology is believed to be obstruction of sinusoidal flow by the masses of sickled
erythrocytes, trapping of red cells with within the liver and compressing the biliary tree. On the
other hand, the pathophysiology could be similar to that suggested by ultrasonography in
splenic sequestration, postulating obstruction at the smaller hepatic veins and/or sinusoids
(Roshkow and Sanders, 1990). Undoubtedly mild episodes are not recognized.

Tests of hepatic function in apparently steady state patients have reported low serum.albumin
and raised serum globulin levels, increased alkaline phosphatase and BSP retention, and
elevation of SGOT, SGPT, and LDH (Green et. al. 1953). There is some evidence that tests of
hepatic function become increasingly abnormal with advancing age, ( Ferguson and Scott
1959,Diggs 1965).

During the painful crisis, liver may enlarge , some patients showing increments as great
as 5 cms, the basis may be sinusoidal dilatation precipitated by a change in the characteristics
of circulating red cells.

Extreme hyperbilirubinaemia
A variety of conditions in sickle cell disease result in acute elevation of conjugated
bilirubin together with hepatic tenderness and enlargement. These include, vital hepatitis, a
spectrum of conditions variously termed intrahepatic cholestasis (Kilon et.al.1964), hepatic
crisis (Diggs 1965). cholangiolar jaundice (Wade, 1960), sickle cell hepatopathy ( Sheehy 1977),
and extahepatic obstruction.

Haemosiderosis is a direct consequence of repeated blood transfusions in SS disease is

the accumulation of iron within.the reticuloendothelial.system and stainable iron is frequently
reported within Kupffer cell and the hepatocytes ( Geen et.al. 1953, Bogoch et.al 1955 Song
1957).

Cirrhosis
A macronodular cirrhosis compatible with a post hepatitic aetiology has been
reported. (Green et.al. 1953, Bogoch et.al. 1955, Song 1955)., and was estimated to affect 20-
40% of the adults with sickle cell disease (Aach and Kissane 1970).

77
 CHAPTER V

Cholelithiasis
The continuously high levels of bilirubin excretion in SS disease result in the frequent
formation of gallstones. A striking feature of the disease is the young age at which cholelithiasis
may occur. ( Mallory 1941, Weens 1945, karayalcin G et. al. 1979).
The prevalence of cholelithiasis in SS disease appears to be lower in West African and
Saudi Arabian patients compared to North Americans.

It is also rare among normal Nigerians. (Da Rocha-Afodu and Adesola 1971). The low
prevalance of gallstones in Saudi Arabian patients with SS disease ( Perrine 1973) probably
results from the mild hemolyt ic rate and lower bilirubin load characteristics of that population.

Gallstones in haemolytic disease are typically of the pigment type and result from
elevated bilirubin excretion. McCall et. al.(1977) noted higher reticulocyte counts and lower
fetal haemoglobin level s in patients with cholelithiasis compatible with accelerated haemolysis.

Extrahepatic obstruction: The prevalence of choledocholithiasis observed in normal

populations with chlolelithiasis has been estimated at approximately 10% and some reports
quote figures between 20-60% in SS disease. (Appleman et.al.1964,Cameron et. al. 1971).

Although there is a recognized association between gall bladder disease and intestinal
salmonella carriage in otherwise normal people virtually no information is available in sickle cell
disease, which is surprising. More research is needed here.

Cholecystectomy
Laparoscopic cholecystectomy on an elective basis in a well-prepared patient has
become the standard approach to symptomatic patients. However, symptoms attributed to
cholecystitis often persist after cholecystectomy. Because intra-operative cholangiography has
a false positive rate estimated at 25%, ERCP at the time of laparoscopic cholecystectomy is
preferred. However, IOC is still useful in delineating the anatomy of the cystic duct and its
artery. The decision to proceed to cholecystectomy should be based on the natural history of
the disorder in this patient population. The Jamaican data (Walker et al, 2000) provide the
strongest argument for a conservative approach, but Jamaican patients seem to be
substantially less symptomatic than those in North America. An aggressive approach provides
the benefit of reducing the risk of the morbid complications of cholelithiasis, as well as
eliminating gallbladder disease as a confounding item in the differential diagnosis of RUQ pain.
For asymptomatic patients, the data support a conservative approach, but considerable
controversy exists.

78
 CHAPTER V

The Spleen

The Spleen played a prominent role in early observations of sickle cell anemia. The first
autopsy reported a striking spleenic atrophy (Sydenstricker et. al. 1923) which led tothe belief
that spleenic involvement might account for the repeated attacks of abdominal pain, and the
proposal that the disease was familial and hereditary defect of the spleen. Rich.(1928)
interpreted spleenic pathology as a malformation of the.spleenic sinuses allowing free escape
of blood into the pulp, but this concept was contested by Tomlison (1945b) who considered the
spleenic pooling to be the result of circulating sickled cells.

Atrophy was characteristic of many early autopsy reports (Yater and Mollary 1931,
Grahm 1924, Jaffe 1927, Bennett 1929). Clinical splenomegaly was common in young patients
(Jamison.1924, Archibald 1926) . intermittent in others (Dreyfoos 1926) and in some the spleen
appeared to enlarge during painful crisis (Stewart 1927, Josephs 1928). Occasionally marked
enlargement extended up to the iliac crest (Hahn and Gillespie 1927). Fig.5.2.

Order emerged from this confusion with.the recognition that the spleen was frequently
enlarged in young children and became smaller with age (Wollstein and.Kreidel 1928). Hahn
and Gillespie (1927) anticipated later thinking when they commented that the spleen'
nevertheless, greatly injured by it's long continued overuse and completes it's life history as an
atrophic remnant of a once enlarged organ.

Clinical syndromes

Acute splenic sequestration

Acute splenic sequestration (ASS) is the first major clinical manifestation of the disease
and important cause of mortality. The Spleen undergoes acute enlargement trapping significant
proportion of the red cell mass, causing a precipitate fall in haemoglobin level and the risk of
death from peripheral circulatory failure. There is usually a hypercellular marrow and marked
increase of reticulocytes in the peripheral blood.

Hypersplenism

Although progressive splenic atrophy.is characteristic of older children and adults with
SS disease, some children manifest gross splenic enlargement and hypersplenism. Excessive red
cell destruction associated with splenomegaly and alleviated by splenectomy, was noted by
several early workers ( Hahn.and Gillespie 1972, Hahn 1928, London.and Lyman 1929) and
splenic sequestration of labelled red cells was confirmed by Jandl et. al. (1956).

79
 CHAPTER V

Fig. 5.2 Hypersplenism in Jamaican child aged 5 years

The Spleen measured 16 cms, weighed 460 gms at
Splenectomy. (Photo courtesy Serjeant GR)

Splenectomy
Possible indications for splenectomy in SS disease are in the prophylaxis of attacks of
acute splenic sequestration and the treatment of hypersplenism. As with any therapeutic
decision the risks of the therapy must be compared.with those of other potential therapies and
weighed against dangers of the complications.

The dangers of splenectomy are those of anaesthesia surgery, and the loss of a potential
immune contribution from the spleen (Sergeant GR, 1985).

The Kidneys

The kidney in SS disease is affected by both the haemodynamic changes of a chronic

anemia and by the consequences of vaso-oclusion which are especially marked within the renal
medulla. As a result there are many abnormalities in renal structure and function. (Serjeant GR,
1985).

Sickle cell disease (SCD) substantially alters renal structure and function, and causes various
renal syndromes and diseases. Such diverse renal outcomes reflect the uniquely complex
vascular pathobiology of SCD and the propensity of red blood cells to sickle in the renal medulla
80
 CHAPTER V

because of its hypoxic, acidotic, and hyperosmolar conditions. Renal complications and
involvement in sickle cell nephropathy (SCN) include altered haemodynamics, hypertrophy,
assorted glomerulopathies, chronic kidney disease, acute kidney injury, impaired urinary
concentrating ability, distal nephron dysfunction, haematuria, and increased risks of urinary
tract infections and renal medullary carcinoma. SCN largely reflects an underlying vasculopathy
characterized by cortical hyperperfusion, medullary hypoperfusion, and an increased, stress-
induced vasoconstrictive response. Renal involvement is usually more severe in homozygous
disease (sickle cell anaemia, HbSS) than in compound heterozygous types of SCD (for example
HbSC and HbSβ+-thalassaemia), and is typically mild, albeit prevalent, in the heterozygous state
(sickle cell trait, HbAS). Renal involvement contributes substantially to the diminished life
expectancy of patients with SCD, accounting for 16–18% of mortality. As improved clinical care
promotes survival into adulthood, SCN imposes a growing burden on both individual health and
health system costs. This Review addresses the renal manifestations of SCD and focuses on
their underlying mechanisms.

Fig. 5.3 Hypercellular enlarged glomeruli,in SS disease, a necropsy showing two large,
Congested, hypercellular glomeruli, and one sclerosed glomerulus. (lower left)
(Photo courtesy Dr. D. Shah)

Renal involvement can occur throughout the life of a patient with SCD. Manifestations
such as hyperfiltration, hypertrophy, and impaired urinary concentrating ability are described
as early as in infancy. Microalbuminuria is observed in some patients in childhood, whereas
haematuria and acute kidney injury (AKI) can occur at any age. Macroalbuminuria tends to

81
 CHAPTER V

occur in early to middle adulthood, and can be accompanied by regression of the glomerular
filtration rate (GFR) to the normal range. In the later decades, the risk of chronic kidney disease
(CKD), progressive reduction of GFR, and end-stage renal disease (ESRD) increases.

This steady, age-dependent accrual of adverse renal sequelae shortens the average
lifespan of patients with SCD. Proteinuria and a reduced GFR are risk factors associated with
increased mortality among patients with SCD;16–18 approximately 16–18% of overall mortality in
this patient group is ascribed to kidney disease.19,20 Once ESRD is reached, the mortality of
patients who are on haemodialysis and have SCD is increased severalfold relative to the
mortality of patients who are on haemodialysis but do not have SCD.21Thus, although the
average lifespan of patients with SCD has increased during recent decades owing to improved
management of complications outside the kidney, the age-dependent accrual of kidney disease
contributes substantially to the still increased mortality in SCD.

Fig.5.4 Reduplication of the basement membrane (indicated by arrow) in the Glomerular tuft of a 46
years female with SS disease. Photo courtesy Dr.D.Shah

• Sickle cell nephropathy (SCN) largely reflects an underlying renal vasculopathy characterized
by the coexistence of cortical hyperperfusion, medullary hypoperfusion, and an increased
renal vasoconstrictive response to systemic and regional stress

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• Renal involvement is usually more severe in sickle cell anaemia (HbSS) than in other types
of sickle cell disease (HbSC and HbSβ+-thalassaemia), and is typically mildest in sickle cell
trait
• Proteinuria and decreased glomerular filtration rate are independent risk factors for
increased mortality in sickle cell disease; 16–18% of overall mortality is estimated to arise
from kidney disease
• The most frequent glomerulopathy in SCD is focal segmental glomerulosclerosis, a lesion
considered to be mediated by alterations in glomerular haemodynamics
• Haematuria and loss of concentrating ability are among the most frequent renal syndromes
caused by sickle cell disease, both of which are also common in sickle cell trait
• Angiotensin-converting-enzyme inhibitor therapy is recommended for patients with SCN
and microalbuminuria and proteinuria, even in the presence of normal blood pressure

Renal manifestations of sickle cell disease

Alterations in renal haemodynamics

• Increased renal blood flow rate

• Increased renal plasma flow rate
• Increased glomerular filtration rate
• Decreased renal vascular resistance
• Decreased filtration fraction
• Decreased medullary perfusion

Renal and glomerular enlargement

Hyperfunction of the proximal tubule

• Increased reabsorption of phosphate and β2-microglobulin

• Increased secretion of creatinine and uric acid
• Increased transport maximum of para-aminohippurate

Glomerulopathies

• Focal segmental glomerulosclerosis

• Membranoproliferative glomerulonephritis
• Thrombotic microangiopathy

Tubular deposits of iron

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 CHAPTER V

Chronic tubulointerstitial disease

Impaired function of the distal nephron

• Decreased urinary concentrating ability

• Partial distal renal tubular acidosis
• Impaired renal potassium excretion
• Increased susceptibility to acute kidney injury
• Chronic kidney disease and its progression to end-stage renal disease
• Haematuria
• Renal papillary necrosis
• Increased susceptibility to urinary tract infections
• Renal medullary carcinoma

The aetiology of renal failure in SS disease is multifactorial with contributions from

progressive glomerular sclerosis tubular changes, cortical and medullary infarction and
infection. The glomerular changes in.SS disease resemble those observed in subtotally
nephrectomized esta (Shimamura and Morrison 1975) raising the intriguing possibility that
progressive renal failure in SS .disease is sequel of many years of nephron hyperfunction
(Morgan and Shah 1985).

