Sickling test and Hb solubility test

Principle

• Deoxygenated HbS polymerises. In the sickling (slide) test a reducing agent, 2% sodium metabisulfite, removes oxygen so HbS-containing cells sickle.
• In the solubility test, saponin lyses cells and sodium dithionite reduces Hb in a concentrated phosphate buffer; reduced HbS is insoluble and makes the solution turbid.

Specimen

• EDTA blood (heparin or citrate acceptable for most kits).

Procedure

• Sickling: mix one drop of blood with one to two drops of freshly prepared 2% sodium metabisulfite on a slide, apply a coverslip and seal with petroleum jelly or paraffin. Examine at about 1 h; if negative, re-examine up to 24 h.
• Solubility: add the stated volume of blood (commonly 20 µL) to 2 mL of reagent, mix and stand for the time stated by the kit (commonly 5–6 min); view against a white card with black lines.

Results

• Sickling test positive: sickle or holly-leaf shaped cells.
• Solubility positive: turbid solution, lines not visible. Negative: clear pink-red solution, lines visible.

Quality control and pitfalls

• Run known positive (AS) and negative (AA) controls with each batch; metabisulfite must be fresh, dithionite reagent within expiry.
• Neither test distinguishes sickle cell trait (AS) from disease (SS) or compound states (SC, S-beta thalassaemia); confirm with HPLC or electrophoresis.
• False negatives: infants under about 6 months (high HbF), Hb below about 7 g/dL, recent transfusion, outdated reagent.
• False positives (solubility): hyperlipidaemia, high plasma proteins, very high WBC, high Hct, and rare sickling variants (e.g. HbC-Harlem).

Clinical use

• Rapid screening for HbS before surgery, in antenatal and population screening, and in emergency settings; not a definitive diagnostic test.