Pappenheim's Methods

PAPPENHEIM'S METHODS (Pappenheim), methods proposed by Pappenheim for staining cellular elements of the blood in blood smears and histological sections from hematopoietic organs. These methods, called Pappenheim's panoptic stains, serve primarily for the differentiation of chromatin and plastin of cells, spongioplasm and paraplasm, and also for the detection of cellular granulation. (For the May-Giemsa stain according to Pappenheim and the methyl green-pyronin stain according to Pappenheim-Unna, see Blood—morphological composition of blood and methodology of its investigation.) Panchrom stain according to Pappenheim. Panchrom consists of a mixture: Methylene blue 1.0, Toluidine blue 0.5, Azure I 1.0, Methylene violet 0.5, Eosin 0.75, Methyl alcohol 250.0, Glycerin 200.0, Acetone 50.0. Staining method for blood smears: preparations fixed for 3 minutes are stained for 15 minutes with a diluted solution of Panchrom (15 drops per 10 cm³ of distilled water). Preliminary staining with May-Grünwald solution is recommended, as in the Pappenheim-Giemsa stain (see Blood—morphological composition of blood and methodology of its investigation). Staining method for histological sections: fixation according to Maximow or Helli. 1) Preliminary staining in diluted May-Grünwald solution (1 part of stock solution to 8 parts of distilled water)—10 minutes. 2) Staining with Panchrom solution (10 drops per 10 cm³ of distilled water)—20 minutes. 3) Differentiate in a 0.1% aqueous solution of picric acid until the sections take on a pinkish hue. 4) Rinse in distilled water (5 minutes). 5) Dry with filter paper. 6) Acetone-xylene (3:7). 7) Xylene. 8) Canada balsam. Results are the same as with May-Giemsa. Staining according to Pappenheim-Kardos. Kardos mixture: immediately before use, 5 drops of Methyl green-Orange solution and 15 cm³ of distilled water are added to 10 drops of Panchrom solution. After shaking, the solution is poured off without foam. 1) Fix a freshly prepared dry blood smear with May-Grünwald solution—3 minutes. 2) Add an equal amount of distilled water—1 minute. 3) Stain with the Kardos mixture—15 minutes. 4) Rinse briefly in distilled water. 5) Dry. The azurophilic granules of lymphocytes are bright red, the granulation of neutrophils is brownish-violet. Staining with polychrome methylene blue and carbol-fuchsin according to Pappenheim. Fixation by heat (7–10 seconds) or with a mixture of osmic and acetic acid vapors according to Weidenreich. For staining, Unna's polychrome methylene blue is used: dissolve 1 g of methylene blue in 100 cm³ of distilled water and 200 cm³ of 96% alcohol, then add 1 g of potassium carbonate and slowly evaporate on a water bath to 100 cm³. To 10 cm³ of distilled water, add 8–10 drops of the above-described methylene blue solution and 8 drops of Ziehl's carbol-fuchsin. Stain for 5–8 minutes. Rinse the preparation several times with distilled water and then dry. Result of staining: nuclei are blue, lymphocyte protoplasm rich in spongioplasm is blue, spindle-shaped cells of lower animals are red. All the above-mentioned staining methods produce very beautiful images, but in essence, they do not offer any particular advantages compared to the May-Giemsa method.