Principle
Sickle cell hemoglobin is insoluble in the deoxygenated state in a high molarity phosphate buffer.The crystals that form refract light and cause the solution to be turbid.
Reagents
Phosphate buffer
Sodium dithionate powder
Normal saline
Preparation of Phosphate buffer
K2HPO4-250gm
KH2PO4-143.5gm
Saponin-2gm
Benzoic Acid-2.5 gm
Distilled water-1 litre

Note This reagent buffer is stable for 6-7 days at room temperature and for 1 month at 4C.Sodium dithionate powder should be kept in air tight container at room temperature.
-Required blood collection-2 ml in EDTA bulb
-Preparation of packed cell
-Fill the small test tube with approximately 2-3 ml Normal saline
-Add 2-4 drops of well mixed whole blood from EDTA tube.
-Mix thoroughly and centrifuge it at 3000 rpm for 2 minutes\\. -Discard the supernant and add 2-3 ml Normal saline again for cell washing.
-Repeat cell washing three times and after third washing discard supernatant.

Method
-Take a pinch of sodium dithionate powder in three test tube.
-Add approximately 1 ml of phosphate buffer and mix well till sodium dithionate powder is dissolved.
-Add approximately 10-20µl of three times washed red blood cell of patients in first test tube,10-20µl of three times washed red blood cells from a known sickle cell disease subject as a positive control to the second tube and 10-20µl of three times washed red blood cells from a normal subject as a negative control to the final tube and shake gently.
-Allow it to stand for 5 minutes.
Interpretation of solubility/DTT Test Results
Positive control Negative control

-The colour will be pinkish violet.
-Hold tube 2.5 cm in front of a white card with narrow black lines and read for turbidity,in comparison with the positive and negative control samples.
-If Turbidity seen-solubility test is positive-black lines do not appear clearly through tubes.
-If Turbidity not seen-solubility test is negative-black lines appear clearly though tubes.
-If tubes are allowed to stand for more than half an hour or centrifuge at 1200 rpm for 5 minutes ,a ring formed by insoluble Hb-S is seen on the surface of the test tube.

Interferences
-False positive results have been reported in severe leucocytosis, hyperparaproteinaemia and in the presence of a unstable hemoglobin ,especially after splenectomy.
-False negative results can occur in patients with a low Hb,old or outdated reagents are used and if the dithionate/buffer mixture is not freshly made,infant under the age of 6 months and in case of post transfusion,when the Hb S level is under 20%.

References
-DACIE AND LEWIS PRACTICAL HAEMATOLOGY(NINTH EDITION)