The Structure of Unstained Reticulocytes

Abstract

Brilliant cresyl blue and certain other basic dyes, if applied to fluid blood, bring out a reticulum in young red blood cells. This reticulum fails to appear if brilliant cresyl blue or other basic dyes are applied to dried and alcohol fixed blood smears. Moreover, no reticular structure could hitherto be demonstrated in the young red blood cells by microscopic examination of unstained blood with direct or oblique illumination, dark field illumination or in ultraviolet light. 1

While examining unstained wet blood films of albino mice with the phase microscope, we found that an occasional red blood cell showed a very delicate, hardly discernible reticulum. The cells showing this reticulum were fewer in number than the reticulocytes demonstrable by supravital staining with brilliant cresyl blue. However, when certain hypotonic salt solutions, preferably potassium oxalate 0.8% or ammonium oxalate 1.2% were mixed with the fresh blood, a larger number of cells, corresponding to the percentage of reticulocytes, showed distinct granules and short rods within their cytoplasm.

Most of these rods and granules showed prominent Brownian movement. In such preparation, varying number of cells showed marked loss of hemoglobin. Granules predominated in hemolyzed cells, while rods were more prominent in cells containing ample hemoglobin. The percentage of cells showing rods or granules was higher in those areas of the preparation in which hemolysis was marked. Smears, dried and fixed in alcohol, failed to show these cytoplasmic structures. However, dried but unfixed smears, if mounted in 10% formalin or 1.2% ammonium oxalate, (Fig. 1), showed rods and granules resembling those seen in the fresh preparations. Also, fixation of dried smears in 3% aqueous potassium dichromate did not interfere with the demonstration of the rods and granules. It is noteworthy that both formalin and dichromate applied to the dried smear hemolyzed the red blood cells.