Different Membrane Buffers and Hemoglobin Electrophoresis

LETTER TO THE EDITOR

The Effect of Different Membrane Buffers on Hemoglobin Electrophoresis

ZE-JUN LIU, HAI-HONG JIANG

Department of Laboratory Medicine, Southwest Hospital, Third Military Medical University, Chongqing, People's Republic of China

Laboratory Hematology 7:101-102
© 2001 Carden Jennings Publishing Co., Ltd.

Although capillary zone electrophoresis is faster, more sensitive, and more selective for the detection of hemoglobin variants [1], the exorbitant cost of this method limits its use, and conventional hemoglobin electrophoresis with cellulose acetate remains the routine screening method in clinical laboratories [2,3]. Many factors affect the results of hemoglobin electrophoresis, such as the pH and concentration of the electrophoretic buffer, the moisture content of the carrier membrane, the voltage used, and the immersing buffer. We performed hemoglobin electrophoresis with Tris-EDTA-boracic acid (TEB) buffer at pH 8.6 on a cellulose acetate membrane and found the hemoglobin to be composed of hemoglobin A2 (3.7%), hemoglobin A (66.3%), and an unidentified fast-moving band (30%) (Figure 1).

To identify the fast-moving band, we performed another electrophoresis using 0.1 mol/L phosphate buffer at pH 6.5, and several bands migrated toward the anode (Figure 2, lanes 4 and 5). Because the isoelectric point of hemoglobin H is 5.6, it migrates toward the anode in pH 6.5 buffer, whereas all other hemoglobin types migrate toward the cathode under these conditions. However, it was discovered that the pH 8.6 TEB buffer had been used as the immersing buffer, not the pH 6.5 phosphate buffer. We then repeated the electrophoresis using the pH 6.5 phosphate buffer as the immersing buffer. This time, only 1 band migrated toward the anode, and we were able to show that the fast-moving band we had seen in the electrophoresis with the pH 8.6 TEB buffer was hemoglobin H. These results demonstrate the effect of different immersing buffers on hemoglobin electrophoresis results, and the importance of careful selection of proper test reagents, including buffers, when hemoglobin variants are being analyzed by electrophoresis.

FIGURE 1. Densitometric pattern of hemoglobin electrophoresis using TEB buffer, pH 8.6.

FIGURE 2. Electrophoretic patterns of hemoglobin using 0.1 mol/L phosphate buffer (pH 6.5). Arrow indicates application point. In lanes 1, 2, and 3, phosphate buffer (pH 6.5) was used as the immersing buffer, and in lanes 4, 5, and 6, TEB buffer (pH 8.6) was used as the immersing buffer. Lanes 3 and 6 represent normal control samples and lanes 1, 2, 4, and 5 represent the patient's sample.

REFERENCES

1. Lin C, Cotton F, Fontaine B, Gulbis B, Janssens J, Vertongen F. Capillary zone electrophoresis: an additional technique for the identification of hemoglobin variants. Hemoglobin. 1999; 23:97-109.

2. Jenkins MA, Hendy J, Smith IL. Evaluation of hemoglobin A2 quantitation assay and hemoglobin variant screening by capillary electrophoresis. J Capillary Electrophor. 1997;4:137-143.

3. Satwekar MM. Reuse of cellulose acetate membrane strips for protein and hemoglobin electrophoretic analysis. Indian J Med Sci. 1997;51:113-114.

Correspondence and reprint requests: Ze-Jun Liu, Department of Laboratory Medicine, Southwest Hospital, Chongqing 400038, People's Republic of China; telephone and fax: 8623-68754691 (e-mail: a65424208@public.cta.cq.cn).
Received and accepted January 15, 2001.

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