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(a) (b)

Fig.5.5 Micro radiographic studies of the renal vasculature’

(a) Normal vessel system (b) Grossly abnormal vessels with
Complete obliteration of the vasa rectae system in patient
With SS disease. ( Photo courtesy Dr.Statius van Eps)

From Central India, Renal manifestations of 29 cases of sickle cell anemia and 20 cases
of sickle cell trait were studied. Gross haematuria was infrequent although microscopic
haematuria was observed. Renal papillary necrosis was present in two cases. Fresh and old
focal haemorrhages were present in.renal parachymal.tissues. Chronic pyelonephritis was
frequently noted in this series and this was confirmed by pyelographic studies. Tubular
dysfunction predominated as was observed by the renal function tests. It is postulated that
sickling of erythrocytes occurs in peritubular plexes us and vasa recti. (Fig.5.5) This leads to
thrombosis, obliteration and rupture of these vessels which is reflected in tubular dysfunction
as well as in histological lessions.(Chaubey BS, Waikar SM, et. al.1975).

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The heart

Adaptation to anaemia
In any chronic anaemia adaptations occur to maintain oxygen delivery to the tissues.
Haemo-dilution reduces blood viscosity and in combination with peripheral vaso-dilatation , the
peripheral resistance is lowered with a resulting increase in cardiac output, the increase is
achieved in acute cases by increase in pulse rate, and in chronic anaemia by increased stroke
volume. (Porter and James, 1953).

Typically, there is cardiomegaly with hypertrophy affecting both left and right ventricles.
(Fig.5.6). The heart valves and pericardium are usually normal. The coronary arteries are wide
and lacking.in atheroma (Gerry et. al. 1978). Intramural fibrosis has been observed in older
patients Bardoli (1969). Pathological confirmation of myocardial infarction is even less frequent
Diggs (1973).

Sickle cardiomyopathy
Congestive cardiac f failure is not uncommon in SS disease over the age of 50 years and
occasionally much younger patients experience severe unexplained cardiac failure (Seeler 1972,
Karayalcin et.al.1975. Recognizable causes of cardiac failure such as systemic or pulmonary
hypertension, ischemic heart disease and valvular heart disease are rare. After excluding all
these potential causes, there remain a group of patients with unexplained congestive cardiac
failure . These observations have led to the hypothesis that a sickle cardiomyopathy may result
from oclusion or slugging within the small cardiac vessels, although the evidence is
circumstantial. The conclusion may need to be revaluated in the light of the echocardiograph ic
studies indicating functional impairment in th e presen ce of ECG evidence of ischaemia. (Alpert
et.al.1981, Covitz et. al.1983)

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 CHAPTER V

Fig. 5.6 chest radiograph of patient with SS disease showing

Cardiomegaly with biventricular hypertrophy

Cardiac manifestations in sickle cell anemia are common indeed. Heart failure may
occur, secondary to multiple occlusion of small and medium sized pulmonary arteries by
masses of sickled erythrocytes. A systolic murmur of variable intensity is usually heard. ( Gupta
VL,1986). The study group consisted of 289 patients with sickle cell anaemia, age range from 6
months to 18 years. Data were collected using a questionnaire which include full history, clinical
examination findings, chest x-rays, and Electro-cardiography. Tachycardia, systolic murmurs,
and cardiomegaly were detected in 28%, 61%, and 54% of patients with SCA respectively. Left
ventricular dilatation was observed in 51% of the study group, while right ventricular dilatation
was observed in 22% of the patients. Left and right atrial dilatations were observed in 16% and
6% of the patients respectively. Contractility, ejection fraction (EF) were found almost always
normal in all study subjects. Chamber dilatations were not associated with any abnormality in
Left ventricular functions. Hemglobin (Hb) levels correlated negatively with cardiomegaly. Left
Ventricular End Diastolic Dimension (LVEDD) correlates negatively with Hb levels and positively
with the severity index. Only four patients (1%) had abnormal valves. In conclusion, cardiac
abnormalities in patients with SCA correlate with the age of the patients and the severity of the
disease.( Ghada O. M. Ali,( Yahya S. Abdal Gader et al 2012)

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 CHAPTER V

Cardiovascular system abnormalities are nearly always apparent in sickle cell anemia.
Increased cardiac output is necessary to compensate for reduced oxygen content of arterial
blood. This is the consequence of anemia and of the lungs' inability to load the deficient
remaining hemoglobin with oxygen. The circulatory system is strained by the sickled
erythrocyte's propensity to occlude small blood vessels. Pulmonary vascular bed obliteration
produces the most obvious effects. Abnormal ventricular performance is probably present but
has not yet been adequately documented. Evidence for a specific myocardial lesion attributable
to sickle cell anemia is tenuous. These mechanisms yield clinical findings associated with a high
output state. In some, the pulmonary vascular bed obliteration is sufficiently advanced to
produce clinical features of cor pulmonale. Heterozygous sickle states have few or no
cardiovascular manifestations. This is true since anemia is less severe and vaso-occlusive events
are less common in these disorders. (Joseph Lindsay Jr., et al 1974)

The Lungs

The Lungs are affected in several.ways by the pathological processes of sickle cell
disease. Mechanical limitations may be placed upon lung.volume and movement by the
differences in the chest shape and by presence of cardiomegaly.The entire content of a
markedly increased cardiac output must pass through pulmonary circulation expanding the
major vessels and feeling many of the peripheral branches diminishing lung compliance and
disturbing ventilation/ perfusion balance . The increased susceptibility to pulmonary infection
and to pulmonary thrombo-embolism may further impair ventilation/perfusion balance and gas
transfer. (Serjeant GR, 1985). Low lung volumes have been reported. (Fowler et. al. 1957,
Sproule et.al. 1958, Young et.al. 1976). Significant.reductions.in total lung capacity, forced vital
capacity, vital capacity, and in the ratio of residual volume to total lung capacity. (Miller and
Segment 1971, Miller et. al. 1978).

Clinical manifestations
Acute pulmonary episodes
Acute pulmonary episodes compatible.with.either acute pulmonary emboli or lobar
pneumonia are common in SS disease and difficulty in clinical differentiation has given rise to
the use of the term 'acute chest syndrome' to cover this spextrum . Such episodes have been
reported in 24% of 353 patients in Jamaican series (Serjeant 1974a).

Different conclusions were reached in a study of 52 acute pulmonary.episodes in 28

adults by Charche et. a!. (1979) who considered that many episodes resulted from pulmonary
infarction. Similar observations were made by Davies et. al. 1984 in review of 25 consecutive
episodes of acute chest syndrome. Further support for the diagnosis of pulmonary infarction
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 CHAPTER V

came from a study comparing features of acute chest syndrome in 28 adults with SS disease
with those of acute lobar pneumonia. ( Petch and Serjeant 1970). Observations seem to suggest
that the acute chest syndrome is more frequently attributable to pneumonia in young children
and to pulmonary embolism or thrombosis in adults.

Pulmonary thrombo-embolism

Pulmonary thrombo-embolism in.Sickle cell disease was reported.by Steinberg (1930)

and the first review of the pathology.in the condition noted an increased frequency of thrombi
and infarcts in the lungs (Diggs and Ching 1934). Recent thrombi.occlusion of arterioles by
organized thrombi, and recanalization of such.vessels have been reported. (Yater and Hansman
1936, Mose r et. al. 1960, Aach and Kissane 1966, Diggs and Barreras 1967).

Fig.5.7 Pulmonary emboli,showing characterstic laminated appearance

(Courtsey, LW Diggs)

Chronic pulmonary thrombo-embolism and cor pulmonary

Progressiveobliteration of the pulmonary vascular bed may occur resulting in pulmonary
hypertension and increase in right ventricular work. Critical review of literature (Collins and
Orringer 1982) yielded only one case with confirmation of pulmonary hypertension by cardiac
catheterization (Durant and Cortes 1966) and two with clinical and autopsy findings. These

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observations tend to suggest that pulmonary hypertension and cor pulmon ale are infrequent
events in sickle cell disease.

Haupt et. al. (1982) were unable to document a significant increase in thrombo-emboli
in sickle cell disease with the exception of necr otic marrow embolism (Table. 5.1) Focal
alveolar wall necrosis often associated with.masses of sickled cells distending and
disrupting.the alveolar capillaries was the prominent finding in latter study.

Table 5.1: Pulmonary pathology in 72 patients with sickle cell disease and in age -sex-race matched
controls ( Haupt et. al. 1982)

Feature. SS %. AA%. Significance

Alveolar wall necrosis 12(17) 4(6) <0.05

Bronchopeumonia 11(15) 21(29) "

Oedema. 54(75) 32(44) <0.001

Focal mesenchymal Scar 19(26) 6(8) < 0.005

Interstitial Pneumonitis 1(1) 3(4) NS

Mucous plug. 3(4) 2(3) NS

Necrotic marrow Emboli 9(13) 0 <0.01

Thrombo-emboli. 18(25) 11(15). NS

Thrombo-embolic Infarct 4(6) 1(1). NS

Bone marrow embolism

The classic syndrome is one of severe pain associated with pyrexia as well as
neurological, respiratory, and renal complications. Pathologically the vessels may be obstructed
by necrotic bone marrow containing fat globules and occasionally spicules of bone.

Pulmonary manifestations are serious complications in patients with sickle cell disease.
Pulmonary infarction may result from primary occlusion of small vessels. Multiple pulmonary
thrombosis leads to increased pulmonary resistance−pulmonary hypertension−cor pulmonale
(Gupta VL, 1986).

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 CHAPTER V

The Brain and Nervous system

The first description of central nervous system involvement in sickle cell disease
reported a left hemiparesis in a 3 year old boy (Sydenstricker et. al. 1923). The most common
major neurological complication in SS disease. Gupta VL et.al . (1981) in their series of 75 cases
reported hemiplegia in one case at presentation. Estimates of it's frequency in hospital based or
clinic population with SS disease may vary 8-10 percent ( Greer and Schotland 1962, Portnoy
and Herion1972). Powars et. al. (1978) calculated risk of stroke in first two decades as 0.761 per
100 person years and after the age of 20 years as 0.524 episodes per 100 person years.
However the pathology was strikingly different, cerebral infarction being almost confined to
children (mean age 8 years) and intracranial hemorrhage predominating in adults. (mean age
25 years).

Intracranial hemorrhage
Subarchnoid haemorrhage may arise from ruptured berry aneurysm ( Wertham et.al.
1942, Hitchcock et. el. 1983, Caprioli et.al.1983), subarchnoid extension of a cerebral
haemorrhage (Bloch et.al.1951), bleeding associated with major cerebral vessels (Bridgers
1939), or without other obvious pathology (Greer and Schotland 1962).

Covulsions, coma, and stupor

Convulsions occurred in 14/86 (16%) and in.11/89 (12%) in two.series. They were
recurrent in half the cases described by Greer and Schotland (1962).Convulsions occurred as an
isolated neurological manifestation in 7/11 cases (Portnoy and Heroin 1972). Generalized
attacks are most common, but focal attacks occur especially in association with cerebral-vas c
ular damage when they are difficult to control.

Coma and stupor as frequent presenting features of severe cerebral-vascular disease. Portney
and Herion noted a history of coma or stupor in 8/89 (9%) SS patients with neurological
complications and in three, it was an isolated neurological finding.

Brain stem lesions

Attacks of vertigo with signs of vestibular dysfunction were reported in 3 patients by
Greer and Schotland (1962). Symptoms in one patient with a haemoglobin level.of.4g/dl were
immediately relieved by transfusion and in two other cases with SC disease resolved gradually
over 2 weeks. Transient ataxia occurred in one described by Portney and Herion (1972).

Others

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Visual disturbances secondary to retinal vessel changes as discussed under ocular

manifestations. Sudden hearing loss, independently of meningitis has been reported in SC
disease ( Morrison and Booth 1970), in SS disease. (Orchik and Dunn 1977).Clinical feature s
suggestive of cord lesions are uncommon. A transient paraplegia with urinary retention during
aplastic crisis occurred, was presumed to be on an ischemic basis. (Gray - personal
communication). Peripheral neuropathy is rare, and is attributed to other co-existing cause
like lead poisoning (Seto and Freeman 1964).

Pathology of cerebrovascular complications

Since sickle cell disease is primarily a cause of small vessel obstruction, it has been
assumed that the cerebral pathology results from multiple small vessels obstruction. However it
is now recognized that punctate hemorrhagic lesions confined to the white cortex, generally
results from arterial embolism by necrotic bone marrow. There is now abundant evidence that
partial or complete occlusion of major vessel is common.

