Histochemical Studies on Developing Mast Cells ’ ALEXIS L. BURTON Department of Anatomy, University of Texas.. Medical Branch, Galveston, Texas ABSTRACT By means of a new staining procedure (using a mixture of Astra blue and safranin 0 ) , studies on the polysaccharides of the mast cell granules were made in adult and embryonic connective tissues of the rat. According to their staining reactivity toward these dyes, it was found that, in the adult tissues, mast cells can be classified into three groups: (1) Astra blue positive mast cells, or “blue” cells; ( 2 ) safranin positive mast cells, or “red” cells, which are the most numerous cells; ( 3 ) intermediate forms, which exhibit affinity for both dyes, or “mixed” cells. In the embryo, mast cells appear first on the fifteenth-sixteenth day of development as small round cells, the cytoplasm of which contains flakes of Astra blue positive material; rapidly, this material becomes granular, and on the twenty-first day (just before birth), a large mast cell population exists in the subcutaneous connective tissue of the embryo, containing only cells of the “blue” type. Eight to 15 days after birth, “red” and “mixed” cells are visible. Progressively, the mast cell population becomes of the adult type. It is thought that these variations i n the staining reactivity correspond to the evolution of the cell: the “blue” forms are the younger cells and the “red” forms are the adult mast cells. This is probably related to a progressive increase of the number of strong acidic groups which are present in the polysaccharidic molecule. In ’50, Steedman introduced Alcian blue as a specific dye for acid mucopolysaccharides. In ’56, Mowry showed that the stain was not absolutely specific, but that its specificity could be greatly increased when the dye was used in a strongly acidic solution, in the range of pH3. Later, Spicer (’60) developed a double staining procedure using Alcian blue and Safranin 0; in this latter technique, safranin stains polysaccharidic components which are not readily stained with Alcian blue. In this way, Spicer (’60, ’63) was able to demonstrate important variations in the staining reactivity of mast cells. He concluded that the vast majority of these cells in the rat have “a surprising lack of affinity for Alcian blue,” and that they preferentially stain with safranin; however, he did note that a few mast cells have a strong affinity for Alcian blue; this effect is especially marked in the cervical lymphnodes of the rat and in the uterus of the mouse. Results of his very extensive comparative studies which employed this technique, Azure A metachromasia, and periodic acid Schiff (P.A.S.) reaction, indicated that Alcian blue positive mast cells (blue mast cells) correspond to the “orthochromatic” or to the slightly metachromatic forms and are slightly P.A.S. positive. ANAT. REC., 150: 265-270. In ’56, Gedick reported that Astra blue, which is similar to Alcian blue, and also a member of the phtalocyanin group, was very useful for the demonstration of polysaccharides. Fasske (’57) and Pioch (‘57) studied this dye and found it specific for acid mucopolysaccharides. Bloom and Kelly (’60) completed a comparative study of the staining properties of Astra blue for mast cells and concluded that this dye seems to be more specific than Alcian blue in that it produces no staining of the nuclei and keeps good staining properties at very low pH. This paper reports a series of tests using a simplified technique using a mixture of Astra blue and safranin in order to study mast cell populations in various adult and embryonic tissues. MATERIAL AND METHODS Two types of tissues were examined: adult and embryonic tissues of rat. 1. Adult tissues were obtained from Cheek-Jones white laboratory rats, male and female, weighing approximately 150 gm. Organs studied were : lymphnodes, muscles of the abdomen, skin of the dorsal region, of the foot and of the lip. The 1This work has been supported by United States Public Health Service Grant 5 T1-459-04. 265 266 A L E X I S L. BURTON animals were killed by chloroform asphyxia. Specimens were dissected out and fixed in a mixture of 10% formalin in absolute alcohol. They were then rinsed in absolute alcohol, cleared in amyl acetate and embedded in paraffin. Sections were cut 8 - l o p thick and mounted on microscope slides by means of an egg albumin solution. 