Quantitation of Serum Hemoglobin-Binding Capacity Using Cellulose Acetate Membrane Electrophoresis

A method is described for the quantitation of serum hemoglobin-binding capacity (haptoglobin level) using cellulose acetate membrane electrophoresis. Electrophoresis is carried out for 90 mm. Eight membranes are dried, stained, and scanned within 45 mm. The reproducibility of this technic is within ± 10%. I F NO INTRAVASCULAR liberation of intracellular hemoglobin occurs with the transfusion of thawed, recOllstituted frozen erythrocytes, the amount of free supernatant hemoglobin in units of frozen blood that may be in-fused without producing henioglobinuria is determined principally by the recipient’s plasma llenloglobin-billdillg capacity (1, 2). Oweii et al. (3) reported a method for quantitation of serum hapto-globin which was dependeiit upon the peroxidase activity of methemo-globiii-haptoglobin complex in which the color of the tetra guaiaco-quinone generated is unstable.,Javid and Horowitz’s modification (4) of Lathem and Worley ‘s method (5) for seruni haptoglobin uses paper electrophoresis and requires 14 hr. for the separation of fi-ee (unbound) hemoglobin, haptoglohin (alpha-0 globulin) bound hemoglobm, and methemalbumin (albumin-bound acid hematin) prior to staining with the stable peroxidase stain o-dianisidine (3,31 diniethoxy beiizidine, Eastman Products, Rochester, N. Y.). We have developed a method for the quan-titation of serum hemoglobin-binding capacity by the use of cellulose acetate membrane electrophoresis which requires approximately 90 miii. for separation and an additional 45 mm. for drying, staining with o-dianisidine, and scanning of 8 membranes