Precipitating Factors for neurological event are, Young age, low HbF levels, family
history, preceding meningitis, hyperventilation. (Serjeant GR, 1983)

A high prevalence of hemiplegia was found in 89 patients with sickle-cell disease seen
over a 5-year period. Twenty-three patients (26%) had neurological manifestations; hemiplegia
occurred in 15 (17%). During the same period, hemiplegia occurred in only 5% of patients with
sickle-hemoglobin C disease and in 1.7% of patients with sickle-cell trait; the latter is the same
as that in the negro population with normal hemoglobin (1.8%). A review of the English-
language literature shows that the high frequency of hemiplegia in the natural history of sickle-
cell disease has not been sufficiently emphasized, despite its previous documentation in
isolated case reports. Unless better methods for controlling the clinical expressions of this
hemoglobinopathy can be found, stroke will remain one of the commonest serious
complications in patients with sickle-cell disease. (Portnoy and Heroin)

Neurological complications of SCD may be more frequent in Africa than currently

reported. The rarity of prevalence studies of these complications makes it difficult to manage
them. The most commonly reported neurological complication of SCD is overt stroke. However,
the frequency of silent infarcts, intracranial bleeding, leukoencephalopathy, cerebral atrophy
and impaired cognition is most often underestimated since these are usually revealed by
neuropsychological and neuroimaging screening studies in otherwise asymptomatic patients
with SCD. The high prevalence of these findings in young patients with SCD underscores the fact
that central nervous system injuries begin early in life. The strength of this review is that it will
be the first to quantify the magnitude of neurological complications of SCD across Africa where

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this haematological condition is highly prevalent. Furthermore, this review might identify
specific research challenges to be addressed in future studies of neurological complications of
SCD in Africa.

The main possible limitations of this review could be the scarcity of studies on the
subject and the predominance of poor quality studies (due to lack of population awareness and
inadequate screening or follow-up) that might result in an underestimation of the real
prevalence of the neurological complications of SCD in Africa. Regional differences in the
standard of care could also lead to significant heterogeneity among studies, thus precluding the
meta-analysis and making it difficult to get a global picture of the burden of neurological
complications of SCD across Africa. (Michel K Mengnjo, Joseph Kamtchum-Tatuene et al. 2016)

The Bones and Joints

Narrowing of the marrow cavity and abnormal cortical thickening of long bones was
noted in an early autopsy report. (Grahm 1924). Since then there have been many reviews of
the radiological features of the disease (Golding 1956, Diggs 1967, Ennis et.a l. 1973, Bohrer
1981, Gupta VL et. al.1982).

Skull
Marrow expansion in the vault of the skull leads to widening of the diploic space and
thinning of the outer table.

The trabeculae in the diploic space may be either parallel to the inner table giving rise to
curvilinear or 'onion peel' lamellation (William et.al. 1975) or perpendicular to the inner table
producing 'hair on end' appearance (Fig. 5.8) or both (Lagundoye 1982).

Vertebrae
Marrow expansion in in vertebrae leading to radiolucency and the prominence of the
remaining vertical trabeculae is characteristic and may allow the diagnosis of sickle cell disease
to be suggested on radiological ground. The vertebrae may be flattened with an increased
width to height ratio. (Diggs et.al.1937). The upper and lower surfaces of the vertebral bodies
may form smooth biconcavoties or a steplike depression. (Fig.5.9)

Pelvis shows a generalized osteoporosis, and recently 'protrusio acetabuli' has been
described (Martinez et.al.1984).

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Fig.5.8 Diploic thickening with rare “hair-on end” appearance

In patient with SS disease. (Photo courtesy. Dr. LW Diggs)

Fig.5.9 Biconcave depression of vertebral bodies

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Long bones: Marrow expansion in long bones is manifest by widening of the medullary cavity,
thinning of the cortex, and occasionally endosteal scalloping of the inner cortical margins. There
may be loss of the normal concave medial margin of the lower femur, the so called 'flask
shaped' deformity. (Fig5.10) and the 'squaring ' of the metacarpels. (Fig.5.11).

Fig.5.10 The ‘flask shaped’ deformity resulting from

Medullary expansion. (Courtsey, Serjeant GR)

Dactylitis (hand -foot syndrome)

The syndrome was first reported by Danford et.al. (1941) and subsequently by Macht
and Roman (1948). The term 'hand -foot ' syndrome appears to have been used by Smith
(1960). Clinically the child presents with acute painful, non-pitting swelling of one or more
extremities. (Fig. 5.12), usually with fever pathological studies of dactylitis confirm complete
necrosis of the bone marrow and inner third of the cortex (Weinberg and Currarino 1972).
because of intracellular sickling.

Commonly reported in African and Jamaican population (Stevens et. al. 1981b) and also
in USA. (Konotey Ahulu, 1971b, Watson et.al.1963). Not that commonly reported from Indian
subcontinent. (Gupta VL, 1982).

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Fig.5.11 Prominent nutrient foraminae and loss of

of the normal mid-metacarpel constriction

Fig.5.12 Dactilytis right hand in patient with SS disease

(Courtsey Dr LW Diggs)

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Long bone involvement

Acute diaphyseal infarction of long bones affects most frequently.(Fig.5.13) the
intermediate segment between the diaphysis and metaphysis at the point furthest from the
entry of nutrient vessel.

At the later ages, acute diaphyseal infarction is rare, lesions taking the form of
permanent medullary or cortical changes. Cortical infarction may cause periosteal new bone
formation with progressive cortical thickening and narrowing of medullary space (Golding 1956,
O'Hara 1967). Occasionally changes are so gross as to obliterate the medullary cavity or
produce the appearance of a generalized osteoporosis. (Kraft and Bertel 1947). The "bone
within A bone"

Fig. 5.13 Multiple infarcts femur tibia

appearance ( Moseley 1963, Reynolds 1965, Gupta VL, 1980) may be a sequel to either cortical
infarction or to calcified medullary.infarcts parallel to the endosteal cortex.(Fig. 16.14)

Avascular necrosis of the femoral head

Avascular necrosis of the femoral head has been diagnosed as early as 6 years and the
incidence in the Jamaican cohort study is between 3-5 percent by the age of 10 years. Adult
patients with SS disease in the USA, the prevalence has varied between 4-19 percent .

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(Sebes and Kraus 1983). In the cooperative study in the US, this complication occurred in 19
percent patients with SS disease, compared to 9 percent patients with SC disease. (Sebes and
Kraus 1983).

Fig.5.14 Abnormal medullary calcification secondary to medullary infarction, “bone within a bone”

The symptoms are persistent pain in the hip, worse on walking and relieved by rest.
Necrosis of the immature capital epiphysis leading to a flattening of the femoral head with
rebounding of the acetabulum. Later stages may include resorption of the femoral head leaving
the acetabulum articulating with femoral neck, features of osteoarthrosis, and total destruction
with fibrous or bony ankylosis. Such Progres ion only occurs in a small proportion of patients,
the majority limiting the extent of bone damage and frequently healing well.

Two basic patterns of radiological change are discernible according to the maturity of
the femoral head at the time of necrosis. In children involvement of unfused epiphysis widened
flattened epiphysis, epiphyseo-metaphyseal overlap, a wide femoral neck, and a mushroom
shaped deformity of the mature femoral head (Fig.5.15) In older patients AVN is typically
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segmental, earliest sign is lucent subcortical line, subarticular trabecular fracture and resorption
which precedes collapse of the articular cortex. (Norman and Bullough 1963, Lee et.al. 1981)
(Fig. 5.16)

Fig.5.15 Avascular necrosis showing remodeling of femoral head and smooth articular surface, (c) age
11 yrs, (d) age 15 yrs

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Fig.5.16 Segmental infarction of mature left femoral head in 20 yrs old patient with SS disease

Avascular necrosis of humeral head

AVN commonly affects the humeral head but because of it's absence of weight bearing
articular surface deformity and persistent symptoms are less frequent than in hip. Limitation of
abduction and occasionally severe persistent pain together with restricted movements may
occur (Carrington et.al., 1958).

Radiological features include patchy or diffuse sclerosis, cystic areas, and spotty
decalcification within femoral head with occasionally collapse of the articular surface (Fig.5.17).

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 CHAPTER V

Fig.5.17 Avascular changes in humeral head with gross Deformity of articular surface in 40 yr old
patient with SS disease

Osteomyelitis
In general population the staphylococcus is the com m onest cause of haematogenous
osteomyelitis, but salmonella osteomyelitis appears to ac count for over half the cases in sickle
cell disease. (Diggs 1967). Gupta VL et.al. (1981) reported two cases of salmonella
osteomyelitis in sickle cell disease. In one case there was involvement of vertebrae. In young
children osteomyelitis may be superimposed upon the features of dactylitis.

(Noonam 1982) and in older children upon the features of long bone diaphyseal in farcts. The
commonest bones affected in order are the humerus, radius, tibia femur, and ulna
(Adeyokunnu and Hendrickse 1980, Bohrer 1981) (Fig.5.18).

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Fig.5.18 Osteomyelitis in patient with SS disease

Ocular

Sickle cell disease (SCD) is the most common genetic disease worldwide. The increase in
life expectancy of SCD patients in recent years has led to the emergence of more complications
of the disease, e.g. ocular, which in the past were uncommon. This review describes current
knowledge of the ocular manifestations of patients with SCD. SCD can affect virtually every
vascular bed in the eye and can cause blindness in the advanced stages. The most significant
ocular changes are those which occur in the fundus, which can be grouped into proliferative
sickle retinopathy, and non-proliferative retinal changes based on the presence of vascular
proliferation. This distinction is important because the formation of new vessels is the single
most important precursor of potentially blinding complications. Although various systemic
complications of SCD are known to be more common in patients with the Hb SS genotype,
visual impairment secondary to proliferative sickle retinopathy is more common in patients
with the Hb SC genotype. There is also an increase with age in the incidence and prevalence
rates of all ocular complications of SCD. It is therefore recommended that all patients with SCD
undergo periodic ophthalmological screening from the age of 10 years. (Fadugbagbe AO,et al,
2010).

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Fig. 5.19 Sickle cell retinopathy showing ischaemic areas, arteriovenous shunts and neovascularisation
(CQM’s personal archive)

Early reports
The earliest reference to ocular changes in sickle cell disease was of fresh retinal
haemorrhages in a 7 year old patient dying with subarchnoid haemorrhage. (Cook 1930).
Dilatation and tortuosity of major retinal vessels was feature of several early reports (Harden
1937, Klinefelter 1942, Ray and Cecil 1944), and also the retinal microaneurysms and vitreous
haemorrhage. ( Edington and Sarkies 1952).

Conjunctival vessel abnormalities, sludging of blood, Sacular and sausage like dilatations.
(Knisely et. al. 1947, De Quevedo 1952). and multiple, short, comma shaped or curlicued
capillary segments, pathognomic of sickle cell disease. (Paton, 1962)

Anterior segment ishaemia is characterized in the acute stage conjunctival congestion, corneal
oedema, keratic precipitates, white deposits in the necrotic lens capsule, a low intra-ocular
pressure, and a dilated and unresponsive pupil

Glaucoma secondary to obstruction of the anterior chamber outflow tract can occur in sickle
cell disease and trait following.relatively minor hyphemas. (Goldberg et. al. 1979, 1979a).

Post segment manifestation s include virtuosity of major retinal vessels, central artery and
major branch oclussion, abnormalities of macular vasculature and posterior ciliary artery
oclussion.

Punctate red dots on or close to the disc were reported in several series. (Goodman et.al. 1957,
Condon and Serjeant 1972c, Goldbaum et. al. 1978).
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The presence of angioid streaks in SS disease was first mentioned by Goodman et.al.
(1957),and the frequency of subsequent observations (Geeraets and Guerry 1960b, Nagpal
et.al. 1976a) suggested relationship.

Peripheral retinal changes include whitening of the peripheral retina with poorly defined
margins, pale areas with clearly defined margins( Nagpal et.al. 1976b). Haemorrhage s in
peripheral retina are common.

Figure 5.20. Sea fan formation with neovascularization. Fluorescein angiogram image of an individual
with sickle cell retinopathy showing sea fan formation with neovascularization. This image was taken
using an Optos P200MA ultra-wide field imaging device. (This image was originally published in the
ASRS Retina Image Bank by Michael P. Kelly, FOPS Director, Duke Eye Center Labs, Duke University
Hospital, Sickle Cell Retinopathy. 2012; Image Number, 721. © the American Society of Retina
Specialists)

The most striking feature of ocular involvement in sickle cell disease is the development
of arteriolar occlusion and the capillary loss at periphery. Extensive remodelling of the
peripheral vasculature may occur. (Galinos et.al. 1975a). Abnormal arterial-venous
communications develop at the border. It is from these abnormal vessels that the lessions of
proliferative sickle retinopathy (PSR) develop. These abnormal vessels leak intravenously
administered fluorescein. The size and configuration vary widely from small well defined vessel
loops to large co mplex lessions that may occupy a quadrant of the retinal periphery.

The clinical significance of PSR lies in the frequency of vitreous haemorrhage causing
transient visual loss and of retinal detachment causing permanent visual loss. The incidence of
these events in the.representative samples of the patients is entirely unknown, although visual
loss is sufficiently frequent that a suitable treatment for PSR should be sought.

Retinal changes were first demonstrated by Goodman G. et. al. 1957. Five stage
classification of ocular manifestation of sickle cell disease is given by Mansoor F. Firmaly 1974.
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 CHAPTER V

Stage I : Sluggish blood flow with.minimal evidence of obstruction or occlusion of arteries and
capillaries.

