Test Tube Method Screens for Variant Hemoglobin

The novel test-tube method for HbE screening uses diethyl aminoethyl (DEAE)-cellulose resin and it is based on the principle of anion-exchange liquid chromatography.

Scientists at Chiang Mai University (Thailand) used the principle where DEAE-cellulose resin was used as the stationary phase, and glycine buffer with sodium chloride as the mobile phase. However, the method was set to be completed inside the tube instead of in an open-ended column. Equilibrated DEAE-cellulose resin is added to polystyrene tubes, followed by the addition of 2 mL eluting buffer. The tubes are capped and stored at 4 °C.

For HbE screen, packed red blood cells are mixed with a hemolysis reagent to obtain the hemolysate. An aliquot of hemolysate is added to the tube containing DEAE-cellulose resin suspension in eluting buffer, mixed vigorously and centrifuged. The color of the supernatant was then visualized after centrifugation. If the color of the supernatant was red, the result was positive, whereas a colorless or markedly pale red supernatant was negative. A positive result indicated the presence of HbE, while a negative result suggested the absence of HbE in the blood samples.

One hundred and thirteen blood samples were screened for HbE by the test tube method and by standard cellulose acetate electrophoresis (CAE). Seven of 113 samples were found to be positive for HbE by the tube method, in complete agreement with the findings from the CAE. The test-tube method is cheap and simple with no requirement of sophisticated equipment. The reagent could be stored at 4 °C for up to five months. Hemolysate samples aged up to five months were still suitable for this test.

HbE is a β-structural variant common worldwide. This Hb disorder can form a compound heterozygous state with the β-thalassemia gene, leading to life-threatening hereditary hemolytic anemia, HbE/β-thalassemia. The authors concluded that the novel test-tube method using the DEAE-cellulose resin (Sigma-Aldrich, Saint Louis, MO, USA) could be an alternative method of mass population screening for HbE, particularly in small health care facilities. The study was published online on August 23, 2011, in the International Journal of Laboratory Hematology.

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