2. Embryonic tissues. Pregnant female rats were sacrificed on days 15, 16, 17, 18, 19, 20 or 21 after mating. The embryos were removed and tissues were prepared and fixed in the same formalin-alcohol mixture as above. These specimens were embedded in paraffin and sections were cut in the same way as previously mentioned. Staining techniques. In our first experiments, a two step staining technique according to Spicer (’63) was used. It involved a 30 minutes staining in a 0.5% Astra blue solution in 3% acetic acid, followed by a five minute rinse in tap water and finally a one minute immersion in 0.5% safranin solution in 1.125N HC1. Under these conditions it has been possible to obtain results very similar to those described by Spicer and to confirm the existence of two types of mast cells: Astra blue positive mast cells (blue mast cells) and safranin positive mast cells (red mast cells). However, during the first step of this technique, all mast cells stain with Astra bIue, even those which have a poor affinity for the dye. Differentiation begins during the washing period and is completed during the second step. Safranin thus acts as a counterstain and simultaneously as a differentiating agent. It is obvious that this differentiation could introduce a slight factor of inconsistency in the final result. For this reason it has been attempted, and then preferred, to use these dyes as a mixture in a single step staining procedure. Our technique can be summarized as follows : A. Paraffin is removed from the sections in xylol. B. The sections are hydrated by passing through a series of 100, 95 and 70 etanols, and then in distilled water. C. Slides are then stained for 15 minutes, at room temperature in the following formula : Astra blue (Chroma 10110) 0.5% solution i n distilled water 50 ml Safranin 0 (National Aniline 688) 0.5% solution i n distilled water 1 ml Glacial acetic acid 20 ml Distilled water 29 ml D. Sections are rinsed in distilled water, to remove the excess of stain and E. Then dehydrated in 100% ethanol, cleared in xylol and mounted in Permount. The stain is very resistant to dehydration. The staining solution has a pH of 2.3; it does not precipitate and keeps well for an indefinite period of time. If desired, a nuclear staining with Harris hematoxylin can be performed between steps D and E. Comparative studies were made, using other classical staining techniques : A. Metachromasia. The metachromatic reaction was assayed by immersion of sections in a 0.02% solution of Azure A (or a 0.02% toluidine blue solution) at various pH between 3 and 0.5, for 30 minutes. B. Gomori’s aldehyde f u c h s i n (’50) was also applied following the original procedure of this author, but with the omission of the pre-oxidation steps. C. The P.A.S. reaction was performed, according to the technique of McManus (Pearse, ’60), either alone or in combination with Astra blue (derived from the Alcian blue- P.A.S. method of Mowry and Winkler, ’56). D. Finally, a large number of sections were submitted to the “ c o l d sulfation technique (Moore and Schoenberg, ’57) and staining reactions with each of the above mentioned procedures were studied. RESULTS 1. Adult tissues. With the mixture Astra blue-safranin (ABS), nothing is intensely stained, except mast cell granules, cartilage, mucous glands and goblet cells. Nuclei exhibit a very pink shade, and can be better demonstrated either by a counterstain with hematoxylin or by phase microscopy. Three types of mast cells were observed (fig. 1 ) : A. Astra blue positive m a s t cells (blue mast cells). These stain in a greenish blue shade, resembling very much the “cyan” blue of the photographic color processes. They are generally small (8-12 u ) and round; their granules are not as well STUDIES ON DEVELOPING MAST CELLS defined as are those in the other types; their nuclei are round to ovoid, sometimes slightly indented and may look very much like nuclei of fibroblasts. Most often, these cells are located in the dermal papillae of the skin or right under the basal layer of the epithelium in the tongue. Occasionally however, some of them can be seen in the deepest parts of the organs, in the connective tissue or in the muscular layers. In the rat tongue, rough enumerations were made which show that these cells represent approximately 10-12% of the total number of mast cells; this percentage seems to vary from organ to organ and further studies are necessary to obtain more accurate data on this point. Fig. 1 Section from the tongue of the rat. A.B.S. mixture. Magnification = 1,200 x. This picture was taken through a blue filter in order to enhance the photographic black and white contrast between “blue” and “ r e d cells. Three mast cells are visible here: the two located on the sides of the frame are “red” cells and appear black. The one in the center which appears