Stage II : Vascular occlusion

Stage III : Neovascularization

Stage IV : Proliferative neovascularization of the vitreous

Stage V : Retinal separation

Non-proliferative Sickle retinopathy (NPSR):

The retinal changes in NPSR occur secondary to vaso-occlusion and local ischemia.
Occlusion of retinal vasculature first appears in the peripheral retina as “salmon patches”,
which represent retinal hemorrhage from superficial blood vessels. These lesions are round or
oval in shape and can be elevated or flattened (Figure 5.21). (Emerson GG, Lutty GA, 2005).
Although they are initially red, they become salmon-colored due to the hemolysis of red blood
cells. Over time, the hemorrhage is resorbed and the area appears normal with refractile
deposits, which are iridescent spots of hemosiderin and macrophage deposition just beneath
the internal limiting membrane. Migration and proliferation of retinal pigment epithelium (RPE)
leads to the development of black sunburst spots which likely occur in response to hemorrhage.
(Jampol LM, Condon P, et al,1981). Reduction in visual acuity is usually from the occlusion of
perifoveal capillaries, causing a concave macular depression from RPE degeneration. Occlusions
in the choroidal circulation with associated breaks in Bruch’s membrane are known to cause
angioid streaks, a phenomenon seen in many retinal diseases including NPSR and PSR.
Proliferative Sickle Retinopathy (PSR):

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As noted above, the ocular complications of PSR are more characteristically seen in HbC (SC)
and SThal disease, rather than in SS disease. As localized vascular occlusion leads to the changes
described above in NSPR, chronic changes of local hypoxia and ischemia lead to the
upregulation of vascular growth factors, which may result in retinal neovascularization, pre-
retinal or vitreous hemorrhage and tractional retinal detachment.

Figure 5.21. Salmon-patch hemorrhage in sickle cell retinopathy. (This image was originally published
in the ASRS Retina Image Bank by Larry Halperin, MD, Retina Group of Florida. Sickle Salmon-Patch
Hemorrhage. 2012; Image Number, 1789. © the American Society of Retina Specialists)

In a study of 19 cases of sickle cell anemia, ophthalmic examination including fungus

examination was carried out. 73.6% of cases had venous tortuosity and fullness, 21.05% had
normal fungus. 5.2% had retinitis proliferans with neovascularization (Anuradha Raut 1986).

Other manifestations

Leg ulceration

Leg ulceration occurred in the first four cases of sickle cell disease but was not
recognized as a specific complication until Cummer and La Rocco (1940) suggested a casual
relationship. Leg ulceration is most common In SS disease and less frequent in other variant s
of sickle cell disease. In SS disease estimates of the prevalence have varied up to 75 percent
(Serjeant 1974b).

Leg ulceration is rare before the age of 5 years and uncommon in the first decade. In
Jamaica, leg ulceration for the first time occurs most frequently between ages of 10-19 years

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and becomes increasingly unusual after the age of 30 years. Poorer patients in Jamaica having a
greater duration of ulceration although not apparently greater incidence. (Ashcroft and
Sergeant 1981)

Ulceration has been reported in Indian patients with.SS .disease (Beohar et. al. 1963,
Gupta VL et. al. 1987) but appears to be uncommon. (kar quoted.by Serjeant G R, 1985). Leg
ulcers typically affect the areas around the ankles above the medial and lateral malleoli. (Fig. 5.
22).

Fig.5.22 Chr.leg ulcer in patient with SS disease (Gupta VL,1987)

Two broadly different patterns of leg ulceration are recognized in sickle cell disease at
the time of initial ulceratiion, some patients give a clear history of trauma. Secondary infection
occurs and a large infected ulcer may develop. In other patients there is no history of trauma
but a spontaneous painful area develop often within normal skin, following which ulcer
develops.

The second type of leg ulcer is presumed to result from skin infarction and although
impossible to prove pathologically is more common in patients with low HbF levels (Serjeant
1974b).

The treatment of leg ulceration is unsatisfactory. Use of local antiseptic to cl ean., Local
or systemic antibiotics if there is evidence of secondary infection, debridement if indicated are
some of the measures employed.

Oral zinc sulphate 200 mhm three times a day has been to significantly improve leg
ulcers healing but the rate is very slow. (Serjeant et. al. 1970, Gupta VL et . al. 1987) (Fig.5.23).

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Surgical approaches to leg ulceration have included full thickness grafts, split grafts,
pinch grafts, and myocutaneous grafts.(Gueri and Serjeant 1970).

Fig.5.23 Improvement in leg ulcer with zinc therapy

(A) Before zinc therapy (B) After zinc therapy (Gupta VL,1987)

Priapism

The corpora cavernosa of the penis are specialized blood reservoirs characterized by the
slow percolation of blood through a complex network of septa. As with the spleen and bone
marrow, such circulations are prone to localized stasis, sickling and obstruction of venous
drainage. When affecting the corpora cavernosa this result in a sustained painful erection or
priapism.

Probably the first reported case of priapism in a patient with.SS disease was presented
to the New York Society for clinical psychiatry in 1932 as a 'castration fear complex' (Obendorf
1934) although Diggs and Ching (1934) were first to recognize the association. Hasen and
Raines (1962) reviewed the literature and added their experience in 12 patients seen over a 20
year period.

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Most series show a wide range in age of onset from 5-35 years. In 100 Jamaican children
with.SS disease aged under 12 years, priapism ocuured in only one child aged 4 years (Gray
1971) and the age of onset varied from 5-40 years (median age 21 years) (Emond et.al.1980). It
is rare in this part of world i.e. Indian subcontinent. (Gupta VL, et.al. 1983).

One of two attacks occurred in 19/61 (31%) whereas multiple episodes (>10) affected 35
(57%). Single attack.tended to be severe exceeding 24 years while multiple attacks were
typically brief (< 3 hours). Major attacks are characterized by extremely severe pain in the
penis, lower abdomen, and perineal region. Precipitating Factors have included normal
erections during sexual intercourse ( Krauss and Fitzpatrick 1961) or masturbation (Karayalcin
et.al. 1972b).

In Jamaica major attacks of priapism were @associated with painful crisis?

Relief was most commonly obtained by gentle exercise such as walking or cycling, or by
cold water or ice applied directly to the penis. Other relieving factors included analgesics,
urination, or drinking cold water, alkalinization, stilboestrol, and blood transfusion.

Surgical measures include direct aspiration of the corpora through a wide bore needle
(McKay and Colston, 1928). Shunting of blood from the corpora to the adjacent saphenous vein.
(Grayhack et.al.1964).

A much simpler, quicker, procedure amenable to out patient use is the creation of
shunts between the corpora cavernosa and corpora spongiosum. (Ebbehoj 1974, Winter 1976,
1979).

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CHAPTER VI
MANAGEMENT OF SICKLE CELL
DISESAE

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General supportive measures

Early identification
Perhaps the most critical aspect of optimizing SCA management is early identification of
affected patients, before the onset of signs and symptoms of disease. Without early diagnosis
and intervention, SCA often acts as a swift and invisible killer, with many infants dying suddenly
of bacterial sepsis or acute splenic sequestration crisis (ASSC) within the first few years of life
(Pearson et al. 1969, 1977; Rogers et al. 1978; Powars et al. 1981). Sometimes fatal
complications occur even before families or medical providers are aware the infants have SCA.

With the recognition that infants with SCA have greatly increased risk of bacterial sepsis,
the landmark multicenter double-blinded placebo-controlled PROPS trial proved that penicillin
prophylaxis significantly lowered the risk of bacteremia and death (Gaston et al. 1986). This
simple intervention provided the justification needed for newborn screening of SCA, to identify
affected infants soon after birth and to allow lifesaving prophylactic antibiotic therapy.
Although a 1987 NIH Consensus Conference recommended newborn screening for SCA,
universal screening was not accomplished in all U.S. states and territories until 2006.

Newborn screening programs in the United States, Jamaica, and Europe have
documented the utility of early identification of SCA, with a marked reduction in morbidity and
mortality, especially in the first 5 years of life (Rogers et al. 1978; Vichinsky et al. 1988; Almeida
et al. 2001; Bardakdjian-Michau et al. 2001). It illustrates that early identification of SCA
through neonatal screening programs has contributed to the improved survival rates (Quinn et
al. 2010).

Education

Parental and family sickle cell education should begin once the diagnosis is made and
continue throughout childhood. Given variable parental education and literacy, education
should be provided in written and spoken form, and should be repeated with each visit to
ensure information is comprehended. When possible, both parents should receive education,
plus extended family members and other caregivers, to become knowledgeable about SCA.

Education in the early newborn period should focus on the basics of SCA, including its
genetics and inheritance, need for penicillin prophylaxis, and benefits of protein-conjugated
pneumococcal immunizations. At each visit these key points should be repeated to parents and
caregivers. The importance of regular medical care should be emphasized, especially the need
for prompt medical evaluation for fever. Antipyretics should never be given for fever at home,
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because this treatment can mask a serious infection. Education for young patients should also
include signs and symptoms of ASSC: pallor, fussiness or irritability, and tender splenomegaly.
Teaching parents to palpate their baby’s spleen regularly, ideally several times a day during
routine diaper changes, allows early diagnosis of ASSC and potentially prompt and lifesaving
intervention.

As the child grows, education should focus more on recognition and early medical
intervention for acute vaso-occlusive complications such as dactylitis and other painful events,
respiratory distress, acute chest syndrome, and stroke. Parents should learn to manage mild
pain at home with oral hydration and analgesia. At an early age, families should be introduced
to possible treatment options including hydroxyurea and transfusions, and even stem cell
transplantation, if available. As affected children grow up and enter adolescence, it is critical to
provide ongoing education about SCA and its complications, to provide young patients with the
skills necessary to understand and advocate for their own medical care. Such self-awareness
and investment in their medical care becomes critically important on transition from pediatric
to adult hematology care. Unfortunately, evidence suggests increased morbidity and mortality
in late adolescence and early adulthood following this transition of care (Brousseau et al.
2010; Quinn et al. 2010).

Coping and support

If you or someone in your family has sickle cell anemia, you may want help with the stresses
of this lifelong disease. Consider:

• Finding someone to talk with. Sickle cell centers and clinics can provide information and
counseling. Ask your doctor or the staff at a sickle cell center if there are support groups
for families in your area. Talking with others who are facing the same challenges you are
can be helpful.
• Exploring ways to cope with the pain. Work with your doctor to find ways to control your
pain. Pain medications can't always take all the pain away. Different techniques work for
different people, but it might be worth trying heating pads, hot baths, massages or
physical therapy.

• Learning about sickle cell anemia to make informed decisions about care. If you have a
child with sickle cell anemia, learn as much as you can about the disease. Ask questions
during your child's appointments. Ask your health care team to recommend good sources
of information.

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Support Groups/Psychiatric Support

The psychosocial dynamics of sickle cell disease are complex. As with any other chronic,
and often debilitating, illness, patients face a plethora of social problems that greatly influence
their clinical condition (Whitten and Fischhoff, 1974). Loneliness, isolation, self-resentment, loss
of self-esteem, and simple anger are common in patients with sickle cell disease. These factors
can profoundly influence the patient's ability to cope with pain. Patient support groups and
psychological counseling often are very useful. The positive results in studies in which children
were taught psychological coping skills for pain, reinforce the importance of this component of
patient care (Gil et al., 1991).

Nutrition

The child with sickle cell disease has greater caloric requirement because of the
moderately hypermetabolic state secondary to the increased tissue turnover in the bone
marrow and to the the increased cardiac work. Total body protein turnover is increased
because of the greater erythrocyte activity and the structurally unbalanced nature of the
rapidly turning over haemoglobin molecule .
High protein, high caloric diet is recommended for these patients with sickle cell disease.
Folic acid requirements are increased by haemolysis, and should be.adequately supplemented.
Zinc deficiency in SS disease and clinical manifestation of.the disease will be discussed
separately in this chapter.
Because of concept of iron overload and some protection iron deficiency state offers.
Iron supplements better be avoided, unless there is continuous iron loss or marked iron
deficiency. Quite few patients in Indian settings, are iron deficient because of various reasons
and may need to be supplemented to improve clinical outcomes. (Gupta VL, et. al. 1982).
Lifestyle and home remedies
Taking the following steps to stay healthy may help you avoid complications of sickle cell
anemia:

• Take folic acid supplements daily, and choose a healthy diet. Bone marrow needs folic
acid and other vitamins to make new red blood cells. Your doctor might recommend a
folic acid supplement. Choose a diet that focuses on a variety of colorful fruits and
vegetables, as well as whole grains.
• Drink plenty of water. Dehydration can increase your risk of a sickle cell crisis. Drink water
throughout your day, aiming for about eight glasses a day. Increase the amount of water
you drink if you exercise or spend time in a hot, dry climate.

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• Avoid temperature extremes. Exposure to extreme heat or cold can increase your risk of
a sickle cell crisis.

• Exercise regularly, but don't overdo it. Talk with your doctor about how much exercise is
right for you.

