Sickle cell Anemia

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SICKLE CELL
ANEMIA
Notes on sickle cell anemia…By Dr. Ashish V. Jawarkar M.D.
Contact – pathologybasics@gmail.com Website – www.pathologybasics.wix.com/notes

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OVERVIEW
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Pathology
Types
Distribution
Pathogenesis
Factors that influence sickling
a. concentration of HbS
b. Other types of Hb
c. MCHC
d. Oxygen tension
e. Temperature
f. Low pH
Clinical features (sickle cell anemia)
Lab diagnosis
a. Red cell indices
b. P.S.
c. ESR
d. Bone marrow
e. Sickling test
f. Hb electrophoresis
g. Solubility test
h. Neonatal screening
i. Prenatal screening
Principles of therapy
Sickle cell trait


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* PATHOLOGY
Caused by mutation in beta globin gene – at sixth position, glutamic acid is replaced by
valine

* TYPES
1. Sickle cell anemia
Homozygous state for HbS (βs βs)
>70% Hb is HbS
2. Sickle cell trait
Heterozygous carrier state for HbS (βs β)
25-40% of Hb is Hbs
3. Sickle cell – β thalessemia
Double heterozygote in which sickle cell gene is inherited from one parent and beta thal
gene from other parent
(βs β0) or (βs β+)
4. Combination of HbS with other abnormal hemoglobin
(HbSD, HbSC, HbSO (arab disease), HbSE disease)

* DISTRIBUTION
1. Prevalent in Africa, middle east and Central and south India
2. High prevalence in areas of high malaria endemicity
Sickle cell is said to provide protection against P. falciparum – P. falciparum infested
RBCs are rapidly phagocytosed and destroyed due to rapid sickling
The theory of balanced polymorphism – Because sickle cell provides protection
against malaria, sickle cell genes are preferentially selected over normal genes in
endemic areas, giving a high prevalence of sickle cell patients.


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* PATHOGENESIS
Red cells with HbS
Passage through microcirculation in spleen
Low O2 tension
Arrangement of fibres along the long axis of the red cell
Delay time/ lag time
Sickling of the cell on formation of a polymer of critical size – k/a nucleation phase

Cells passing through tissues with good O2 tension
Desickling

Repeated cycles of sickling and desickling
Irreversibly sickled red cell

Lyse by themselves in
Circulation

macrophage phagocytosis
in spleen

Intravascular hemolysis
vascular stasis

adherence to
endothelium

extravascular hemolysis

Platelet activn

Chronic hemolytic anemia

Autosplenectomy

Spleen

in hands and feet, head of femur
And renal papillae

Hyposplenism
(decreased fn of spleen)

Infection by capsulated
org like pneumococcus

Vasculr occlusion

Damage and necrosis of various
organs
Dactylitis, necrosis of femoral head
Renal papillary necrosis

Typhoid
Osteomyelitis


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* FACTORS THAT INFLUENCE SICKLING
(i)

INTRACELLULAR CONCENTRATION OF HbS
Sickle cell trait patients have less concentration of HbS and so there is less tendency
to sickle.
Sickle Cell anemia patients on the other hand have greater tendency of their red cells
to sickle

(ii)

OTHER TYPES OF Hb
HbF does not participate in sickling
Hence infants, heterozygotes for HbF and Hereditary persistence of fetal hemoglobin
patients donot have significant sickling.

(iii)

MCHC
Increased MCHC due to cellular dehydration favours increased contact between HbS
strands and favours sickling.
This factor is responsible for sickling in renal medulla (hyperosmolar mileu causes
dehydration)

(iv)

OXYGEN TENSION
HbSS cell sickle at PO2 – 40 mm Hg
HbAS cell sickle at PO2 – 15 mm Hg

(v)

TEMPERATURE
Cold induces vasoconstriction and may cause sickling episodes

(vi)

LOW Ph
Acidosis increases sickling


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* CLINICAL FEATURES
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Symptoms start around 3-4 months of age as the level of HbF falls
Anemia of variable degree is present, more in sickle cell anemia, less in sickle cell trait
Splenomegaly in infants and young children
: due to RE system hyperplasia
Splenectomy later in life
: due to autoinfarction
Increased risk of infection due to capsulated organism like s. pneumonia and H.
influenzae – due to impairment of phagocytic function
Salmonella osteomylitis
Jaundice and hepatomegaly
Signs of vasoocclusive events
a. Recurrent leg ulcers
b. Dactylitis
c. Acute abdominal pain due to visceral infarcts
d. Renal papillary necrosis
e. Priapism
f. Pulmonary infarction and acute chest syndrome
g. Sickle retinopathy – salmon patches due to intra retinal hemorrhages
h. Spontaneous abortions
Crises in sickle cell anemia
a. Sickling crises
Pain in abdomen, bone pain, chest pain and stroke
Precipitated by fever, dehydration and infections like malaria
b. Hemolytic crises
Sudden lowering of Hb
Reticulocytosis
Increased bilirubin (indirect)
Sudden increase in rate of hemolysis
c. Aplastic crises
Due to infection with Parvovirus B19
Bone marrow shows giant proerythroblasts with intranuclear inclusions
d. Sequestration crises
Seen in sickle β thal or HbSC disease
Massive sequestration of sickled red cells in spleen
Decreased blood volume
Shock


