Protocol:Hb E detection by DCIP test

Principle
The Hb E mutation occurs from the mutation on the beta globin gene at codon 26, GAG-AAG, resulting in the amino acid change from Glu-Lys. This results in a mildly unstable haemoglobin and exposure of the -SH group, which can be oxidized by certain chemical agents including the dye DCIP (dichlorophenolindophenol) at the neutral pH (7.5). Hb E and other unstable haemoglobin molecules such as Hb H will be precipitated when exposed to this dye at 37oC (20).

Reagents

 * DCIP Reagent:

Dissolve in distilled water and adjust pH to 7.5 by 6 M HCl and adjust the volume to 500 ml. This working solution should be kept in 4oC.

Method

 * 1) Add 30 &mu;l of whole blood or 20 &mu;l packed red cell into 5 ml DCIP solution.
 * 2) Gently mix and incubate at 37oC for 1 hour.

Interpretation
Precipitated hemoglobin can be visualized by the naked eye at the bottom of the tube.

Results and comments
In homozygous Hb E, a heavy sediment will form at the bottom of the test tube. In Hb E trait, and Hb E/&beta;-thalassaemia, the precipitation of Hb E produces a cloudy or an evenly distributed particulate appearance. The test is positive also in HbH disease and other unstable haemoglobins. To overcome these false positive results a modified DCIP test has been described (21) and more recently the CMU-E (Chiang Mai University-E) has been found to have 100% sensitivity and 99.1% specificity (22).