grey is a “blue” form. B. Safranin positive m a s t cells (red mast cells). These mast cells lack any affinity for Astra blue and stain red with safranin. They can be found everywhere in the sections, being by far the most numerous type. They are ovoid or elongated in shape and larger than the “blue” cells. Their granules are very distinct and exhibit the so-called “safranin metachromasia,” which means that they stain in a red-orange shade which differs from the original bluish-pink of safranin. C . Mixed cells. This third category is represented by elements which have a dou- 267 ble affinity for both Astra blue and safranin. In these cells, the granules are generally stained in a dirty brownish blue; in some instances, both red and blue granules may be seen inside the cytoplasm, side by side. These elements, probably, represent a transitional form between the two other types. 2. Embryonic tissues (fig. 2). The development of mast cells in the rat embryo has been studied by Schauer and Eder (’62) in relation to the appearance of the enzymatic activity of these cells. These authors found that mast cell polysaccharides are first demonstrable on the sixteenth-seventeenth day of pregnancy. In the first stages of development, around the fifteenth day, these polysaccharides are diffusely distributed in the cytoplasm and, according to the tests made with toluidine blue in solutions of graduated pH, only a few of them exhibit a strong acidic reaction. At this time, however, most of them show a diffuse PAS positive reaction. Fig. 2 Section of the skin of a newborn rat, showing a large mast cell population, consisting only of “blue” cells. A.B.S. mixture. Magnification = 90 X. In our sections, stained with the ABS mixture, it has been found that mast cells can be identified in the embryos by the sixteenth day after mating. They are few in number and are located in the dermis or in the hypodermis. They appear as small round cells with a round nucleus and a cytoplasm which contains flakes of Astra blue positive material. Progressively, granules appear to be formed from this material and by the nineteenth day, mast 268 ALEXIS L. BURTON cells are now present which exhibit a few blue granules. At the twenty-first day (i.e. immediately before birth), a large mast cell population exists which is entirely composed of small round cells with blue granules. After birth, this situation remains practically unchanged for the first week. “Red” mast cells then begin to appear and progressively increase in number so that at about 12 days, the mast cell repartition, with its three types of cells, is similar to that found in the adult. 3 . Comparative staining procedures. The skin of the fetus of the rat at birth containing a large and pure population of “blue” cells, whereas the adult skin contains a majority of “ r e d cells. These tissues thus provide excellent material for a comparative study of these two types with regard to their reactivity toward other classical staining procedure. Aldehyde fuchsin was of no use for differentiating these various kinds of mast cells. Both cell types are aldehyde fuchsin positive. Following staining for testing metachromasia, no true “orthochromatic” cells were observed. Slight variations in the metachromatic shift could be noted in some cells, but this is perhaps not chemically significant. In all cases, however, after the use of a combination Astra blue-P.A.S., all these cells show a definite and strong affinity for Astra blue. This is true whichever is the order in which the two dyes are applied. A slight P.A.S. positivity was observed in some cells, when P.A.S. was used separ ately . It has also to be noted that, after a sulfation procedure, all blue mast cells change their affinity and stain red with safranin in the A.B.S. mixture. DISCUSSION In adult tissues “blue” mast cells are smaller in size and less granulated than are the “ r e d ones. It was suspected that these “blue” cells were younger forms. This view has been supported by the finding that, in the Embryo, mast cells appear as Astra blue positive elements, and that safranin positive cells are to be found only later, i.e., a few days after birth. In this respect, the staining procedure using the A.B.S. mixture is of great interest for the demonstration of young and adult forms of mast cells. The combined use of the two dyes in a single mixture proves that we are dealing here with true affinities of the respective dyes for granules constituents. All artefacts due to variations in concentrations, pH, time of staining, temperature and differentiation can be definitely eliminated by this method. It