Sickle cell anemia is suggested when the abnormal sickle-shaped cells in the blood are identified under a microscope. Testing is typically performed on a smear of blood using a special low-oxygen preparation.
This is referred to as a sickle prep. Other prep tests can also be used to detect the abnormal hemoglobin S, including solubility tests performed on tubes of blood solutions. The disease can be confirmed by specifically quantifying the types of hemoglobin present using a hemoglobin electrophoresis test.
Prenatal diagnosis (before birth) of sickle cell anemia is possible using amniocentesis or chorionic villus sampling. The sample obtained is then tested for DNA analysis of the fetal cells. The hemoglobin electrophoresis test precisely identifies the hemoglobins in the blood by separating them. The separation of the different hemoglobins is possible because of the unique electrical charges they each have on their protein surfaces, causing them each to move characteristically in an electrical field as tested in the laboratory.
Some of the Investigations carried out in the Haematological Laboratory includes:
- FBC and blood film: the haemoglobin level is in the range 6-8 g/dL with a high reticulocyte count of 10-20%; the blood films may show sickled erythrocytes and features of hyposplenism.
- Sickling of red cells on a blood film with 2% sodium metabisulphite.
TITLE: Sickling test
AIM: To Screen for an abnormal type of. haemoglobin called Hb S in patients’ blood sample
PRINCIPLE: Blood is mixed on a slide with a chemical reducing agent such as sodium metabisulphite or sodium dithionite, covered with a cover glass, and incubated at room temperature for up to 1hour or more. The reducing agent deoxygenates the haemoglobin in the red cells providing the conditions for cells containing Hb S to sickle
SAMPLE: Fresh blood in any anticoagulant
REAGENTS: 0.2g of sodium metabisulphite in 10ml of distilled water, stir until dissolved, prepare fresh each time
1. Deliver 1 drop of patients capillary blood or well mixed venous blood on a slide, add an equal volume of fresh reducing reagent, mix and cover with a cover glass. Exclude any air bubbles
Note: if patient is severely anaemic, use 2 drops of blood or preferably use a plasma reduced blood sample
2. Set up a negative control: deliver one drop of blood from a person that does not have sickle cell disorder on a slide marked 'Neg control' Add an equal volume of fresh reducing reagent and mix. Cover with a cover glass. Exclue any air bubbles
Note: if a blood from a known sickle cell trait person is available, set up also a positive control
3. Place the slides in a container or petri dish, with a damp piece of blottin paper or tissue in the bottom to prevent drying of the preparations. Close the container and leave at room temperature
4: After 10-20 Minutes,examine the patients preparation microscopically for sickle cells. Focus the cells first with X10 objective and examine for sickling using X40 objective.
Examine several parts of the preparation. Sickling often occurs quicker in one area than the other. The sickle cells may appear crescent shaped with pointed ends or holly leaf shaped especially in sickle cell trait
5: Report the patient’s preparation as Sickle cell test positive or Sickle cell test Negative
Note: the -ve control preparation should show no sickle cells
Interpretation of test result on SICKLE CELL DISEASE
A positive sickle cell test indicates that a person's red cells contain HbS. A stained blood film and clinical history may enable sickle cell disease to be differentiated from sickle cell trait. Blood should be sent for haemoglobin electrophoresis to confirm the disorder.
The test is not sufficiently sensitive to detect small amounts of HbS which may be present in the blood of infants below 6 months.
Haemoglobin S Solubility Filtration test
Sickle solubility test: a mixture of HbS in a reducing solution such as sodium dithionite gives a turbid appearance because of precipitation of HbS, whereas normal haemoglobin gives a clear solution.
TITLE: Solubility filtration test
AIM: To differentiate Sickle cell anaemia from sickle cell disease in patients’ blood sample.
PRINCIPLE: Blood is mixed in a phosphate buffer-saponin solution containing sodium dithionite and filtered. HbSS is indicated by a red precipitate on a filter paper with a pale yellow filtrate. Other forms of haemoglobin are soluble when in a reduced state.
- Phosphate buffer saponin PH 7.1 Reagent No.67. Store at 2-8 degree celsius. Renew every 3months or if it becomes turbid.
- Sodium dithionite powder.
To make working reagent:
- Measure 20ml of buffer-saponin solution.
- Add 0.2g sodium dithionite and mix gently until the chemical is dissolved
Note: the working reagent is not sable,it can be used only on the day it is prepared.
