Cellulose Acetate Electrophoresis and Hemoglobinopathies
Abstract
The identification of hemoglobin variants is essential in the diagnosis of hemoglobin disorders that can be inherited. Three patient samples were analyzed using cellulose acetate electrophoresis. The hemoglobin variants were able to separate, showing hemoglobin S, A, and F. Two of the patients had hemoglobin S, indicating having sickle cell trait, but further testing was required to confirm the diagnosis.
Introduction
Electrophoresis is a technique that is used in the identification of different variants of hemoglobin, a protein most abundant in the blood. Electrophoresis can differentiate the structural variants of hemoglobin basing on their net negative charge (Wada, 2002). Cellulose acetate electrophoresis is done using an alkaline buffer, and the samples are left to migrate from the cathode to anode as they separate (Marengo-Rowe, 1965). The method is also used for the quantification of the variants.
The hemoglobin protein has many variants that occur due to point mutations that occur on either of the chains that make it, resulting in hemoglobinopathies. Another defect affecting hemoglobin causes defects on the alpha and beta globulin during synthesis, causing thalassemia (Kim, et al., 2011). The differing compositions of the hemoglobin globulin result in hemoglobin A, hemoglobin F, hemoglobin A2, hemoglobin S, and hemoglobin C, although many more variants keep being identified a few have been found to be of clinical importance. Structural mutations in the chains of hemoglobin can cause disorders that are inherited, such as sickle cell anemia, which in the homozygous state can be severe and fatal.
Cellulose acetate electrophoresis of hemoglobin in newborn babies can be used to identify whether an individual has the sickle cell trait or has sickle cell disease. Occurrence of hemoglobin A, S, and F, where the proportions of S and A are nearly equal, could indicate sickle cell trait. While if only hemoglobin S and F occur, it could be the homozygous state (Jacobs, et al., 1986). Follow up tests should be done in adulthood to rule out other disorders, though. In healthy adults, the most significant percentage of hemoglobin A, with lower percentages of A2 and F.
Materials and Methods
Four samples of hemolysates were prepared, one being a control while the other three from different patients. An acetate strip soaked in the buffer was picked from the shaker, and excess buffer drained using filter papers. The strip was then secured on the bridge using plastic pegs. One compartment of the tank was filled with the buffer and tilted to ensure its level is the same on both sides. A pipette was then used to transfer 15 microliters of the samples to the semi-micro drop holder. The bridge having the strip attached was then placed in the electrophoretic chamber, and an applicator used to add the samples on the strips, the samples were added at the cathode. The chamber was then covered, and the power supply switched on and allowed to run for 45 minutes.
After the time had elapsed, strips were removed from the bridge and were stained using Ponceau S stain for 5 minutes, washed in detaining solution followed by placing then in a clearing solution. Excess clearing solution was removed using a glass rod, the edges cut and placed in the oven to dry.
Results
After the 45 minutes and drying of the strip, it was noticed that the control sample was able to separate into four bands, indicating the presence of Hb A, Hb A2/ Hb C, Hb F and Hb S. This can be seen on the strip in Figure 1 below.
Figure1: Cellulose Acetate Electrophoresis on four samples: control, patient 1, patient 2 and patient 3.
Hemoglobin in patient 1 separated into three bands; Hb A, Hb F, and Hb S. Patient 2 hemoglobin separated into two bands; Hb A and Hb S, while that of patient 3 had only one band Hb A.
Discussion
Hemoglobin A makes about 98% of hemoglobin found in adults, and normal adults could also have a small percentage of hemoglobin F, basing on this, it is right to say that patient 3 is a normal adult, given that he image only shows one band of Hb A, that accounts for more than 90 % (Olufemi, et al., 2011).
Patients 2 and 3 have bands indicating the presence of Hb S that is found in individuals having sickle cell diseases or sickle cell trait. Patient 2 lacks Hb F while patient 1 has Hb F. In healthy adults, small percentages of hemoglobin F can be present, but when together with hemoglobin S indicates the presence of hemoglobinopathy. In patients that present with sickle cell trait or disease, the levels of hemoglobin F could be elevated as its one of the compensatory mechanisms (Olufemi, et al., 2011). Diagnosis in patient 1could be confirmed using further tests, to rule out thalassemia. The proportions in patients one is seen to be about 80% HbA, 15% Hb F and 5 % or less Hb S.
Patient 2 hemoglobin proportions are about 85% Hb A and 15% Hb S. The patient is likely to be having sickle cell trait. Proportions can be confirmed using densitometry as it is more accurate for clinical setups. Low-Pressure Liquid Chromatography can also be used in the identification and quantification of different variants of hemoglobin. LPLC is a very sensitive method and can separate fractions that are below 10% of the total composition of hemoglobin (Chambers, Phillips, & Chapman, 1993).
References
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Kim, L.-E., Kim, B.-R., Woo, K.-S., Kim, J.-M., Park, J.-I., & Han, J.-Y. (2011). Comparison of capillarity electrophoresis with cellulose acetate electrophoresis for the screening of hemoglobinopathies. Korean Journal of laboratory medicine, 31, 238-243.
Marengo-Rowe, A. J. (1965). Rapid electrophoresis and quantitation of hemoglobins on cellulose acetate. Journal of clinical pathology, 18, 790-792.
Olufemi, A. E., Sola, O. B., Oluwaseyi, B. E., Ajani, R. A., Olusoji, M. O., & Olubunmi, H. R. (2011). Hemoglobin F level in different hemoglobin variants. The Korean Journal of Hematology, 46(2), 118-122.
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