to specimen collection by approved techniques. Collect whole blood in EDTA, heparin, sodium citrate or ACD anticoagulant. Though fresh blood samples are preferable,
forms, draw 4 mL of blood into a syringe containing 1 mL
of 10% formalin, examine wet preparation.
Acceleration of removal of oxygen from hemoglobin by
Sodium bisulfite or ascorbic acid, 2 g, in 100 mL water (to
Use capillary blood, oxalated or defibrinated. Mix 2 drops
of either reagent with one drop of blood on a glass slide.
Cover, and seal coverslip with petrolatum or nail polish. If
no sickling appears in 4 hours, the test is negative. Positive
test: 10% or more red cells show sickled forms.
The red cells are lysed, hemoglobin deoxygenated,
and the b-globin chains are displaced to provide the
molecular steric fit characteristic of sickling of S and no S
hemoglobins. The test solution becomes turbid if sickling
Add 20 μL (0.02 mL) of well mixed whole blood EDTA
anticoagulated) to 2 mL dithionite working solution in a
12 × 75 mm test tube. Mix by inverting and allow to remain
at room temperature for 56 minutes. Examine for opacity by
holding the tube 2.5–3 cm from a sheet of black newsprint.
Turbidity (newsprint not visible) indicates the presence of
sickling hemoglobin. Clear solution indicates a negative
test. A positive test (turbidity) indicates the presence
of any of the hemoglobins S (SS or AS), C (Harlem), C
(Georgetown), Barts and perhaps Alexandra. All positive
tests should be followed by hemoglobin electrophoresis.
Hemoglobin S can be separated from other hemoglobins
To 50 mL of working solution (above: from previous test),
add 6 g of urea. Mix until dissolved.
Add 20 μL (0.02 mL) whole blood to 2 mL of urea dithionite
solution in a 12 × 75 mm test tube. Mix by inverting tube
and allow to remain at room temperature for 5 minutes.
Read with tube 2.5–3 cm away from newsprint background.
Turbidity produced by the sickled liquid crystal system
Hemoglobin Dithionite Urea-dithionite
Refer to Table given under HbF Estimation for
Percentage of HbS in Various Disorders
The presence of HbA guards, to a modest extent, against
tactoid (polymerization) formation, HbF exerts a very
strong protective effect. It is probable that the cells of
patients with sickle cell disease undergo intravascular
sickling and unsickling many times as they circulate.
Repeated shape changes, however, stresses the cell
membrane and causes the loss of small membrane
fragments. This reduces the ratio of surface to volume,
making the cell less flexible and less responsive to physical
changes. Some cells gradually lose their mechanical and
osmotic resistance and undergo intravascular dissolution,
whereas others are removed early by reticuloendothelial
system. Red cells in HbS disease experience a chronically
The likelihood of sickling increases with low oxygen
tensions, lowered pH and increased body temperature.
HbS protects against Plasmodium falciparum infection.
Sickle cell anemia rarely becomes clinically apparent
before 6 months of age because protective amounts of HbF
remain in cell; for the same reason, it is difficult to screen
(Courtesy: Tulip Group of Companies)
Hemoglobin S (HbS) differs from the normal hemoglobin
A (HbA) by a single amino acid mutation at position 6 of
the beta chain, wherein glutamic acid is replaced by valine.
During low oxygen conditions, the RBC morphology
may range from mild elongation to irreversible elongated
tactoid. This elongated filamentous tactoid formation
results in the typical “sickle” appearance of the RBC.
Individuals with sickle cell anemia (homozygous S/S)
may have early mortality with vascular occlusions of
multiple organ systems, severe hemolytic anemia and
Individuals with the sickle cell trait (heterozygous
A/S) are usually asymptomatic. However, under certain
conditions of reduced oxygen tension such as hypoxia
during anesthesia, fight in poorly pressurized airplanes,
severe pneumonia, they can experience a sickle cell crisis.
a. Sicklevue reaction tubes: Prefilled with sodium
b. Phosphate buffer: A concentrated, ready-to-use
solution containing a red cell lysing agent.
Store the reagents at RT (15–30°C). Do not refrigerate or
Do not expose to light for excessive periods. Best stored
The shelf-life of the reagents is as per the expiry date
mentioned on the Sicklevue carton.
Sicklevue is based on the solubility difference between
HbS and HbA in concentrated phosphate buffer solution.
RBCs under test are lysed by a powerful hemolytic agent
and the released hemoglobin is then reduced by sodium
dithionite in a concentrated phosphate buffer.
In the presence of sodium dithionite, HbS precipitates
causing turbidity of the reaction mixture. Under the same
conditions, HbA, as well as most other hemoglobins, are
HbS when reduced in the phosphate buffer forms a
turbid solution, which is easily visualized. Normal HbA
and most other hemoglobins remain in solution resulting
1. Reagent for laboratory use only.
3. Phosphate buffer does not contain preservatives.
Aseptic conditions should be followed to avoid
contamination. However, as a powerful hemolytic
agent is included in the composition, avoid contact
with skin or mucosa. Wash hands after use.
4. The reagent can be damaged due to microbial
contamination or on exposure to extreme temperatures.
5. Use reagent of same lot numbers. Do not interchange
reagent of different lot numbers.
Sample Collection and Preparation
No special preparation of the patient is necessary prior
to specimen collection by approved techniques. Collect
whole blood in EDTA, heparin, sodium citrate or ACD
anticoagulant. Though fresh blood samples are preferable,
the sample can be stored at 2–8°C for up to 24 hours, in
Materials Provided with the Kit
Sicklevue reaction tubes, phosphate buffer and result
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