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OCR for page 176
176 PART' III. ABNORMAL HEMOGLOBINS DISCUSSION Mr. Jbuer R. Rol~i~zson: With respect to the problems of separation and quantitation of hemoglobin ~ to which Dr. Chernoff has referred, I should like to describe a technique which has been devised in our laboratory. We found that this method clearly separates hemoglobin F as well as hemo- globins A, S. and C, and when used in conjunction with filter paper electro- phoresis differentiates hemoglobin D from hemoglobin S. The method consists of preparing a 1~2 per cent agar gel using a pH 6.2, citric-citrate buffer of 0.05 molarity. The agar is poured in a 1-2 mm. layer on a 4 in. x 10 in. glass plate and allowed to gel. The hemoglobin solutions are introduced into the gel by placing small pieces of filter paper soaked with hemolysate in slits made with a razor blade. Paper wicks are placed on the ends of the plate and the plate is sprayed with plastic. The plate is then placed in a horizontal type of electrophoresis apparatus and a current of 20 ma. at 350 volts is allowed to flow for 16 hours. The plastic is then stripped off and the plate stained with amino-schwartz 10B. FIG. 1.- Agar gel electrophoresis. Com- parison of two Hb AS and two normal Hb A individuals. Figure 1 demonstrates the type of pattern obtained in two individuals known to be of hemoglobin type AS and two normal individuals with 2.5 per cent alkali-resistant hemoglobin determined chemically. Note the order of separationhemoglobin F fastest, followed by hemoglobins A and S. At this pH hemoglobin should migrate toward the cathode and it does. How- ever, the order is the reverse of that obtained by free electrophoresis. This reversal clearly indicated to us that the method depended upon absorption on the agar as well as upon electrophoresis and should be called an electro- chromatographic method. Note also the presence of a distinct spot just ahead of hemoglobin A and also the increased density in the position of hemoglobin S in the two normal individuals. The spot between hemoglobin A and F seems to be present in most bloods examined. This minor component could be identical with Dr. Kunkel's "A2." The slight increase in density in the

OCR for page 176
DISCUSSION FIG. 2. Ag a r gel e l ectrop ho r es is. Comparison of hemoglobins with va ~ ying degrees of alkali resistance. 177 hemoglobin S position, on the other hand, apparently occurs only rarely,- ill normal individuals. Figure 2 illustrates the results obtained with a sample containing 87 gel cent alkali-resistant hemoglobin as determined chemically. Note the spots in the positions of hemoglobins A and C, on sample 1, obtained from an infant possibly representing genotype AC. Sample 2 was a typical SC combination on paper. The method clearly shows additional minor components in the positions of hemoglobins A and F; the alkali resistance was 2 per cent chemi- cally. Sample 3 appeared as only hemoglobin S on paper and had 1.5 per cent alkali resistance. Note the faint spot in the hemoglobin A position. Sample 4 illustrates what happens when too much hemoglobin is placed on the plate. Figure 3 is a paper electrophoretic pattern at pH 8.6 of an interesting Greek family in which the father gave a typical AS pattern, and the mother gave a typical A pattern, but manifested thalassemia minor hematologically. Two of the children, Catherine and George, showed an SF pattern and have 10 and 15 per cent alkali resistance and hematologically are classified as .... ... .~.... FIGS. 3 and 4. Comparative results of paper electrophoresis (left) and agar gel electrophoresis ( right) from same family.

OCR for page 176
178 PART III. ABNORMAL HEMOGLOBINS sickle cell thalassemia. One other child, Gus, gave a typical hemoglobin A pattern. Figure 4 presents the picture obtained for this family on agar. Note the presence of small amounts of fetal hemoglobin in the mother, Gus, and the father, as well as a hemoglobin-like component in the hemoglobin A position in the two children with sickle cell thalassemia disease. Figure 5 illustrates a typical paper electrophoretic pattern at pH 8.6 of two typical AS individuals and two individuals whose hemolysates had pre- viously been shown to contain hemoglobins A and D, by solubility tests, lack of sickling, and free electrc~phoresis method. Note the distance of separation of hemoglobin S and D from A. Figure 6 shows the agar patterns for the ..~......... ...~..~. a. 2:': f "":"' .,, ~.,....~.............. . ...... .. ....... . .... . . ~ ~ . ~ FIGS. 5 and 6.- Comparative results of paper electrophoresis (left) and agar gel electrophoresis (right) from same individuals. same individuals. Note the failure of hemoglobin D to separate from hemo- globin A. This provides another means of differentiating hemoglobin D from hemoglobin S. The ability of this technique to detect low concentrations of hemoglobin F makes this method a useful tool. It also is able to separate clearly hemo- globins A, S. and C, and detects the presence of small quantities of other hemoglobin-like components. Indeed, in the light of the possible genetic im- plications, these findings of an A-like hemoglobin in the S and C combination and in the S alone may be the method's most valuable aspect. Dr. A. M. Josephson: I would like to make a point with relation to Mr. Robinson's discussion. We have been doing the same thing in our laboratory. We are using a similar technique, however using pH 6.5, which completely separates the S. A, F. and the other hemoglobin fractions, giving a very simple techr~ique to identify this form of the sickle cell thalassemia disease, rather than through the complicated mechanism of Tiselius electrophoresis.