sickle Cell Disease
With High concentration of phosphate buffer, it give hemoglobin S (Hb S) insoluble in the reduce state.
When in the buffer, it form a shape like water crystal. The crystal refract and deflect light ray, as such yield a turbid solution. With this process allowed solubility test to be carried out.
Reagent for Hb S solubility Test
The test for Hb S solubility test have 3 reagent, the reagent and it production are discuss below;
Stock of phosphate buffer 2.58M
- Distilled water 1litre
- K2HPO4 66g
- KH2PO4 164g
Dissolved K2HPO4 and KH2PO4 in a 500ml distilled water, and make up the solution to 1litre with distilled water, monitor the solution pH and make sure the pH did not exceed 6.5 or below 6.5.
High molarity buffer 2.24M
- Stock buffer solution 434ml
- Distilled water 500ml
Measure 434ml of produce stock solution and dilute it with 500ml of distilled water, mix the solution very and stopper.
Low molarity buffer 1.1M
- Stock buffer solution 213ml
- Distilled water 800ml
Measure 213ml of stock buffer solution produced, and dilute it with 800ml of distilled water, mix the solution very well and stopper.
Note: buffer solution can be store at room temperature and be used, as long as the solution did not have any change of colour or contamination.
Also test kit for the test is now available with brand name; Sickledex, Sickle prep.
- 12 x 75mm test tube
- 3ml Pipette
- Carbon tetrachloride (CCL4)
- Pasteur pipette
- Sodium dithionite powder
- Label 12 x 75ml test tube for two
- Mark one test tube low molarity and the second test tube high molarity
- Pipette 1ml of low molarity buffer into a test tube mark low molarity
- Pipette 1ml of high molarity buffer into a test tube mark high molarity
- Using a disposable Pasteur Pipette CCL4 hemolysate, drop two drop into low molarity buffer tube, and two drop into high molarity buffer tube.
- Label the Pasteur pipette
- Mix each of the test tube very well.
- Add about 20 to 20mg of sodium dithionate powder to first test tube and to the second test tube.
- Mix the solution a read your result immediately.
Make sure to run positive and negative control via the same step given above.
Interpret your result
When precipitate is form on the two test tube, then Hemoglobin S is present in that sample. Non present hemoglobin S sample must be free of any kind of precipitate.
But in case of cellulose acetate electrophoresis show a band in the Hb S position, the solubility test should read as shown in the following table;
|Low Molarity||High Molarity||Result|
|No precipitate||Precipitate||Hb S|
|Non precipitate||No precipitate||Hb D or G|
Poor reagent or specimen not concluded
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