The Supravital Method in the Study of the Cytology of Blood and Marrow Cells

Abstract

1. Dependable results with supravital staining can be obtained only with apochromatic objectives, compensating oculars, an achromatic condenser, controlled illumination and a transparent daylight filter on the light source. 2. Proper technic is of the utmost importance in making supravital slides. 3. Pinacyanol is preferable to Janus green as a mitochondrial stain. 4. Supravital preparations should be studied in a darkened room to increase the apparent brilliance of the staining. 5. More accurate counts of the number of lobes in the nucleus of neutrophils can be made with the supravital method than from dried stained smears. 6. Myelocytes C in supravital preparations correspond to the myelocytes seen on dried smears, while myelocytes B are the promyelocytes. 7. Myelocyte A has been redefined. The neutrophil granules of these cells do not as a rule stain on dried smears, and these cells are therefore classed as myeloblasts or leukoblasts in the latter type of preparation. They are peroxidase-negative. 8. Myeloblasts may or may not contain neutral red vacuoles, and may or may not contain a rosette. 9. Supravital staining is a good technic for the identification of monocytes from human blood and marrow, and is the method of choice for the study of macrophages. 10. Supravital preparations of the peripheral blood of severe cases of erythroblastosis foetalis show all transition stages between monocytes and macrophages, and there is no reason for regarding these cells as separate types. 11. Lymphocytes contain many more neutral red vacuoles than have been previously described. 12. The lymphocyte of infectious mononucleosis has a characteristic appearance, and can be used as a means of diagnosis. 13. Qualitative changes do not always occur in the lymphocytes of acute lymphatic leukemia. 14. Myeloma cells have a characteristic appearance in supravital preparations. 15. Sarcoma cells in the peripheral blood are more variable in appearance than had been supposed. 16. The precipitation of neutral red in the cytoplasm is a quantitative staining reaction and is not specific for the earliest stages in red cell formation. 17. The appearance of the developing red cells in supravital preparations is described. 18. The megaloblastic line of developing red cells which is present in pernicious anemia cannot be distinguished in supravital preparations from the normoblastic line found in normal marrow or in marrow after hemorrhage or hemolysis. 19. The supravital method therefore cannot be used for diagnostic work involving erythropoiesis. 20. There is no evidence of any kind indicating that a maturation arrest occurs in pernicious anemia. 21. Cells intermediate between pronormoblasts and myeloblasts are occasionally seen in supravital preparations. 22. Differential counts can be made from supravital slides just as accurately as from dried stained smears, and if many fragile cells are present, more accurately. 23. The cytology of the developing blood cells as seen in supravital preparations would support a monophyletic view of hematopoiesis just as well as the polyphyletic theory.

The Quantitative Study of the Leukocytes

“…There seems to be little doubt, at the present time, about the identity of the small lymphocyte in bone marrow, which formerly was confused with the "primitive" cell of Cunningham, Sabin, and Doan (1925); though, at a later date, Sabin and Miller (1938) concluded that the morphological criteria for distinguishing between primitive cell and small lymphocyte are not satisfactory. With this conclusion, Schwind (1950), after a careful study of marrow cells by the supravital technique, was also in agreement.…”

Studies on Leukocytic Secretory Activity

“…It was suggested (Richter, 1942), on the basis of certain cytophysiologic considerations and others, that the neutrophilic granules of the polymorph may be fundamentally equivalent to the osmiophobic component of the lymphocytic golgi complex and to that of other tissue cells. First, there are mitochondria present in these cells, even though they are reduced in number (Richter, 1942; Bloom and Wislocki, 1950;Cowdry, 1914;Tompkins and Cunningham, 1938; Schwind, 1950; Dawson and Spark, 1929;Hall, 1938Hall, ,1931, and others). Second, the specific granules during their genesis first appear in the region of the myeloblastic golgi complex, as reported by Ehrich (1934), Simpson and Deming (1927), Maximow and Bloom, (1930), Schilling (1929).…”

“…In the granulocytes, mitochondria are generally considered to be present in the immature forms as short rods (Butterfield, Heineke, and Meyer, 1909;Downey, 1928;Schilling, 1929;and others). In the mature forms, the mitochondria have been reported to be reduced in number and sometimes lacking, although the majority of workers regularly identify them in supravital preparations (Cowdry, 1914;Bunting, 1938;Cunningham and Tompkins, 1930;Tompkins and Cunningham, 1938;Sabin, 1923;Schwind, 1950;Richter, 1942).…”

“…With dark-medium phase contrast, the droplets appear as discrete milky white spherical bodies (FIGURES 15, 16). They color somewhat metachromatically in the living cell with the supravital dye, neutral red (Rosin and Biebergeil, 1904;Metchnikoff, 1905;Renaut, 1907;Arrigoni, 1908;Dubreuil, 1913;Shipley, 1919;Simpson, 1921;Sabin, 1923;Maximow, 1927Maximow, , 1928aRhoads and Parker, 1928;Dawson and Spark, 1929;Wilson and Cunningham, 1929;Bloom, 1928a, 192813;Hall, 1931;Lewis and Rubin, 1932;Tompkins and Cunningham, 1938;Lawrence and Todd, 1941;Schwind, 1950;Jackson, 1954). It is evident that these droplets have been known to us for a long time.…”

Some Problems of Lymphocyte Production

“…(1) They are morphologically indistinguishable from small lymphocytes, whether in supravital preparations (Schwind, 1950) or in dried smears. This is in agreement with the view expressed by Sabin and Miller (1938); the transitional cell (Yoffey, 1957) was not described as such by Sabin et al (1925) but was implicit in their work, and it introduces an obvious source of dif-…”