Protocol:Hb S detection - sickling and solubility tests

Sickling test
Carrying out the sickling test is part of the diagnostic work up in patients suspected of having a sickle cell syndrome. The test should be also carried out if there is an abnormal electrophoretic or chromatographic haemoglobin fraction in the position of Hb S (eg Hb D or G, see table below). Many tests have been described and several commercial kits are available. Described here is a simple method that has proved very reliable and simple to carry out.

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.
 * 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.



Solubility test
Hb S is quite insoluble when in the reduced state in high phosphate buffer solution. It forms tactoids (water crystals) which refract and deflect light rays and produce a turbid solution, permitting a solubility test (19).

Reagents

 * 1) Stock 2.58 M phosphate buffer:
 * Dissolve 239.66 g K2HPO4 and 164 g KH2PO4 in distilled water; then make up to 1 Litre with distilled water. The pH should be 6.5.
 * 1) High molarity Buffer (2.24 M):
 * Dilute 434 ml of stock buffer above to 500 ml distilled water. Mix and stopper.
 * 1) Low molarity Buffer (1.1 M):
 * Dilute 213 ml of stock buffer above to 500 ml distilled water. Mix and stopper.

Buffers may be kept at room temperature and used as long as they are clear and uncontaminated. Kits for this test are commercially available (eg. Sickledex, Sickle prep)

Method

 * 1) Label two small (12 x 75 mm) test tubes for each patient. Mark one low molarity and one high molarity.
 * 2) Pipette 1 ml of high molarity buffer (2.24 M) into its properly labelled tube and 1 ml of low molarity buffer into other labelled tube.
 * 3) Add two drops CCl4 haemolysate into each tube with a disposable, labelled Pasteur pipette. Mix.
 * 4) Add a pinch (about 10-20 mg) of sodium dithionite powder to all tubes. Mix and read immediately.
 * 5) Run positive and negative control bloods by following the same steps given above.

Interpretation
Haemoglobin S is present if a precipitate forms in the tube labelled “high molarity.” “Low molarity” tubes must all be negative for precipitate. When cellulose acetate electrophoresis shows a band in the HbS position, interpret the solubility test as follows:

False positive results may be due to polycythaemic blood and a variety of abnormal haemoglobins including Hb’s I, Bart’s, C-Georgetown, Alexandra and C-Harlem.

Positive test should be confirmed by haemoglobin fractionation. High concentration of HbF may inhibit the reaction. The solubility test is unlikely to be reliably positive result until after 6 months of age.

Most common haemoglobin variants with S-like mobility after electrophoresis at alkaline pH.