PROCEDURE: set up with the test, a negative control (HbAA) and a positive control using blood from a person with sickle cell trait (HbAS)
1. Pipette 2ml of working reagent into a test tube approximately 13 x 77 mm.
2. Wash in 0.1ml of capillary blood or well mixed venous blood.
Note: when the haemoglobin is below 70g/l (7g/dl), use twice the volume of blood or if a venous blood sample,use plasma reduced blood.
3. Mix well and filter through a small (5.5 cm diameter) No.1 filter paper
4. Note the colour of the solution (pale yellow,pink-red,or dark red) and whether there is any red precipitate (insoluble reduced HbS) on the filter paper.
Results after Filteration
HbSS.....Clear pale yellow filtrate. Abundant red precipitate on filter paper
HbAS.......Clear pink filtrate. Small amount of red precipitates on filter paper
HbAA (normal).......Dark red fluid (soluble reduced Hb) with no precipitate on filter paper
5. Report the HbS solubility filtration test as:
- Positive for sickle cell anaemia when result shows HbSS appearance
- Positive for sickle cell haemoglobin when result shows HbAS appearance
- Negative for HbS when result shows HbAA appearance
Note: confirm the test results by haemoglobin electrophoresis and also examine a Romanowski stained blood film
Haemoglobin analysis, eg by electrophoresis, is always needed to confirm the diagnosis. There is no HbA, 80-95% HbSS, and 2-20% HbF.
Sickle cell trait is diagnosed by the finding of a positive sickling test together with haemoglobins A and S on electrophoresis.
TITLE: Haemoglobin electrophoresis
AIM: To separate the normal and abnormal types of haemoglobin in patients’ blood sample
PRINCIPLE: In an alkaline PH (btw 8.4 - 8.6) Hb is negatively charged, and then on exposure to electrical field, due to their negativity, they migrate to the positive side of the electric field (cathode) and as they move, some move faster, and with these, you can determine the haemoglobin pattern of such individual.
1. Electrophoresis chamber with power pack
2. Cellulose acetate medium
3. Tris buffer, PH 8.4
Boric acid. 3.2g
Distilled water 1 litre
4. Ponceau stain (0.5%)
Ponceau S. 0.5g
Trichloroacetic acid. 5.0g
Distilled water. 100ml
5. Destaining and clearing agents
5% acetic acid
20% acetic acid in absolute methyl alcohol
6. Normal saline
7. Abnormal haemoglobin controls specimens
Specimen: EDTA-anticoagulated blood
STEP 1: preparation of haemolysate
i. Centrifuge the anticoagulated blood at. 2500rpm for 5minutes.
ii. Remove the plasma and wash the packed cells with large volumes of saline 3 times
iii. After the final washing, lyse the red cells by adding an equal volume of distilled water, one-quarter volume of toluene and one drop of 3% potassium cyanide. Mix by inversion and centrifuge to remove cell debris
iv. Transfer the haemolysate to a clean tube.
STEP 2: electrophoresis
i. Pour the buffer into the electrophoresis chamber, soak the wicks and position them. Pre-soak the cellulose acetate plate for 20-30minutes in the buffer, remove excess buffer by keeping the plate between absorbent papers.
ii. Using an applicator, apply 0.5 - 0.6ml of the specimen approximately 3cm from the cathode. Also, apply at least 2 abnormal controls on each plate
iii. Place the plate in the electrophoresis chamber, place a microscope slide over it
iv. Run the electrophoresis at 450volts for 20minutes
v. Remove the cellulose acetate plate and stain with ponceau S for 3minutes
vi. Wash in three changes of 5% acetic acid
- Fix in absolute methyl alcohol for 5minutes.
viii. Clear in 20% acetic acid in absolute methyl alcohol for 10minutes
ix. Dry in oven at 65-degree celsius for 10minutes
x. Scan the cellulose acetate plate with a scanning densitometer.
Compare a relative mobility of abnormal haemoglobin S in the control samples with those of the test sample, both visually and with scanning densitometer. Identify the abnormal Hbs present in the test sample.
Other investigations such as renal function tests, LFTs and lung function tests should also be performed at diagnosis (baseline) and routine monitoring. Other investigations will depend on any complications, eg infection screen, abdominal ultrasound, CT scan of the head (eg if a subarachnoid haemorrhage is suspected)