• Use over-the-counter (OTC) medications with caution. Use OTC pain medications, such as
ibuprofen (Advil, Motrin IB, Children's Motrin, others) or naproxen sodium (Aleve)
sparingly, if at all, because of the possible effect on your kidneys. Ask your doctor before
taking OTC drugs.

Avoiding Exposure to known precipitating factors of sickle cell crisis

Known precipitating factors, like exposure to infections, prophylaxis, prompt treatment,

avoiding exposure to extreme climatic conditions, stress, dehydration, high attitude, extreme
exercise schedule should be avoided as this can precipitate crisis and add to morbidity and
rarely mortality.

Preventive measures
Immunization
Immunization with the pneumococcal vaccine is standard practice both in adults and
children with sickle cell disease. Several studies suggest that immunization provides some
protection, although incomplete, against pneumoccocal infection (Ammann et al., 1977)
(Ammann, 1982) (Schwartz, 1982). The vaccine appears to be effective even in adults where
splenic function has been lost (Wong et al., 1992). The more recently available 23-valent
vaccine provides broader coverage than earlier versions. Although the duration of protection is
unknown, most specialists re-innoculate patients once every 5 to 7 years. A noteworthy
contrary voice comes from a broadbased review of pneumococcal vaccine efficacy that cast
doubt on the role of the vaccine in patients with sickle cell disease (Butler, et al., 1993).

More recently, a vaccine against Hemophilus influenzae has entered the clinical arena
(Rubin et al., 1989). The efficacy of this vaccine in sickle cell disease is unknown. Given the
serious nature of H. influenzae infections in these patients, many specialists, particularly
pediatricians, now routinely immunize their patients against this organism.

Immunization against viral influenza is common practice. Viral influenza per se is not a
special threat for patients with sickle cell disease. Since influenza is often complicated by
bacterial infection and other problems, prevention of the disease by immunization is a very
practical intervention.
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Recently, an effective vaccine against hepatitis B was developed. Since patients with
sickle cell disease are likely to require one or more transfusions in their lifetime, immunization
against hepatitis B is a reasonable precaution (Mok et al., 1989).

Pneumococcal immunization should begin with locally available pneumococcal

conjugate vaccines (7, 10, or 13 valency) and supplemented at age 2 and 5–7 yr with the 23-
valent pneumococcal polysaccharide vaccine (Pneumovax). Additional recommended
vaccinations include the H. influenzae type b series, meningococcal conjugate vaccine
(Menactra), and yearly influenza. When locally feasible, the published immunization schedule
for high-risk children as recommended by the American Academy of Pediatrics
(www2.aap.org/immunization/IZSchedule.html) should be followed.

Infection Prophylaxis
Infection is a leading cause of death in patients with sickle cell disease. Hyposplenism,
due to splenic autoinfarction, is a major contributor. Hyposplenism is not the sole cause of the
defective host defense as evidenced by the fact that overwhelming sepsis is the leading cause
of death of children under three years of age (Gill et al., 1995). Splenic autoinfarction is less
common in these very young children.

Antibiotics
A double-blind study of the use of penicillin prophylaxis for children between the ages
of six months and three years was terminated before the expected time of completion (Gaston
et al., 1986). The trend indicated clearly that penicillin protected patients from infection or
death due to overwhelming infection by Streptococcus pneumoniae. The recommendation now
is that all children be placed on prophylactic penicillin at a dose of 250 mg twice a day. Patients
with allergies to penicillin should be treated with erythromycin. No recommended duration of
treatment with prophylactic penicillin exists.

A second study looking at the role of prophylactic penicillin in older children was
recently completed. No difference in the incidence of severe infection was found in this cohort
of children between the ages of 5 and 12 years (Falletta, et al., 1995). The implication is that
penicillin plays an important prophylactic role only in young children. One caveat to the
interpretation of this study is that the incidence of pneumoccocal infection was strikingly low in
both groups. This could have been a clinical fluke. As such, a true difference in infection rate
between the two groups could possibly have been missed.

The role of prophylactic penicillin in adults with sickle cell disease is unclear. Adults
develop overwhelming sepsis, but at a much lower frequency than do children. No controlled

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study to determine whether prophylactic antibiotics are useful in adults has been done. The
recently completed trial in older children suggest that prophylactic antibiotics may not benefit
adults. Nonetheless, many physicians still prescribe prophylactic antibiotics for adults.

As the result of vascular congestion, intraparenchymal sickling, and hypoxic injury to the
spleen, infants with SCA have early loss of filtrative splenic function and are susceptible to
acute life-threatening infections, particularly from encapsulated bacteria such as Streptococcus
pneumoniae and Haemophilus influenzae type b (Winkelstein and Drachman 1968; Pearson et
al. 1969; Pearson 1977). This risk remains increased throughout life, but is most significantly
increased in the first 5 years, when bacteremia incidence is the highest (Overturf et al.
1977; Powars et al. 1981; Gill et al. 1995).

The PROPS trial showed that prophylactic oral penicillin reduced the frequency of
bacterial infection by 84% among young children (age 3–36 mo) with SCA (Gaston et al. 1986).
However, the follow-up PROPS 2 study was unable to show benefit from penicillin after age 5
yr, primarily owing to their lower incidence of bacteremia (Falletta et al. 1995). After
introduction of protein-conjugated pneumococcal vaccines, the incidence of invasive
pneumococcal disease decreased by 93.4% among young children with SCA (Halasa et al. 2007).
In Africa the dangers of pneumococcal sepsis for children with SCA have been questioned, but
recent data confirm its prevalence and lethality (Williams et al. 2009).

Based on overwhelming evidence, early pneumococcal prophylaxis is recommended for

all infants with SCA. Penicillin 125 mg by mouth twice daily should begin by 3–4 mo of age, as a
liquid formulation or crushed tablet. The penicillin dose should be increased to 250 mg by
mouth twice a day as the child grows, typically at 3 yr of age. Some international programs
recommend monthly IM penicillin to help ensure compliance. Oral erythromycin can be used as
a substitute if penicillin allergy or rash develops, but this is uncommon.

Penicillin prophylaxis should continue through age 5, when the risk of invasive bacterial
disease is lower. Once the immunization series is up to date, including the Pneumovax booster,
children with SCA may discontinue penicillin prophylaxis. However, penicillin should be
continued indefinitely if a child has had culture-positive sepsis or a surgical splenectomy.

The dramatically increased risk of overwhelming and rapidly fatal infection among
young patients with SCA must be understood by all caregivers and medical providers. Fever
>38.5°C is a medical emergency requiring prompt medical evaluation, including physical
examination with vital signs and splenic palpation, blood culture, complete blood count,
reticulocytes, urinalysis, and chest X-ray if clinically warranted. Type and crossmatch should be
obtained if there is extreme pallor, splenomegaly, clinical instability, or acute respiratory or
neurologic symptoms. After obtaining the blood culture, broad-spectrum antibiotics (e.g.,
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ceftriaxone) should be administered intravenously. Addition of another broad-spectrum

antibiotic (e.g., vancomycin) should be considered if the child appears toxic, has high fever, or
suspicion of central nervous system (CNS) infection.

Hospitalization is recommended if clinical or laboratory indicators suggest sepsis, such

as hemodynamic compromise including hypotension, child <1 yr of age, prior history of sepsis,
temperature >40°C, WBC >30 × 109/L, or <3 × 109/L, concurrent symptoms such as pain or acute
anemia, or if close follow-up is not reliable (Lane et al. 2001).

Blood transfusion

Sporadic Transfusion
Patients with sickle cell disease are anemic, by definition. The degree of the anemia
varies. The hematocrit frequently is in the mid- 20's. Some patients have hematocrits in the low
30's while others have values in the high teens. The baseline hematocrit remains relatively
stable in a given patient, however. Patients with Hemoglobin SC disease tend to run
hematocrits in the low to mid 30's. Most patients are conditioned to tolerate their degree of
anemia, and routine transfusion is not necessary. Raising the hematocrit provides no clinical
benefit, unless the baseline value has fallen into the mid-teens, at which point oxygen carrying
capacity can be compromised. Hematocrits in such a low range leave little leeway for further
decline. On the other hand, transfusing patients with sickle cell disease to hematocrits in the
mid- to upper-30's can be dangerous, since blood viscosity increases substantially at higher
hematocrits (Kaul et al., 1983). The increase in viscosity can worsen the sickling propensity by
increasing the time during which the cells remain in the low oxygen tension regions of the
circulation.

Chronic Transfusion Therapy

The best established use of chronic transfusion therapy is in patients who have suffered
strokes and have had initial exchange transfusions. Chronic transfusion therapy is less well
established for the treatment of other complications of sickle cell disease. This modality has
been advocated as a means of treating recurrent severe episodes of sickle pain, priapism, and
as a prophylactic measure inpregnant patients. Variable improvement in these condition
occurs. The utility of transfusion therapy is limited by complications, most notably
alloimmunization and iron overload (Rosse et al., 1990) (Wang et al., 1986) . Clinically
significant iron overload can occur after as few as 30 red cell transfusions. The only treatment
for transfusional iron overload is chelation therapy with desferrioxamine (Cohen and Schwartz,
1979). Marginal iron mobilization with this drug is a frequent problem.

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A major hurdle to the use of desferrioxamine is non-compliance. This is a particular

problem for young people, and occurs in other disorders that require chelation therapy for
transfusional iron overload, including beta-thalassemia and congenital sideroblastic anemia.
Investigation of other chelators, including some orally active drugs, is ongoing. Approval is
imminent for no agent, however

Alloimmunization
Alloimmunization against minor red cell antigens is a major problem for patients with
sickle cell disease who receive frequent transfusions (Rosse et al., 1990). The representation of
minor antigens, such as Kell, Duffy, and Kidd, differs between African-Americans and European-
Americans (Issitt, 1994). For patients who receive only a few transfusions, the problem is not
serious. With repeated transfusion, however, antibodies develop against these minor
determinants complicating typing and jeopardizing further transfusion.

Blacks are substantially underrepresented as blood donors, compounding the problem

of alloimmunization for patients with sickle cell disease. In addition, patients with sickle cell
disease appear to develop alloantibodies more rapidly than other black patients who are
transfused (Vichinsky et al., 1990). Some institutions perform extended panel matching which
includes the most clinically significant minor antigens in an effort to delay the development of
alloantibodies. Some patients develop such severe problems with alloantibodies that
transfusion becomes nearly impossible. A number of institutions have active programs to
recruit blood donors from the black community to lessen the impact of alloimmunization.

Routine use of blood from black donors for black patients with sickle cell disease is not
warranted. The likelihood of finding matched units for patients with sickle cell disease is greater
when black people are in the donor pool. Matching is necessary nonetheless since antigen
variation among black people, like all other humans, is great. An expanded donor pool
substantially improves the chance of a match with antigen testing.

Management of Sickle Cell Crisis

ACUTE VASO-OCCLUSION
Pain Events
The sudden onset of pain that occurs frequently in patients with SCA results from acute
intravascular sickling, so is often referred to as painful VOE or vaso-occlusive “crisis” (VOC).
Although many providers and patients use the simple phrase “pain crisis,” VOE is preferable
because it broadly defines the process and avoids stigma about pain perception and
management.
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The VOE results from erythrocyte sickling, microvascular occlusion, and tissue
ischemia/reperfusion, and is a hallmark clinical feature of SCA. Pain is the most common cause
of acute morbidity of SCA, and is associated with severity of disease and early mortality among
young adults (Platt et al. 1991). Pain often accompanies acute chest syndrome (ACS), a serious
and potentially life-threatening complication of SCA, in 72% of cases (Platt et al. 1994; Vichinsky
et al. 2000). This association usually follows sternal or truncal pain, which leads to splinting and
poor inspiratory effort, and lack of active and complete inspiration following opioid-induced
sedation. The frequency and severity of pain in SCA is more than just episodic and acute,
however; pain in SCA is often chronic, underrecognized and underreported, and therefore
undertreated (Solomon 2008). Pain diaries of 232 adult patients showed that SCA pain is
common and often chronic; pain was present on 54.5% of days and 29.3% of patients reported
pain on >95% of days (Smith et al. 2008).

The pathophysiology of vaso-occlusive pain is multifactorial and complex, and includes

various blood cells including reticulocytes and neutrophils, plus plasma factors and vascular
endothelium (Ware 2010a). Several factors have been identified as triggers of painful VOE, with
individual patients often recognizing their own specific triggers. The most commonly described
triggers include cold temperatures and especially cold water, as well as dehydration,
overexertion, and menses (Redwood et al. 1976; Resar and Oski 1991; Yoong and Tuck 2002).

