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CLINICAL CASE
DISCUSSION
Dr Chinmayee Dhavan
Guide – Dr Richa Shah
Case

10 yr old male belonging to tribal regions of Maharashtra presents with
history of jaundice. He gives history of painful ulcer over medial
malleolus of leg since one month and history of recurrent pain in left
hypochondrium. There is no history of passing high colored urine.

O/E patient appears small for age

Icterus +++

Pallor ++ +

Ulcer over leg

P/A- soft , liver and not palpable.

CVS- Haemic murmur +
Case

10 yr old male belonging to tribal regions of Maharashtra presents with history
of jaundice. He gives history of painful ulcer over medial malleolus of leg
since one month and history of recurrent pain in left hypochondrium. There is
no history of passing high colored urine.

O/E patient appears small for age

Icterus +++

Pallor ++ +

Ulcer over leg

P/A- soft , liver and spleen not palpable.

CVS- Haemic murmur +

RS- NAD

CNS- NAD
Pallor

Pale appearance of the skin and mucous membranes

Causes:
-anemia
-Collapse and shock
-myxodema
-nephritis
Icterus

Yellowish discoloration of skin and mucous membranes.

Causes
Hemolytic anemias

Gilbert syndrome

Crigler-Nagler syndrome

Viral infections – hepatits, herpes simplex etc

Metabolic liver disease – Wilson disease , alpha 1 antitrypsin

Biliary tract disorders- cholelithiasis , cholecystitis

Vascular cause – Budd Chiari syndrome, veno- occlusive disease
Left hypochondriac pain

GERD

Gastritis

Pancreatitis

Splenic infarct

Splenic cyst

Splenic abscess
Positive findings in our patient

history of jaundice

recurrent pain in left hypochondrium

small for age

G/E- Icterus +++

Pallor ++ +

Ulcer over leg

S/E- CVS- Haemic murmur +
How to investigate ?
•
CBC and Peripheral blood smear
•
Reticulocyte Count
•
LFT
•
ESR
•
Iron studies

CBC

RBC morphology
 HGB- 7g/dl Decreased
 HCT- 18% Decreased
 MCV- 85 fl Normal
 MCH- 30 pg Normal
 MCHC- Normal
 RDW- 20% INCREASED


WBC -18,0000
Neutrophilic leucocytosis
PLT- 500,000 mildly raised
MORPHOLOGY-Anisopoikilocytosis ++
Normocytic Normochromic+ sickle cells +, boat shaped
cells +, target cells +
 Polychromatophils + basophilic stippling+ , nucleated rbcs
seen , howel jolly body noted

Reticulocyte count – 8 % Increased
Liver function test

S.BILIRUBIN (TOTAL)-1.6ml/dl INCREASED

S.UNCONJUGATED BILIRUBIN-1.8mg/dl INCREASED

S.CONJUGATED BILIRUBIN-0.2 mg/dl (0-0.2)

Iron studies – normal

Esr – raised
Sickling test

Principle – sodium meta bisulphite reduces the
oxygen tension and induces sickling of red blood
cells

Sample – fresh blood in any anticoagulant

Method –
1 drop of reagent are added to 1 drop of
anticoagulated blood on a slide. Cover glass is put on
and sealed with petroleum jelly /paraffin wax
mixture.

In Hb SS, sickling occur immediately, while it
may take 1 hour in Hb S trait.
HAEMOGLOBIN SOLUBILITY TESTS

Principle -Sickle cell haemoglobin is
insoluble in the deoxygenated state in a high
molarity phosphate buffer. The crystals that
form refract light and cause the solution to
be turbid..

The haemoglobin is added to a solution of
sodium dithionite, a reducing agent, in
phosphate buffer.

If Hb-S is present, the solution becomes
turbid.

Whole blood may be used, but th·e addition
of saponin, a lysing agent, then becomes
necessary.
Hemoglobin electrophoresis

Initial screening test in the evaluation of haemoglobinopathies is
electrophoresis at alkaline pH (8.5) using a Tris EDTA-borate buffer

Various supporting media are used to achieve separation of haemoglobins such
as filter paper, starch gel, or cellulose acetate membranes.

