The identification of mixed granulocytic-erythrocytic colonies in vitro

The identification of mixed granulocytic-erythrocytic colonies in culture poses special problems. First, methods based on the detection of peroxidase-like activity of red-cell hemoglobin using benzidine can give false-positive results with eosinophilic cells in colonies. Secondly, the use of the color of hemoglobin itself to identify mixed colonies prevents the detection of those colonies where the erythrocytic component is small or contains relatively little hemoglobin. We present here a new method using O-Dianisidine (O-D) to detect red-cell peroxidase activity. Subsequent staining with Luxol fast blue showed that eosinophils did not give a positive reaction with O-D. The technique also has the advantage that the whole culture gel is processed and that there is no need to pick off individual clones for identification. Using the combined stain O-D and Luxol fast blue most lineages within clones can be discriminated.