In order to detect basophilic stippled erythrocyte (BSE), Kozukue (one of the authors) et al. (1966) devised a kind of specific staining for ribonucleic acid (RNA) in red cells as follows: The peripheral blood is smeared thinly onto a glass-slide, dried in air, fixed with methanol for 3 minutes, stained with 1:2, 000 acridine-orange phosphate buffer solution (pH8.0) for about 2 seconds, washed with tap water, dried, mounted with paraffine oil, and observed under a fluorescence microscope. By this method, BSE is easily detected by the red fluorescence of its granules (basophilic stipples) while normal reticulocyte (Ret) emits a diffuse reddish orange fluorescence. Using this technique, the authors have found that BSE appeared also in cases of eczema, dermatitis, disorders involving various organochlorine compounds and steroids, in addition to lead poisoning.
The results from examination of this fluorochroming method in cases of abnormal time for drying blood smears before fixation are herein described. For this purpose, smears from rabbits (1) with occurrence of BSE induced by lead poisoning, (2) with post phlebotomy reticulocytosis without BSE, and (3) of normal rabbits were dried in various times by use of a humidity chamber, ventilation at room temperature or with hot air, or heating (30-50°C) of glass-slide.
1) In blood with occurrence of BSE, BSE increased in number with the prolongation of time from 4 seconds to 5 minutes but did not appear within 3 seconds. While in normal and mere reticulocytemic blood, BSE count always stayed within 0.2‰ per whole erythrocytes.
2) The granules showed a tendency to increase in size and number as the BSE count was increased.
3) These phenomena can be explained by the following hypothesis: Visible difference between BSE and Ret, both of which can not be distinguished by supravital staining, appears in the period within which the smear remained with moisture. During this period, in BSE, RNA begins to aggregate around mitochondria forming granules as time passes. This change stops with the competition of drying, while in normal Ret, RNA continues to disperse diffusely in cytoplasm independently of the time. But this continued dispersion is caused by certain properties in the blood (normal or with disorders induced by lead or other causes) so that even when the time is prolonged, BSE count, size and number of granules never exceed a certain limit that varies with the origin of each blood. Such property of the blood might be decided by the distribution of mitochondria and quantity, quarity or abnormality of RNA in each cell.
4) In practicing this fluorochroming technique, smeared blood should be dried slowly spending a minimum of 30 seconds, avoiding hot air, radiant heat or strong wind. A wind-protecting box or humidity chamber should be used under the circumstances where dessication is promoted.
5) On the other hand, in order to merely detect BSE or distinguish from normal blood, it is very useful as a provoking method to dessicate smears in 4.5 to 5 minutes by means of putting them into a humidity chamber for about 4 minutes.