A combination approach of nonpharmacologic and pharmacologic agents should be

used for acute management of vaso-occlusive painful events. Nonpharmacologic interventions
with shown effectiveness include oral hydration, heat, massage, and various cognitive-
behavioral and self-relaxation techniques (Rees et al. 2003; Dampier et al. 2004). Cold packs can
increase local sickling and may exacerbate pain, so should be avoided. Pharmacologic
interventions should begin at home with nonopioid analgesics, including acetaminophen and
nonsteroidal anti-inflammatory drugs (NSAIDs); ibuprofen (10 mg/kg or 800 mg for adults >40
kg every 6–8 h) is an effective oral agent given its potent analgesic and anti-inflammatory
properties. Corticosteroids may reduce the duration of painful VOE but on discontinuation, are
associated with an increased frequency of rebound painful episodes requiring readmission
(Griffin et al. 1994), and so are relatively contraindicated for routine pain management. If pain
is not controlled with increased hydration, oral analgesia, and other conservative measures,
opioids should be used. Oral narcotic therapy such as codeine and its derivatives can often be
used effectively at home, in selected settings and patients.(Fig.6.1)

In the event of severe vaso-occlusive pain requiring formal medical evaluation,

aggressive pain management should be implemented promptly with intravenous hydration and
opioid (morphine or hydromorphone) analgesia, and adjuvant intravenous NSAID therapy.
Historically, the most painful VOEs have been evaluated and treated in the local emergency

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room, but given the relative lack of sickle cell knowledge and familiarity among some
healthcare providers, such evaluations have delays in initiating appropriate analgesia, poor pain
control, and a high rate of hospital admission. A sickle cell day hospital approach, which
features staffing by experienced sickle cell providers, is increasingly used and results in
improved pain management, better patient satisfaction, and decreased rates of hospitalization
for both adults and children with SCA (Benjamin et al. 2000; Raphael et al. 2008).(Fig.6.1)

During treatment of VOE, frequent evaluation of pain is important to assess the degree
of relief and potential side effects of narcotic analgesia. When hospitalization is required,
continuous opioid infusion by patient-controlled analgesia (PCA) is recommended (van Beers et
al. 2007; Jacob et al. 2008). Figure 3 illustrates a convenient algorithm to consider for
management of mild to severe painful VOE, but flexibility should exist for individual patient
preferences. When opioids are used, an aggressive bowel regimen should be used concurrently
to reduce gastrointestinal complications, especially hypomotility (O’Brien et al. 2010). Teaching
and encouraging frequent incentive spirometry with ambulation can reduce the risk of
developing complications including ACS (Bellet et al. 1995; Ahmad et al. 2011).

Acute Splenic Sequestration Crisis

ASSC remains an important cause of morbidity and mortality for young children with
SCA. Most ASSC events occur in infants or toddlers before age 2 yr. In some cases, ASSC may be
the first clinical manifestation of SCA, and hence should be emphasized in the education of
families during the first year of life.

The pathophysiology of ASSC involves erythrocyte sickling and rapid accumulation

within the spleen. ASSC is clinically defined as a decrease in baseline hemoglobin concentration
by ≥2 g/dL, in the presence of active reticulocytosis and splenomegaly; mild thrombocytopenia
is common. The acute sequestration event can result in severe anemia, occasionally with
hypovolemia, and even can evolve to circulatory shock or death (Topley et al. 1981; Emond et
al. 1985; Powell et al. 1992).

Medical management of ASSC begins with early recognition and diagnosis; parents and
caregivers must be educated about early signs and symptoms and the need to seek urgent
medical evaluation. After initial assessment including vital signs and physical examination,
laboratory studies should include complete blood count with reticulocytes, blood culture if
febrile, and type and crossmatch. Transfusion volumes should not be excessive because a
transfusion “overshoot” phenomenon can occur when the spleen abruptly unloads trapped
erythrocytes, raising the hemoglobin level above the target goal. Small aliquots (from the same
unit) should be administered every 12–24 h to treat anemia and hypovolemia, while avoiding
hyperviscosity following splenic release.

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Recurrent ASSC events are common, occurring in about half of the children who survive
the first episode. Chronic transfusions can be implemented after the first episode, but their
benefits on reducing recurrent events or avoiding splenectomy are limited (Kinney et al. 1990).
The benefits of splenectomy for ASSC must be compared to its infectious and other
postoperative risks; surgery is usually recommended only after one severe or life-threatening
ASSC event, or after several recurrent ASSC events. Pneumococcal immunizations should be
completed before surgery, and then lifelong penicillin prophylaxis is recommended (Ammann et
al. 1977; Deodhar et al. 1993). Partial or subtotal splenectomy could potentially preserve some
filtrative and immunological splenic function, but published reports in SCA are sparse and
anecdotal experiences have been unsuccessful (Rice et al. 2003).

Stroke

Cerebrovascular accidents are a relatively common and devastating complication of

SCA, with an overt stroke incidence rate of 11% by age 20 years and 24% by age 45 (Ohene-
Frempong et al. 1998). Clinical stroke events represent only a fraction of the cerebrovascular
complications of SCA, which include silent cerebral infarctions (Miller et al. 2001b; Pegelow et
al. 2001; DeBaun et al. 2012) and other neurocognitive deficits (Schatz et al. 2001; Thompson et
al. 2002).

In the setting of acute clinical stroke, quickly reestablishing cerebral blood flow is
crucial; new-onset weakness or aphasia suggests stroke and intervention should never depend
on confirmatory radiological imaging. Modest IV hydration can help acutely, and should be
provided while blood is being crossmatched for transfusion. Particularly for children with severe
anemia, a simple PRBC transfusion can rapidly reduce intravascular sludging and help improve
cerebral blood flow, which is critical to help reverse acute symptoms and prevent stroke
progression. When available, exchange transfusion should be performed promptly to reduce
HbS <30%, with a target hemoglobin concentration of ∼10 g/dL (Swerdlow 2006). A
retrospective analysis of children with overt stroke suggested children receiving exchange
transfusion had a significantly lower risk of recurrent stroke, compared to children receiving
only simple transfusion (Hulbert et al. 2006).

After an initial stroke, the risk of recurrent stroke events is 47%–93% without specific
treatment (Powars et al. 1978; Balkaran et al. 1992; Pegelow et al. 1995). Chronic transfusions
provided every 3–4 wk to maintain HbS of ∼30% are recommended to prevent recurrent events
(Lusher et al. 1976; Pegelow et al. 1995; Strater et al. 2002; Platt 2006). Once initiated,
transfusions should be continued indefinitely, because discontinuation of transfusions is
associated with increased risk of recurrent events (Wang et al. 1991; Adams and Bramilla 2005).
Although efficacious, 10%–20% of patients will develop a second stroke despite transfusions
(Pegelow et al. 1995; Scothorn et al 2002). Recent evidence further shows progression of
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vasculopathy and silent cerebral infarctions among chronically transfused patients (Hulbert et
al. 2011). Hydroxyurea for the prevention of recurrent stroke, coupled with phlebotomy to
remove iron overload, has shown efficacy (Ware et al. 1999, 2004), but in a phase III
randomized clinical trial was inferior to transfusions and chelation therapy (Ware and Helms
2012). In certain clinical settings in which chronic transfusions are unsafe or otherwise not
feasible, however, hydroxyurea may be a viable treatment option (Ali et al. 2011).

Fig. 6.1 Pain management during Vaso-oclussive Crisis in Sickle Cell Disease

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Therapeutic attempts to inhibit sickling

Approaches inhibiting sickling,Inhibition of gelation of Hb s

Ureas, Alkylureas, Fagara Zanthoxyloids, Sodium cynate, Nitrogen mustard

Decreasing concentration of deoxy Hb s

Alkalies, Carbamylation and acetylation , Modification of sulphydryl. groups. Promoting
synthesis of other haemoglobins.

Agents with proposed membrane effects

Phenothiazines, Steroid hormones, ZINC, Cetiedil.

Inhibition of microvascular entrapment

Vasodilators, Anti-sludging agents, Anticoagulant

*Dean and Schechter, 1978, Serjeant GR, 1985.

All these therapeutic interventions were tried with some success, which was not
reproducible and had poor external validity. Some agents were not recommended because
therapeutic benefits were outweighed by side effects and toxicity, these agents did not stand
the test of time. The search for ideal antisickling agent continued.

Zinc
In vitro incubation of SS red cells.with.Zinc of 0.3 mM concentration.resulted in
improved filtration characteristics. (Brewer and oelshlegel 1974) and.oral administration of zinc
has been reported to significantly lower the ISC counts (Brewer et.al. 1977). The mechanism of
this inhibition.is unclear although.is presumed to be a membrane.effect. Clinical claims of a
general supportive improvement or a decrease in the frequency of painful.crisis (Brewer et. al.
1975) have not been confirmed in controlled studies.

Zinc deficiency in sickle cell anemia is a known fact. (Serjeant GR, Galloway RE, 1970 Greaves
MW, Skillen AS 1970,). Earlier studies showed that zinc deficiency is a common finding in cases
of severe anaemia, polycythemia and certain.leukaemias, (Tallot TR, and Jr.Ross JF, 1960).
Persistent reports of low serum zinc levels and encouraging results of oral zinc sulphate in
chronic non h ealing ulcers in sickle cell anemia made workers to work.more in.this direction
(Hussain SL 1969).. Acceleration.of healing process is well documented with.oral zinc sulphate.
Auckland G.(1970) suggested direct application of zinc at ulcer site and thought.that it will be
more helpful. Karayalcin G. et.al. (1974) Reported lower serum.Zinc levels in sickle cell trait and
still lower levels in sickle cell disease. This correlates with.RBC survival in.sickle cell anemia
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which is low in heterozygous and lowest in homozygous sickle cell anemia (Gupta VL and Borse
SD, et.al. 1981). Zinc maintains cell wall integrity. This trace element is lost during.repeated
sickling process (James G White 1974. Can zinc deficiency at cell membrane level be a one of
the causes for short life span of erythrocytes in sickle cell anemia was the thought.

Gupta VL et. al. (1982) studied the effect zinc sulphate solution (1%) in perheral blood
on sickling . In stead of sodium meta bi sulphite. ZnSO4 (1%) was used reagent in sickling test
described by Donold and Castle. For comparison reagent sodium meta bi sulphite (2%) was
used. Number of sickled erthrycytes were significantly less in slides where zinc was used as
reagent. Difference was statistically significant when the slide was examined for delayed
sickling. (P < 0.05) , indicating inhibitory effect.of. Zinc over process of sickling.

Zinc exerts a protective effect in maintaining the integrity of both cellular and organelle
membranes. Zinc deficiency lead to decreased membrane peroxidation and subsequently
membrane damage and abnormalities of cellular transport with decreased activity of
enzymes.(Burin RE, and James F. Sullivan 1976). Milos Chvapil (1976)proposed mechanism for
the effect of zinc. on various cells.

Zinc deficiency in sickle cell anemia is established fact. Higher levels of zinc are reported
in sickle cell trait . This coincides well with red blood cell survival which is lower in sickle cell
trait and lowest in sickle cell anemia.

Table – 6.1
RBC survival and Plasma zinc levels

Group. RBC survival. Plasma Zn levels

G.Karayalcin et al Gupta VL et al
AA 25.25 days. 177 mgm/dl 110 mgm/dl
SA 17.88 days. 158 mgm/dl 90 mgm/dl
SS 13.08 days 116 mgm/ dl 78 mgm/dl

*Gupta VL et. al. 1987, *Karayalcin G. et. al.

Gupta VL and Choubey BS (1987) studied serum zinc levels and electrokinetic potential
(EKP) of red blood cells (Zeta Potential) in patients with sickle cell disease who were under
follow up for one year before study. Serum zinc levels were significantly low in sickle cell
anemia. (P < 0.05). Electrokinetic potential was significantly low in patients with sickle cell
anemia in each blood group (p< 0.025) . A significant improvement in serum zinc levels and EKP
was seen in patients who received zinc therapy (p< 0.05) for four months. The difference in

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post therapy values of EKP in patients of sickle cell anemia and normal.subjects was not
significant. A one and half year's post therapy follow up which included objective assessment in
the form of number of crisis duration of hospitaliazation.and number of work ing days lost with
subjective assessment showed marked and significant improvement. This coincided with
normalization of serum zinc levels and EKP of RBCs in sickle cell disease.

In a double blind randomized controlled clinical trial, 145 patients of sickle cell disease
were recruited, while 130 patients completed the trial schedule (Table- 6.2).

Patients were randomized to receive Zinc sulphate cap. 220 mgm three times a day or
the identical placebo. Major outcome variable was " sickle cell crisis ". After a follow up of 1.5
years, the mean number episodes of crisis was 2.46 ± 1.04 in the intervention group and 5.29 ±
2.58 in the control group (p< 0.0 25, 95% CI for the difference between the groups : 1.98, 3.42).
(Table-6.3). Mean duration of hospital stay was 4.3 ± 2.2 days in the intervention group and
3.9 ± 1.6 days in the control.group. The difference is not significant ( P > 0.05). There was
significant reduction of the mean number of infective episodes (intervention group. 1.7,
control group 3.1, p< 0.01). There was no mortality in either group . Zinc is effective in
prevention of crisis episodes and associated morbidity in patients with sickle cell anemia (Table-
6.4).