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* LABORATORY DIAGNOSIS
1. RBC Indices
Hb – 6-9 gm/dL
TLC on counter – elevated with lymphocytosis
Actually count is falsely elevated due to normoblasts in peripheral blood
Platelet count may be elevated – splenic trapping is lacking
2. Peripheral Smear
a. moderate to severe anisopoikilocytosis
b. Normocytic, normochromic anemia
c. 5-10 % cells may be irreversibly sickled
d. Polychromasia
e. Few RBCs demonstrate Howell Jolly bodies
3. Retic count
Reticulocytosis 3-10%
4. ESR
Is low – because sickled cells fail to rouleaux
5. Bone marrow
a. erythroid hyperplasia
b. Normoblastic reaction
c. Iron stores are increased
d. Myelopoeisis and megakaryopoeisis are normal
6. Laboratory tests

a. Sickling test
Sickling can be demonstrated by use of a reducing agent like 2% sodium
metabisulfite or sodium dithionite
Principle
Sodium metabisulphite reduces the oxygen tension inducing the typical sickle-shape
of red blood cells.
Sample
Fresh blood in any anticoagulant.
Reagents
0.2 g of sodium metabisulphite in 10 ml of distilled water.
Stir until dissolved. Prepare fresh each time.
Method
1. Mix 1 drop of blood with 1 drop of 2% sodium metabisulphite solution on a
microscope slide.


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2. Cover with a cover slip and seal the edge with wax/vaseline mixture or with nail
varnish. Allow to stand at room temperature for 1 to 4 hours.
3. Examine under a microscope with the dry objective.
Interpretation
In positive samples the typical sickle-shaped red blood cells will appear.
Occasionally the preparation may need to stand for up to 24oC. In this case put the
slides in a moist Petri dish.
False negative results may be obtained if the metabilsulphite has deteriorated or if the
cover slip is not sealed properly.
A positive test does not distinguish the sickle cell trait from sickle cell disease. It is
important to examine the preparation carefully and in particular near the edge of
cover slip.
adding Sodium metabisulfite

Before

after

Limitations
1. does not differentiate between sickle cell anemia and sickle cell trait
2. cannot be used for mass screening (microscopy is needed)


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b. Hb electrophoresis (only diagnostic test)

1. Variant haemoglobins such as haemoglobin S often differ in surface charge from
each other and from normal adult haemoglobin, haemoglobin A. They can thus
be differentiated from each other by electrophoresis on a starch block or cellulose
acetate paper.
2. This slide shows the results of haemoglobin electrophoresis of the blood from a
normal adult, a patient with sickle cell anaemia (SS) and people with sickle cell
trait (AS) and haemoglobin C trait (AC). Haemoglobin C is another variant
haemoglobin which, like haemoglobin S, is fairly common in people originating
in West Africa or the Caribbean.
Sickle cell anemia
HbS : 80-95 %
HbF : 5-15%
HbA2: Normal

Sickle cell trait
HbS : 60%
HbA : 40%


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c. Solubility test (screening test)
PRINCIPLE
1. Test is based on the solubility difference between Hb S and Hb A in concentrated
phosphate buffer solution. Red blood cells under test are lysed by a powerful
hemolytic agent and the released hemoglobin is then reduced by sodium
dithionite in a concentrated phosphate buffer.
2. In the presence of Sodium Dithionite, Hb S precipitates causing turbidity of the
reaction mixture. Under the same conditions, Hb A, as well as most other
hemoglobins, are soluble.
3. When subjected to a centrifugal force the precipitated hemoglobin (Hb S) forms a
red precipitate on top layer leaving the lower solution clear and colourless. The
soluble hemoglobin (Hb A) gives a clear red lower solution with a grey
precipitate on the top layer and most HbAS which contains both precipitated and
soluble hemoglobin gives a red precipitate ring on top layer with a light red to
pink colour lower solution.
INTERPRETATION

d. HPLC (high performance liquid chromatography)

Result : Hemoglobin variant with retention time in S window


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7. Neonatal screening
Done to identify babies at risk so that they donot develop complications
Tests done:
Citrate agar electrophoresis
Will develop sickle cell anemia
HbF
HbS
No HbA

Will develop sickle cell trait
HbF
HbS
HbA

Normal
HbF
HbA

8. Prenatal screening
If parents are sickle cell trait, Chorionic villous sampling can be done at 10-12 weeks to
see for sickle cell mutations


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* PRINCIPLES OF THERAPY
1. Prevent crises
a. Treat infections
b. prevent exposure to cold, stress, hypoxia and dehydration
2. Treatment of vasoocclusive episodes
a. analgesics
b. keep warm
c. fluid intake maintainence
d. oxygenation
e. partial exchange transfusion
3. Transfusion therapy
a. PCV transfusion
During aplastic crises
Acute splenic sequestration
b. Regular chronic transfusion
Keep sickle cells to <40%
Prevent cerebrovascular accidents
c. Partial exchange transfusion
Vasoocclusive episodes
d. Folic acid
e. Hydroxyurea
Increases HbF, decreases crises episodes
4. To avoid complications
a. Pneumococcal vaccine
b. Avoid OC pills
c. Avoid radiographic contrast media


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* SICKLE CELL TRAIT
1. Asymptomatic heterozygous state (βs β0)
2. HbS – 25-40%, HbA 65-70%
3. Hb 11-13 gm/dL
Clinical features
1. asymptomatic
2. vasoocclusive episodes may occur at high altitudes
3. Papillary necrosis may occur with proteinuria and hematuria
Peripheral smear
1. normocytic normochromic
confirmed on
1. solubility test
2. sickling test
3. Hb electrophoresis
Treatment
Survival is normal, no treatment required


Sickle cell Anemia

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