Protocol:Hb stability – isopropanol and heat tests

Disruption of the normal structure of the haemoglobin molecule can result in reduced stability, which leads to precipitates in the erythrocyte causing its destruction. The clinical consequence is often a haemolytic anaemia of variable severity.

Amino acid substitutions in the globin chains, particularly those involving the non-polar amino acids that constitute the haem pocket, may result in an unstable haemoglobin. In general the amino acid substitution also produces a modification of electrophoretic mobility. However, if the substituted amino acid is internal or the total charge of the molecule is unchanged, the haemoglobin variant will not be detected by conventional electrophoresis. Therefore, if an unstable haemoglobin is suspected clinically, a specific test for unstable haemoglobin should be performed even if the electrophoretic pattern is normal. Milder instability may also be associated with perturbations of bonding to the haem group (Hbs) or between subunits (eg. Hb H or variants with altered oxygen affinity). There are two stability tests: isopropanol test and heat test.

Principle
The presence of isopropanol makes the buffer less polar, weakening the haemoglobin hydrophobic bindings that facilitate its denaturation and precipitation (9).

Sample

 * 1) One ml of fresh blood (in any anticoagulant) is washed twice in 0,9% NaCl. The packed cells are lysed by the addition of one or two volumes of distilled water. Mix gently and wait few minutes. Centrifugation to remove the erythrocyte ghosts is not necessary. For some Hb variants early precipitation and binding to the red cell membrane may take place.
 * 2) Therefore the use of organic solvents, such as toluene or carbon tetrachloride, which dissolve the red cell membrane should be avoided.
 * 3) Prepare a fresh normal sample in the same conditions as a control.

It is advisable to test the reagents by using a positive control (eg a Hb H blood sample)

Reagents

 * 1) Tris-HCl 0.1 M, pH 7.4:
 * 1.21 g tris-hydroxymetyl-amino-methan
 * 100 ml distilled H2O,
 * Bring to pH 7.4 with 4 M HCl
 * 1) Tris-isopropanol buffer:
 * Tris-HCl 0.1 M, pH 7.4 solution: 83 ml
 * Isopropanol: 17 ml
 * Keep stoppered at room temperature

Method

 * 1) Two small stoppered tubes (one for the sample the other for the control) each containing 2 ml of tris-isopropanol buffer and 0.2 ml of haemolysate are mixed by inversion and placed at 37 oC in water bath.
 * 2) Check for the presence of flocculent precipitate at 5, 20 and 30 minutes.

Interpretation
Flocculent precipitate indicates the presence of an unstable haemoglobin. The control solution should remain clear.

''Comments: The presence of Hb F levels higher than 3% may give false positive results for its mild instability. Storage of the blood sample for several days may results in methaemoglobin formation and possible false positive results. The hemolysate should be fresh prepared. False negative results may be obtained adding KCN to the lysate.''

Principle
Normal haemoglobin precipitates only in a very minimal amount at 50 °C for 30 minutes, while unstable haemoglobins in these conditions are completely denatured.

Sample

 * 1) Fresh blood sample in any anticoagulant.
 * 2) A control blood sample must always be tested at the same time.

Reagent and solutions

 * 1) Phosphate buffer 0.01 M pH 7.4:
 * Solution A: NaH2PO4.2H2O: 15.6 g in 1 litre distilled H2O
 * Solution B: Na2HPO4: 14.2 g in 1 litre distilled H2O
 * 1) Add 19.2 ml of solution A to 80.8 ml of solution B.
 * 2) Mix and leave for 10 minutes at room temperature. Check pH.

Method

 * 1) Lyse as previously described (see isopropanol test).
 * 2) Transfer 5 ml of the lysate and 5 ml of phosphate buffer pH 7.4 to test tube.
 * 3) Mix and centrifuge at 3000 rpm for 10 minutes
 * 4) Transfer 2 ml of supernatant to a glass test tube, which should then be heated at 50 °C for 30 to 60 minutes.

Interpretation
A precipitate easily visible to the naked eye will be formed if a heat-unstable haemoglobin is present. The control sample should remain clear.

''Comments: Slight precipitation is of doubtful significance and, if present, the test should be repeated. The presence of Hb F levels higher than 3% may give false-positive or doubtful results.''