Principle: The migration of molecules having a net charge in an electric field is
known as electrophoresis.

Different haemoglobins have different net charge because of variations in
their structure.

In an alkaline buffer solution, haemoglobins migrate from cathode (-) to
anode (+) and various haemoglobins have different rates of migration due
to differences in their charge.

Identification of different haemoglobins is based on their relative
positions on cellulose acetate strip
Procedure

1. Red cells are haemolysed and a solution is prepared
(haemolysate).

2. Haemolysate is applied near one end of cellulose
acetate strip (point of origin).

3. Cellulose acetate strips are placed in the
electrophoresis chamber containing Tris -EDTA-borate
buffer with point of origin towards the cathode.

4. Electric current is applied till adequate separation is
achieved.

5. The cellulose acetate strips are removed from the
chamber, stained with a protein stain such as Ponceau S,
and dried.
Citrate Agar Electrophoresis at Acidic pH

Citrate agar electrophoresis at acid
pH provides separation of
haemoglobins that have similar
mobilities on cellulose acetate at
alkaline pH.

HbS can be distinguished from HbD
and HbG, and HbC from HbE and
HbO-Arab.

However, haemoglobin variants D, E,
G, Lepore, and H have migration
identical to HbA.
Hplc

Screening test for


(1) detection, identification, and quantification of haemoglobin variants
(2) quantitation of HbA2 and HbF.
Sickle cell anemia
DIAGNOSIS
Sickle cell anemia
Sickle cell anemia

Sickle cell disease is a common hereditary hemoglobinopathy caused by a
point mutation in β-globin that promotes the polymerization of deoxygenated
hemoglobin, leading to red cell distortion, hemolytic anemia, microvascular
obstruction, and ischemic tissue damage.
Hemoglobin

Tetrameric structure is composed of 2 pairs of
globin each with its own heme pair.

Human haemoglobin exists in a number of types,
which differ slightly in the structure of their globin
moiety.
Normal haemoglobin types

In the embryo:

Gower 1 (ζ2ε2)

Gower 2 (α2ε2)

Haemoglobin Portland (ζ2γ2 )

Haemoglobin Barts
 In the fetus:
•Hemoglobin

F (α2γ2)
In adults:
Hemoglobin A (α2β2) The most common with a normal amount over 95%
Hemoglobin A2 (α2δ2) ‐ δ chain synthesis begins late in the third trimester
and in adults, it has a normal range of 1.5‐3.5%
• Hemoglobin F (α2γ2) ‐ In adults Hemoglobin F is restricted to a limited
population of red cells
•
•
Variant forms of hemoglobin which cause disease

Hemoglobin S ‐β‐chain gene, causing a change in the properties of
hemoglobin which results in sickling of red blood cells.

Hemoglobin C‐ is formed by substitution of lysine for glutamic acid at
position 6 of globin chain (β6 Glu→ Lys). Crystallisation of HbC
increases rigidity of red cells, which are destroyed in spleen. This
variant causes a mild chronic hemolytic anemia.

Hemoglobin AS ‐ A heterozygous form causing Sickle cell trait with
one adult gene and one sickle cell disease gene

Hemoglobin SC disease ‐ Another heterozygous form with one sickle
gene and another encoding Hemoglobin C.
Variant forms of hemoglobin which cause disease

Haemoglobin H (β4) ‐tetramer of β chains, which may be present in variants of α
thalassemia

Hb D-Punjab- a point mutation in the beta-globin gene (HBB) in the first base of
the 121 codon (GAA→CAA) with the substitution of glutamine for glutamic acid
(Glu>Gln) in the beta globin chain.

