Osmotic Fragility Test

The osmotic fragility test measures the resistance of red cells to lysis in graded hypotonic saline. Cells with a reduced surface area to volume ratio, such as spherocytes, swell and lyse at saline concentrations that normal biconcave cells tolerate. Cells with excess membrane, such as target cells and hypochromic microcytes, resist lysis. It answers one question: is a spherocytic or membrane deficient red cell population present?

It was the traditional confirmatory test for hereditary spherocytosis. Flow cytometric eosin-5-maleimide (EMA) binding is now preferred where available, because it is more sensitive and more specific, needs only a small sample and is quicker.

Hereditary spherocytosis affects about 1 in 2000 people of Northern European ancestry and occurs in all populations. About 75% of cases are autosomal dominant, from defects in ankyrin, band 3, spectrin or protein 4.2. The unincubated test detects only about two thirds of cases, and incubated blood detects about 80%.

Indications. Spherocytes on the film with a negative direct antiglobulin test, haemolysis with a family history, splenomegaly, early gallstones, prolonged neonatal jaundice, and separation of hereditary from immune spherocytosis when the antiglobulin test is negative or equivocal.

Specimen. Heparinised or defibrinated blood, with a normal control collected at the same time and tested in parallel. Fresh blood is tested within 6 hours. A second aliquot is incubated sterile at 37 °C for 24 hours before testing, which unmasks mild defects.

Method. Blood is added to a series of tubes of buffered sodium chloride, from isotonic to almost distilled water, and incubated for 30 minutes at room temperature. After centrifugation, supernatant haemoglobin is measured at 540 nm and expressed as a percentage of complete lysis. The percentages are plotted as a sigmoid curve and compared with the control curve. The median corpuscular fragility is the saline concentration giving 50% lysis.

Reference values (unincubated). Lysis begins at about 0.45 to 0.50 g/dL sodium chloride and is complete by about 0.30 to 0.35 g/dL. The median corpuscular fragility is about 0.40 to 0.45 g/dL.

Incubation. Incubation shifts normal curves to the right, so incubated results must be compared with an incubated control and the local range.

Increased fragility (curve shifted to the right).

• Hereditary spherocytosis, typically with a tail of fragile cells.
• Immune haemolysis with spherocytes: warm autoimmune haemolytic anaemia, ABO haemolytic disease of the newborn, delayed haemolytic transfusion reaction.
• Hereditary elliptocytosis variants and hereditary pyropoikilocytosis.
• Thermal burns and clostridial sepsis.
• After incubation only: pyruvate kinase deficiency and some other red cell enzyme defects.

Decreased fragility (curve shifted to the left). Iron deficiency, thalassaemia, sickle cell disease, haemoglobin C and E disease, liver disease with target cells, hyposplenism and splenectomy, and marked reticulocytosis.

Abnormal fragility persists after splenectomy in hereditary spherocytosis, although haemolysis stops.

Mixed populations. Coexisting iron deficiency or thalassaemia can pull a spherocytic curve back towards normal.

Critical findings needing immediate action. A sudden fall in haemoglobin with reticulocytopenia in a patient with known hereditary spherocytosis indicates aplastic crisis, usually from parvovirus B19.

Pitfalls.

• A normal result does not exclude hereditary spherocytosis, particularly mild forms.
• Sample age, temperature, pH, saline accuracy and haematocrit alter the curve.
• Recent transfusion dilutes the abnormal population.
• The test is not specific, so interpret it with the film, reticulocyte count and antiglobulin test.

Special groups. Neonates need age specific interpretation, since foetal haemoglobin and ABO incompatibility change the pattern, and EMA binding is preferred. First degree relatives of a proven case can be tested, and mild disease is common in carriers.

Follow up tests. EMA binding by flow cytometry, blood film, direct antiglobulin test, reticulocyte count, haemolysis screen, red cell membrane protein analysis, osmotic gradient ektacytometry, sequencing of ANK1, SPTB, SLC4A1, SPTA1 and EPB42, parvovirus B19 testing, abdominal ultrasound for spleen size and gallstones, family testing.