Osmotic fragility test

Principle

• Red cells placed in hypotonic saline absorb water and swell; they lyse when their capacity to swell is exceeded.
• Spherocytes have a reduced surface area to volume ratio and lyse at higher saline concentrations (increased fragility). Thin flat cells lyse at lower concentrations (decreased fragility).

Specimen

• Heparinised or defibrinated venous blood; test fresh (within a few hours) and after incubation at 37 °C for 24 h.
• Always test a normal control sample in parallel.

Reagents and equipment

• Buffered sodium chloride series, typically 0.9% down to 0.1% (0.9, 0.75, 0.65, 0.6, 0.55, 0.5, 0.45, 0.4, 0.35, 0.3, 0.2, 0.1 g/dL) plus distilled water.

Procedure

• Add a fixed small volume of blood (e.g. 50 µL) to 5 mL of each saline tube; mix gently.
• Stand at room temperature for 30 min, remix, then centrifuge.
• Read supernatant absorbance at 540 nm; 0.9% is the blank (0% lysis) and the lowest concentration or water is 100% lysis.
• Plot % lysis against NaCl concentration.

Results

• Normal fresh blood: lysis usually begins at about 0.45–0.50% NaCl and is nearly complete by about 0.30–0.35%; median corpuscular fragility (50% lysis) about 0.40–0.45 g/dL NaCl. Local ranges apply.

Interpretation

• Increased fragility (curve shifted right): hereditary spherocytosis, immune haemolytic anaemia with spherocytes, burns.
• Decreased fragility (shifted left): thalassaemia, iron deficiency, sickle cell disease, target cells in liver disease, post-splenectomy.

Quality control and pitfalls

• Incubation at 37 °C for 24 h increases sensitivity for mild hereditary spherocytosis.
• Accurate saline concentration and pH are essential.
• Results are affected by recent transfusion and by high reticulocyte counts.
• Normal results do not exclude mild HS; the EMA binding flow cytometry test is now preferred.

Clinical use

• Supports diagnosis of hereditary spherocytosis together with family history, film and DAT.