Hemoglobin E (HbE) is an abnormal hemoglobin with a single point mutation in
the β chain. At position 26 there is a change in the amino acid , from glutamic acid
to lysine
Types of sickle cell disease

Sickle cell anemia: Homozygous state for HbS (βS- βS)

Sickle cell trait : Heterozygous carrier state for HbS (βS -β)

Double heterozygous for HbS- Sickle cell β thalassaemia
Sickle cell hemogobin c disease
Sickle cell hemoglobin d disease
Pathology

Autosomal recessive

Defect in HBB gene , chromosome 11 , short arm

results from inheritance of sickle-cell gene
that codes for abnormal β globin chain.
 There is change of a single base A→ T in the
sixth codon of β globin gene so that there is
substitution of thymine for adenine.
 This in turn results in substitution of valine for
glutamic acid at position 6 of β polypeptide
chain (β6 Glu→Val )
Factors which influence sickling

Intracellular concentration of HbS and of other haemoglobins

Association with thalassaemias

Interaction with other abnormal haemoglobins

Mean corpuscular haemoglobin concentration (MCHC)

Decreased oxygen tension

Temperature

Low pH
Clinical features

Growth and development: These are considerably impaired in children with
sicklecell anaemia.

Splenomegaly: seen in infants and young children and is caused by
reticuloendothelial hyperplasia. In later life, spleen becomes small and fibrotic due to
repeated splenic infarctions, and is not palpable. Spleen, however, remains palpable
in adults in sickle-cell β thalassaemia.
Vaso-occlusive crises
Hematological crises
Hematological crises

Megaloblastic crisis: This results from folate deficiency that may
develop during intercurrent illness or during pregnancy.

Haemolytic (“Hyperhaemolytic”) crisis: Increased rate of red
cell destruction over the chronic haemolytic state is called as
haemolytic crisis. There is a sudden fall in haemoglobin
concentration and levels of icterus and reticulocyte count
increase. Haemolytic crisis is uncommon and coexistence of
G6PD deficiency with superimposed oxidant stress may be
responsible in some cases.

Skin: Chronic leg ulcers are common around ankles on the
medial aspect. They do not heal readily and have a tendency to
recur.
 Proliferative retinopathy: Proliferative retinopathy due to retinal
vascular occlusion is an important complication and is more
common in patients with HbSC disease Arteriovenous
communications and neovascularisation may lead to vitreous
haemorrhage, detachment of retina, and visual loss
 Pregnancy: During pregnancy there is an increased incidence of
spontaneous abortion, prematurity, stillbirth, and intrauterine
growth retardation (due to vasoocclusion of placenta). In the
mother, incidence of infections, chest syndrome, and postpartum
haemorrhage is increased
Genitourinary system
Sickle-cell Trait

This is the asymptomatic heterozygous state for sickle-cell gene (βS /β).

In sickle-cell trait, HbS comprises around 40% of total haemoglobin, the remaining
60% being HbA.

Persons with sickle-cell trait do not have anaemia and are usually asymptomatic.

Some clinical abnormalities: deficient urine concentrating ability, infarction of
spleen and vaso-occlusive crises at high altitudes, and haematuria (renal papillary
necrosis). Instances of sudden death have been reported following strenuous
exercise.

Few target cells may be present on blood films

Diagnosis requires demonstration of HbS by sodium metabisulphite slide test or
solubility test and haemoglobin electrophoresis.

Haemoglobin electrophoresis reveals more HbA (60%) than HbS (40%).

No treatment is required and duration of survival of individuals is normal
Sickle cell β thalassaemia

This disorder occurs when one β gene carries HbS mutation and the other gene carries
β thalassaemia mutation

Sickle-cell β0 thalassaemia (genotype βS β0 ) in which normal β chain synthesis is
completely lacking, and sickle-cell β+ thalassaemia (genotype βS β+ ) in which
normal β chain synthesis is partially deficient.

Clinical manifestations resemble sickle-cell anaemia except splenomegaly that
persists into adult life.

Peripheral blood smear shows features of both sickle-cell anaemia and thalassaemia
such as sickle cells, microcytic and hypochromic cells, and target cells.

MCV and MCH are decreased.

On haemoglobin electrophoresis, HbS is the predominant haemoglobin (70–80%),
HbA is absent, and HbA2 (3–5%), and HbF (10–20%) are elevated

Diagnosis is confirmed by demonstrating that one parent has sickle-cell trait and the
other has β thalassaemia trait(double heterozygous state)
Sickle-cell Hb-C disease

Sickle-cell Hb-C disease results from the inheritance of the Hb-S gene from one
parent and the HbC gene from the other.

resembles homozygous sickle-cell disease clinically, it is less severe.