Table – 6.2: Base line characterstics of patients groups

Parameters Control group Intervention group
(N = 65) (N = 65)
Age
Range 14−19 yrs. 12−27 yrs.
Mean 18 yrs. 16.4 yrs.
Sex
Males 50 ( 77%) 46 (71%)
Females 15 ( 23%) 19 (29%)
Caste
Nav Boudha,Mahars 42 (65%) 39 (60%)
Others 23 (35%) 26 (40%)

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Table – 6.3: Main Outcome Event in the Patient Groups

Type of Crisis Control Intervention

Vaso-oclussive 220 (63.95%) 91 (58.87%)
Mixed 68 (19.76%) 38 (23.75%)
Haemolytic 40 (11.62%) 17 (09.37%)
Sequestration 12 (01.48%) 12 (01.25%)
Aplastic 04 (01.16%) 02 (01.25%)
Total 344 160

Table – 6.4: Measures of major outcome variable in patient group

Control Intervention “P” 95% CI

Mean ± SD Mean ± SD
Crisis episodes 5.29 ± 2.58 2.46 ± 1.64 0.025 1.98-3.42
Hospital stay/crisis 3.9 ± 1.6 4.3 ± 2.2 0.01
Loss of work days/crisis 4.9 ± 2.1 3.4 ± 2.18 0.01

What this study does'nt tell us is, how long zinc therapy should be continued in these
patients, if therapy is discontinued, how long beneficial effect is going to last?

Will zinc therapy give same results in set of patients who are not zinc deficient?

Larger studies with longer follow up and field trials (effectiveness study) will establish
credentials of zinc as therapeutic agent of choice in sickle cell disease, and also that results of
such a trial should be replicated in other places (External Validity) before generalizing the
results.

Newer Therapies
Therapies of Proven Benefit

Hydroxyurea

Hydroxyurea inhibits ribonucleotide reductase, blocking DNA synthesis and cell division.
The drug also enhances fetal hemoglobin by developing erythroid cells (Platt et al., 1984)
(Stamatoyannopoulos and Nienhuis, 1992). Since fetal hemoglobin blocks sickling, hydroxyurea
has been administered to patients with sickle cell disease in an effort to enhance fetal
hemoglobin production (Charache, 1991) (Rodgers et al., 1990). Hydroxyurea induces fetal
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hemoglobin production, increases the red cell mean corpuscular volume, and reduces the
number of dense cells and irreversibly sickled cells in the circulation (Goldberg et al., 1992).

Fig. 6.2 Significantly lesser no. of crisis episodes in hydroxyurea arm

On January 31, 1995, the Multicenter Study of Hydroxyurea in Sickle Cell Anemia (MSH) was
suspended by the NIH because patients on the hydroxyurea arm of the study had significantly
fewer painful crises than did the controls (Charache et al., 1995) ( Fig. 6.2). This made
hydroxyurea the first (and only) drug proven to prevent sickle cell crises. A second major
observation was that 50% fewer episodes of acute chest syndrome occurred in the patients
treated with hydroxyurea. Hydroxyurea does not cure sickle cell disease, nor is it effective in all
patients. A detailed study showed that hydroxyurea modifies the characteristics of red cells in
patients with homozygous HbS disease to resemble those of patients with HbSC disease
(Bridges, et al, 1996). The heterogeneous response seen in the MSH study is consistent with
people with sickle cell disease being "converted" to a HbSC disease physiognomy. Patients
should be carefully screened and meet certain criteria:

1. Age - 18 years or older.

2. Frequent painful vaso-occlusive crises. "Frequent" can reasonably be defined as three or
more crises per year that require hospitalization.
3. Use of accepted modes of contraception to prevent conception while on the drug.

Hydroxyurea is not reasonable therapy for many patients with sickle cell disease.
Patients who have relatively few vaso-occlusive pain crises should not receive hydroxyurea
therapy. Other contraindications for hydroxyurea include:

1. Pregnancy
2. Allergy to the drug
3. Thrombocytopenia or neutropenia

Although thrombocytopenia and/or neutropenia are relative contraindications, some

patients can tolerate the medication despite these pre-existing factors with close monitoring.
Bimonthly blood counts are required when patients are started on hydroxyurea. In some
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patients on hydroxyurea, the hematocrit rises to the high 30's or even low 40's. No evidence
exists to support hydroxyurea as prophylaxis against stroke, chronic leg ulcers, priapism, or
other complications of sickle cell disease.

The dose of hydroxyurea needed to prevent painful crises is unknown. In the MSH
study, patients received the maximum tolerated dose (MTD). The dose administered was
increased stepwise until signs of toxicity, such as mild neutropenia, developed. The dose of
hydroxyurea was then reduced slightly. Whether such intense treatment is required is
unknown. Lower doses of hydroxyurea (e.g., 25mg/Kg/day) are used by some specialists. Most
patients treated with hydroxyurea develop macrocytosis (e.g., MCV=110). Macrocytosis is not a
good treatment gauge, however.

The data on hydroxyurea applies only to patients with homozygous sickle cell disease
(two hemoglobin S genes). Patients with compound heterozygous conditions (e.g., sickle-beta
thalassemia, Hb SC disease) were excluded from the MSH study to eliminate if possible any
response variability in the data. Future trials may address these issues.

Hydroxyurea is not approved for use in children. The MSH study was restricted to
people 18 years of age or older. An NIH-sponsored trial of the drug in children is on-going. A
number of issues have been raised regarding hydroxyurea in children, including neurocognitive
development and bone maturation. The pediatric hydroxyurea study will address some of these
issues.

Hydroxyurea is teratogenic in mice, but its toxicity to the human fetus is unknown. The
drug has not been associated with carcinogenesis. The carcinogenic potential with very long-
term use is unknown, however. The NIH-sponsored Follow-up Study of Hydroxyurea in Sickle
Cell Disease is designed to monitor the 300 people in the original MSH study for long-term side
effects.

Bone Marrow Transplantation

Bone marrow transplantation can cure SCD. This intervention was first used in a patient
with sickle cell disease who also had relapsed acute lymphocytic leukemia. The transplant was
done to treat the leukemia, but cured the sickle cell disease as well. The largest experience with
transplantation for sickle cell disease comes from Belgium and France, where about 80 patients
have undergone bone marrow transplantation (Apperley, 1993). The results have been quite
promising with cure of the sickle cell disease in every case in which engraftment occurred.

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Concerns about problems such as graft versus host disease and interstitial pneumonia,
two potentially fatal complications of bone marrow transplantation, have limited the use of this
modality in the United States (Kalinyak et al., 1995) (Davies, 1993). A recently reported trial of
bone marrow transplantation in children from centers in the US reaffirmed that the procedure
can cure sickle cell (Walters et al., 1996). Ten percent of the children died from the procedure,
and some suffered severe complications, such as graph rejection. A later report by this group
includes thirty-four children under the age of 16 years who have received bone marrow
transplants (Walters MC, et al., 1997). The incidence of complications as lower in the children
who underwent transplant subsequent to the initial report.

The questions of when to perform a transplant and which patients should receive the
therapy are difficult. The optimal time for transplantation is during childhood, since children
fare better with transplantation than do adults. The variable clinical manifestation of sickle cell
disease makes it impossible to predict in childhood which patients will have a more severe
course. This issue is particularly pertinent considering that transplantation is best carried out
prior to the development of end organ damage from the sickle cell disease.

Analysis of data from the Study of the Natural History of Sickle Cell Disease reported by
Platt, et al, suggested that patients with fetal hemoglobin levels of less than 8.6% tend to have
more severe disease over the long run (Platt et al., 1994). This would seem to provide a guide
that could be used in the decision of which patients to transplant. However, the data are only
statistical values. With rare exceptions, statistical data cannot be applied to a particular patient
to predict the clinical course.

An unresolved ethical question surrounds bone marrow transplantation for children

with sickle cell disease. Sickle cell disease is often a debilitating condition that makes life
miserable for its victims. Although the Natural History Study indicates that patients with sickle
cell disease die earlier than actuarial projections for other African-Americans, data collected in
the 1980's showed a life span that extends into the 40's. With the nearly universal use of
prophylactic penicillin in children to prevent overwhelming pneumococcal infections along with
other advances in supportive treatment, this figure is likely to improve.

The question of who should decide to subject a child to this potentially fatal procedure
likewise is complex. Should the decision be left to parents and physicians? In a study at the
University of Chicago, parents were presented with hypothetical data on cure/mortality rates
for their children with sickle cell disease, and asked to indicate when the risks of the procedure
were acceptable relative to the gravity of the disease (Kodish et al., 1991). About one-third of
the parents indicated that a transplant mortality rate of 15% along with a 15% incidence of

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graft-versus-host disease were acceptable odds. However, young adults older than 18 years
were not allowed to participate in the decision process. Should the patients, the ones with the
most to gain and the most to lose, be excluded from the decision-making loop? Should the
courts appoint advocates for the children, to ensure that the parents and physicians indeed are
acting in the "best interest" of the youngsters?

A program of bone marrow transplantation for beta-thalassemia major has been

successfully initiated in Italy (Lucarelli et al., 1993). Although sickle cell disease and beta-
thalassemia major are both hemoglobinopathies, clear differences between the diseases exist.
The most important is the monotonous progression to disability and death that occurs with
beta-thalassemia major. Bone marrow transplantation for sickle cell disease offers promise. The
jury has not returned a final verdict, however.

Experimental Therapies

Erythropoietin

Erythropoietin is a hormone produced by the kidneys that stimulates red cell production
(Adamson and Eschbach, 1990). Usually, the hormone is made in response to hypoxemia (Kario
et al., 1992). Erythropoietin also increases fetal hemoglobin levels in the red cells of many
patients (Nagel et al., 1993). A number of investigators have examined the extent to which
erythropoietin will raise fetal hemoglobin levels in sickle cell disease. The consensus is that the
drug can significantly raise fetal hemoglobin levels, particularly when given in high doses.

In one report, the drug was used in a dose of over 1,000 U/kg three times per week
(Rodgers et al., 1993). The treatment significantly elevated fetal hemoglobin levels. The
quantity of erythropoietin required for this effect was enormous, particularly when compared
to patients on hemodialysis where the typical dose is now about 150 U/kg three times per
week. The cost of erythropoietin at the higher dose is prohibitive. Further, while erythropoietin
can increase the fetal hemoglobin content of red cells, no controlled trial has shown that it
alters the clinical course of sickle cell disease.

Butyrate

Arginine butyrate and similar compounds have been tested in patients with sickle cell
disease (Perrine et al., 1989). The use of this agent stemmed from the observation that babies
born to diabetic mothers with poor glucose control had sustained production of fetal
hemoglobin after birth relative to infants born to normal women. Butyrate produced as a
byproduct of the hyperglycemia produced the phenomenon. A group of investigators
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subsequently examined the use of butyrate as a means of inducing fetal hemoglobin synthesis
in patients with sickle cells disease (Perrine et al., 1993).

Arginine butyrate can increase fetal hemoglobin levels, but the effect is variable (Sher et
al., 1995). Unfortunately, the drug must be given intravenously and has a half-life of only about
5 minutes. Intermittent rather than continuous infusion of arginine butyrate may induce fetal
hemoglobin synthesis more effectively. Side effects that have been seen in patients who have
received arginine butyrate include anorexia, nausea, vomiting, and abnormal liver function tests
(One patient had a seizure on the medication after inadvertently receiving 4 times the
recommended dose.) For most patients, arginine butyrate is well-tolerated, however. The
requirement for intravenous administration limits the use of the agent. However, many drugs
now are administered intravenously to patients at home (e.g., desferrioxamine for iron
overload). Creative strategies are being explored to make arginine butyrate a useful therapeutic
option.

An effort is underway to identify orally active agents with longer half-lives. Several
compounds have been identified, and a couple have been placed into early clinical trials. One of
these is sodium phenylbutyrate, a drug that has been used for patients with urea cycle
disorders (Dover et al., 1994). Unfortunately, patients tolerated the medication poorly, in part
due to the fact up to 40 tablets per day are needed to obtain acceptable blood levels. The
increase in Hb F levels produced by the oral agents studied thus far have been significantly less
than that seen with arginine butyrate (Perrine et al., 1994). The butyrates have a number of
hurtles to leap before they are accepted for more general use, including a demonstration that
they consistently alter the red cell profile and, ultimately, improve the clinical picture in sickle
cell disease.

Clotrimazole

Red cell dehydration contributes substantially to polymerization of sickle hemoglobin in

patients with sickle cell disease. The cell membrane is damaged in part through repeated
physical distortion by hemoglobin polymerization/depolymerization, and in part through
oxidant damage from reactive oxygen species generated by hemichromes and other
hemoglobin byproducts. K-CL co-transport increases, with K+ loss and associated water loss.
Also, sickle cells accumulate Ca2+. As a consequence, the Gardos channel (Ca2+-activated
K+ export) is activated, with further dehydration.