Growth, body habitus, and sexual development are norntal. Most patients have
painful crises and attacks of acute febrile pulmonary disease, but they are
usually- well between the crises, and the disease is compatible with longevity.

Eye complications are often a prominent feature.

Pregnancy occurs more frequently than in homozygous disease, but is almost
as hazardous for mother and child as in the latter condition

Thrombo-embolic episodes and haematuria are particularly common.

Splenomegaly is seen

The patients are usually only mildly anaemic or may have a normal hemoglobin
level. Numerous target cells are seen on the blood film, but irreversibly sickled
cells are often not present. MCV and MCH are usually mildly reduced, and the
reticulocyte count mildly elevated
Sickle-cell Hb-D disease

Sickle-cell Hb-D disease is rare; it results from the inheritance of the Hb-S gene
from one parent and the Hb-D gene from the other.

Clinically, it resembles homozygous sickle-cell disease, but is less severe and
the patients are mildly anaemic.

Most have a normal habitus and lead a relatively normal life with only very
occasional painful crises.

Numerous target cells are seen on the blood film.

The electrophoretic pattern may be confused with that of homozygous sicklecell disease
Neonatal Screening for Sickle-cell Anaemia

Screening can be carried out to identify those newborns who will later develop
sickle cell anemia.

preventive measures can be taken to avert serious complications and reduce
morbidity and mortality in later life.

Widely used test for this purpose is citrate agar gel electrophoresis at acid pH.
Haemolysate from cord blood sample is used.

Newborns who will develop sickle-cell anaemia show predominance of HbF,
some HbS and absent HbA; those with sickle-cell trait have HbF, HbS, and HbA
Prenatal Diagnosis

Mothers from high-risk ethnic group should be screened in early pregnancy for
HbS carrier state.

If prospective mother as well as father are positive, they should be offered the
option of prenatal diagnosis or of newborn screening.

Foetal blood analysis:This involves globin chain synthesis studies in foetal
blood using CM-cellulose chromatography. Abnormal globin chain is separated
from normal globin chain and quantitated. Foetal blood sampling (by
cordocentesis) can only be done after 18 weeks of gestation. Apart from
prolonged waiting period, risk of procedure-related foetal loss is also
comparatively greater.
Prenatal Diagnosis

Foetal DNA analysis: Foetal DNA may be obtained either from amniotic fluid
cells or from chorionic villi (see prenatal diagnosis of thalassaemias). Chorionic
villus biopsy is preferred because, if required, termination of pregnancy can be
performed earlier. Tests preformed are

Southern blot analysis

Restriction fragment length polymorphism (RFLP) analysis

Methods employing DNA amplification

Direct detection of mutation with restriction enzymes

Allele-specific oligonucleotide probe analysis

Colour DNA amplification
Treatment

Treatment of sickle-cell anaemia is symptomatic and supportive.

Hydroxyurea: A mainstay of treatment in sickle-cell anaemia is hydroxyurea
(now called hydroxycarbamide). It has following benefits in sickle-cell anaemia:
(a)
it increases production of HbF and reduces number and severity of crises
(b)
it reduces white cell count thus causing anti-inflammatory effect
(c)
it increases red cell volume and hydration thus reducing sickling and haemolysis
(d)
it decreases adhesiveness of red cells and leucocytes
(e)
it releases nitric oxide that causes vasodilatation.
Blood transfusion in sickle-cell disease

Packed red cell transfusion (10–15 ml/kg) is indicated in—
– Symptomatic anaemia
– Aplastic crisis
– Splenic sequestration crisis
– Before surgery

Exchange transfusion is indicated in—
– Impending stroke
– Acute chest syndrome

Chronic transfusion is indicated for –
– Prevention of recurrence of stroke

Haematopoietic stem cell transplantation is the only form of therapy that can
cure the disease. Since it is associated with significant morbidity and mortality,
it should be reserved for severely affected patients having HLA- matched
sibling donor.

Penicillin prophylaxis

Vaccination against S. pneumoniae, H. influenzae, influenza virus, and
hepatitis B virus


Folate supplementation
Iron chelation (if iron overload)
Thankyou
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