Clotrimazole and other imidazole antimycotics specifically inhibit the Ca2+-activated

K+ channel pathway of normal and sickle erythrocytes. The original report of Alvarez and

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colleagues described the inhibition of the normal human red cell Gardos channel by
clotrimazole (CLT) and other imidazole antimycotics. Dr. Carlo Brugnara showed in sickle
erythrocytes that CLT blocks K+ transport via the Gardos channel, prevents the change in
membrane potential observed when the Gardos channel is activated by internal Ca2+, and
inhibits dehydration induced by either the Ca2+ ionophore A 23187 or cyclic oxygenation-
deoxygenation.

Initial work by Dr. Brugnara and colleagues at Children's Hospital, Boston showed that
clotrimazole reduces the number of dense cells and the number of irreversibly sickled cells in
patients with sickle cell disease (Brugnara, et al, 1996). A study of the agent at Children's
Hospital and Brigham and Women's Hospital evaluated the combined effects of clotrimazole
and hydroxyuea in patients with sickle cell disease. The hope was that the agents, which have
different mechanisms of action on sickle cells, would work at least cooperatively, and perhaps
synergistically, to reduce sickling. Clotrimazole proved to have a number of side-effects that
limited its success, including severe dysuria in many men. A newer imidazole with fewer side-
effects has recently come available. Enrollment in the clotrimazole study has be suspended in
anticipation of this newer agent.

Nitric Oxide

Nitric oxide is one of the newest agents to enter testing for possible treatment of
patients with sickle cell disease. It is an inhaled gas that has been used in a variety of
investigational conditions, including neonatal pulmonary hypertension and adult respiratory
distress syndrome.

Nitric oxide is know primarily for its ability to relax smooth muscle relaxation. However,
the compound also forms a covalent link with hemoglobin, particularly attaching as an S-nitroso
group to the ß-93 cysteine (Gow and Stamler, 1998). This amino acid residue is near the
"acceptor pocket" on the ß-subunit of hemoglobin where the ß-6 valine of Hb S forms a non-
covalent interaction. The hydrophilic S-nitroso cysteine at the ß-93 residue could destabilize the
interaction between deoxy-Hb S molecules in polymerized sickle hemoglobin.

Dr. C. Alvin Head, of Massachusetts General Hospital, and Dr. Carlo Brugnara, of
Children's Hospital, studied the interaction of nitric oxide both in vitro and in vivo in normal
volunteers and patients with sickle cell disease (Head, et al., 1997). Their data suggested that
nitric oxide breathed at a concentration of 80 ppm reduces the polymerization tendency of
sickle hemoglobin. Reduced polymerization was inferred by a fall in the P50 of sickle hemoglobin
(no effect occurred with Hb A). Nitric oxide for acute painful vaso-occlusive crisis is being
studied in an ongoing multicenter trial. Other investigators were unable to reproduce the
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P50 effect (Gladwin, et al., 1999). Further work is needed to determine whether nitric oxide has
a role in the treatment of patients with sickle cell disease.

Fluocor

FluocorTM is a drug manufactured by the CytRx Corporation of Norcross, Georgia. A

phase III clinical trial of the drug for patients with acute sickle cell pain crisis was recently
completed. FluocorTM is a more highly purified version of the drug, RheothRxTM which went
through phase II clinical studies several years ago. Fifty patients were followed in a placebo-
control pilot study designed to evaluate safety and efficacy of the compound (Adams-Graves, et
al., 1997). The investigators infused the drug continuously for 48 hours at the beginning of a
sickle cell crisis. The treated patients required less narcotic analgesic and showed a net
reduction in hospital length-of-stay relative to placebo control patients. The multicenter study
of FluocorTM failed to confirm these preliminary results. The future of the drug in the treatment
of sickle cell disease is unclear.

Future therapies for sickle cell disease

Gene Replacement Therapy

The beta-globin gene was cloned a number of years ago, fueling interest in the
possibility of gene replacement therapy for sickle cell disease. While the idea of simply
replacing the defective gene with a normal one is appealing, a number of major difficulties
must be surmounted. The first is to engineer a construct in which the beta-globin gene is
expressed at high levels. Our understanding of the factors that control globin gene expression
has advanced significantly over the past few years, but many of the nuances are yet to be
worked out. A large segment of DNA upstream of the beta-globin gene cluster, called the locus
control region, is necessary for efficient transcription of beta-globin mRNA. Any attempt at
gene therapy must include the large locus control region.

Another problem is that the inserted gene must be regulated in its expression so that it
produces beta-globin chains at a level roughly equal to the production of endogenous alpha-
globin chains. Failure to achieve such a balance would produce a thalassemia. Further, the
endogenous sickle beta-globin gene likely would have to be silenced, so that it does not
continue to produce sickle globin chains.

Finally, the cloned gene would have to be introduced into pluripotent stem cells so that
the patient would continue to make normal beta-globin in perpetuity. The retroviral vectors
that have been used to this point infect dividing cells, while pluripotent stem cells divide very

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slowly. To overcome this difficulty, a number of researchers have turned to adeno-associated

viruses (AAV) as vehicles for gene therapy since these viruses can infect resting cells. Here a
new barrier, namely immune response to the viral vector, has appeared. In any event, gene
therapy for sickle cell disease, the ultimate cure for the disorder, is not imminent.

Table 6.5 Future therapies for sickle cell disease

Treatment
Cure Maintenance Acute Pain Management
Goal

Bone Marrow
hydroxyurea nitric oxide
Transplant

Gene Therapy clotrimazole FluocorTM

magnesium Inibitiors of endothelial cell

pitolate adhesion

arginine
Antiinflammatory agents
butyrate

Table 6.5 shows how therapies might be combined, using as examples some of the agents currently
under investigation. We cannot say whether the therapeutic algorithm that eventually evolves will
include these approaches or modalities not yet conceived. The only statement that can be made with
confidence is that new vistas in the treatment of sickle cell disease will usher in better and more fulfilling
lives for these patients

Combination therapy currently is not an option for sickle cell disease, since only hydroxyurea
has been proven to alter the course of the condition. Many, if not most of the agents currently
under investigation likely will fall short of investigators' hopes. If a few survive the rigors of
testing and joint the clinical armamentarium, however, we could mix and match drugs for
patients with sickle cell disease. Ideally, the treatment regimens would include drugs with
differing modes of action. Hydroxyurea, for instance, combined with clotrimazole would team a
drug that enhances fetal hemoglobin production (hydroxyurea) with one that reduces
erythrocyte dehydration (clotrimazole). For a particular patient, sickle cell symptoms might be
improved substantially by neither drug alone. The combination, however, might significantly
ameliorate the condition

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A major goal of investigation should be development of interventions that can be used

in very young patients. Many of the problems experienced by adults and adolescents with sickle
cell disease reflect incremental organ damage by bouts of hypoxia. The affected areas may
initially be microscopic. With time, these foci of injury colasce to form regions of macroscopic
injury, such as avascular necrosis of the femur. Prevention must be the watchword as we seek
to improve the management of patients with sickle cell disease.

Without major breakthroughs in gene therapy or bone marrow transplantation that make these
treatments applicable to a large number of patients, drug intervention will remain the major
therapeutic option for sickle cell disease. The likelihood is low of finding a "magic bullet"
medication that substantially improves sickle cell disease for all or even most patients.
Treatment likely will involve the use of different agents alone or in combination to produce
optimal results. Most chronic illnesses, in fact, require combination drug therapy. Hypertension,
for instance, cannot be managed solely with diuretics. Physicians test combinations of diuretics,
beta blockers, calcium channel blockers, and angiotensin converting enzyme (ACE) inhibitors to
acheive opitmal control of hypertension while producing the fewest side-effects.

Reducing burden of illness,Towards eliminating sickle cell gene

Genetic engineering or gene therapy

We know that it's substitution of valine in place of glutamic acid at 6 th position in beta
chain of haemoglobin molecule that leads to sickling, sickle cell and the problem. We also know
that this sequence is controlled by long arm of chromosome 16. Solution is to replace this
defective gene or modify it to have proper sequencing of amino acid in beta chain. This will
certainly do away with sickle cell disease (Fig.6.3).

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ELIMINATING SICKLE CELL GENE

GENETIC ENGINEERING
To modify the long arm of chromosome 16, which controls
the sequence of amino acids in beta chain of Hb, through
genetic engineering

Long arm of chromosome

16

Sequence of amino acids, in beta Hb-S

Dream

Fig.6.3 Genetic engineering, gene therapy

As of now this technology is not available for application. Practical difficulties in

implementing side effects, techno know how and ethical issues are problems. As is discussed in
this chapter earlier.

But whenever this becomes available for therapeutic use it will be boon for sickle cell
disease and also for many other diseases.

Ante natal diagnosis and Medical termination of pregnancy

With advanced technology use, early ante natal diagnosis of homozygous SS disease can
be made in utero so the foetus can be aborted and pregnancy can be terminated safely.( PCR,
using High Performance liquid Chromatography, HPLC).

There are practical difficulties and problems viz. getting consent after vigorous
counselling may not always succeed. Technology and expertise not available or easily,
accessible. But certainly a way towards eliminating sickle gene.

Social engineering and marriage counseling

Requires to sensitize community regarding sickle cell, awareness education are integral
part of this approach. A stable heterozygous (SA) gets a match after counseling in form of
normal AA, then will be the step towards elimination. Avoiding marriages between two
homozygous (SS + SS) or homozygous and heterozygous (SS + SA). Try introducing "A" replacing
"S" will be long term strategy towards reducing burden of illness and elimination.

136
 CHAPTER VI

The strategy has implementation problem and compliance issue. It does not happen like
this in community. Marriages are not fixed that way unless every body realizes and unless it
becomes a social movement, there is a very little hope of success.

Sickle Cell Prevention and Control Program, guidelines

Goals

• To reduce prevalance of sickle cell disease

• To improve quality of life of the patients
• To reduce morbadity and mortality

Objectives

• Carrier detection in the reproductive age group

• Aggressive marriage counseling
• Prenatal diagnosis and termination of affected foetus
• Health education
• Awareness
• IEC material
• Incentives
• Multidisciplinary approach
• Evidence based management modalities to the patients
• Test new treatment modalities
• Regular follow up
• Establish sickle cell clinics at PHCS
• Training of medical and paramedical staff

Based on these guidelines, “Maharashtra Sickle Cell Prevention And Control Program” was
drafted and was partially adopted through NRHM.

137
Recent Insights, developments

Sickle cell patients have begun receiving the first new treatment for the blood disorder in
Over 20 years

Crizanlizumab
The inherited condition can cause severe pain and organ failure, often requiring hospital
admissions. Crizanlizumab is given as a monthly infusion and is thought to cut visits to A&E by
40%. Loury Mooruth, 62, received the treatment at Birmingham City Hospital, having suffered
repeated periods of intense pain for decades.These are called sickle cell crises and are common
in people with the condition."To have a crisis is pain beyond what you could ever experience,"
Loury says.They give you a scale of one to 10 - but it is way beyond that."Sickle cell disease
causes red blood cells to distort and become sticky, blocking vessels and restricting oxygen
supply, which triggers excruciating pain.

Crizanlizumab, a monoclonal antibody, binds to a protein on blood cells, preventing them from
clumping.Sickle cell mostly affects black people.

Fig. 6.4 Loury Mooruth, 62, received the treatment at Birmingham

City Hospital,Courtsey BBC med.

138
GENETIC TRICKERY

Scientists were about to perform an impressive feat of genetic trickery that would turn back
time in Jimi's blood.When we are still in the womb, our bodies use a different type of
haemoglobin called foetal haemoglobin. This grabs hold of oxygen more tightly than adult
haemoglobin and is essential for a developing baby to take oxygen out of its mother's
bloodstream.

After we're born, a genetic switch is flipped and we start making adult haemoglobin. Crucially,
it's only the adult form of haemoglobin that is affected by sickle cell disease.That genetic switch
- memorably named BCL11A - was identified in the mid-2000s. And advances in the field of
gene editing meant scientists now had the tools to flip it.

"Our approach is to turn that switch off and increase the production of foetal haemoglobin
again, basically turning the clock back," says Dr Haydar Frangoul, who treated Jimi at the Sarah
Cannon Research Institute.Jimi's stem cells were sent to Vertex Pharmaceuticals' laboratories
where the genetic editing would take place.

Fig.6.5 Genetic Trickery, how it works, Courtsey BBC med.

139
Fig. 6.6 After success of “Genetic Trickery” cheerful Jimi,
Quite free of symptoms, Courtsey BBC med.

140
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was

Mahar bt1 caste. Both the parents are healthy and showed presence of sickle
cell trait. Two younger brothers and one elder sister of the patSICKLE CELL DISEASE IN INDIA 553
TABLE 1 -Clinical Data and X-ray Findings in 5 Cases of Sickle Cell Disease
I II III 1V V
K.N. W.K. S.R. CM. P.R.
No. Nagpus Chandaagpur Nagpur

Years al Pains ± ±
Age in

Swelling of Joints + - + ±
Pesence of sickle
cell trait. Two younger brothers and one elder sister of the pat

197