has to be emphasized that fixation plays a very important role here. The differential staining of various types of mast cells cannot be observed after fixation in Carnoy’s fluid for instance, but only after fixation with formaldehyde or with a formalin-alcohol mixture. It would be of the greatest interest to know the chemical significance of this double reactivity of mast cells. This point is only partially explained by results of comparative studies with the other staining techniques. Aldehyde fuchsin was described by Gomori as specific for elastic fibers. Later, it was found by many authors that this dye was also excellent for the demonstration of mast cells and polysaccharides. In ’53, Halmi and Davies demonstrated that no correlation exists between aldehyde fuchsin, metachromasia and P.A.S. reactivity in various tissues. They concluded that “no suggestion can be made regarding the histochemical basis of aldehyde fuchsin staining.” In our observations, all types of mast cells were aldehyde fuchsin positive, regardless of their reactivity toward the A.B.S. mixture. The metachromatic shift of thiazine dyes has been related to the presence of sulfate groups in the molecule of the polysaccharide (Lison, ’36). A very extensive literature has been published on this topic, and discrepancies exist among the interpretations of various authors concerning the mechanism and the chemical significance of this reaction. Two important points have to be emphasized here: 1. Metachromasia may occur in the presence of carboxylic groups (when the reaction is performed at relatively high pH), or also in the presence of strong acidic groups (S03H-, P03-strong organic groups?) when the reaction is induced at lower pHs; in the STUDIES O N DEVELOPING MAST CELLS 269 TABLE 1 Stains Aldehyde-fuchsin Metachromasia P.A.S. Astra blue alone Safranin alone A.B.S. mixture “Neutral” polysaccharides Acidic polysaccharides Strongly acidic polysaccharides ++ ++ ++ + blue ++ ++ t ++ ++ +++ + ++ red ? latter case, the procedure can be considered as reasonably specific, if not for sulfate groups, at least for strong acidic groups. 2. In the mast cell granule, it is obvious that the acidic groups of the polysaccharides are not free groups, but are bound to other molecules (histamine, proteins). In this way, the reactivity toward the metachromatic test depends not only on the presence nor on the number of these groups, but mainly on the number of groups which can be made free and so become available for a binding with the dye molecule (Walton and Ricketts, ’54). In our observations, all mast cells exhibited a more or less intense metachromasia, evidencing strong free acidic groups; this is furthermore substantiated by the fact that this reaction was found positive even at very low pH (pH = 0.5). In the embryonic tissues, and in the adult tissues, after a sulfation procedure, all mast cells exhibit a striking safranin positive reactivity with the A.B.S. mixture. It is, however, questionable whether we are really dealing here with a sulfation process and not with a simple oxydation. These variations in the staining properties correspond, most probably, to the number of acidic or sulfate groups in the polysaccharide. The apparent discrepancies which seem to exist between the various staining procedures are due to the fact that these techniques are only relatively specific. Their specificity can be modified by varying the pH, on the other hand, the range of reactivity of each slightly overlaps each other, as shown in table 1, which summarizes our findings. LITERATURE CITED Bloom, G., and J. W. Kelly 1960 The copperphtalocyanin dye “Astrablau” and its staining properties, especially the staining of mast cells. Histochemie, 2: 48-57. + blue/red Fasske, E. 1957 Zur Darstellung der sauren Mukopolysaccharide im Bereich der Zwischensubstanz mesenchymaler Gewebe. Ztbl. allg. Path. path. Anat., 97: 174-179. Gedick, P. 1956 Uber neue Farbeverfahren fur Mukopolysaccharide und Phospholipide. Ztbl. allg. Path. path. Anat., 95: 486. Gomori, G. 1950 Aldehyde fuchsin. A new stain for elastic tissue. Am. J. Cli. Path., 20: 665-666. Halmi, N. S . , and J. Davies 1953 Comparison of aldehyde fuchsin staining, metachromasia and periodic acid Schiff reactivity of various tissues. J. Histochem. Cytochem., I : 447-459. Lison, L. 1936 Histochimie animale. Methodes et problemes. Gauthier-Villars edit. Paris, p. 237 sqq. Moore, R. D., and M. D. Schoenberg 1957 Low temperature sulfation of tissues and the demonstration of metachromasia. Stain Techno., 32: 245-247. Mowry, R. W., and Ch.H. Winkler 1956 The coloration of acidic carbohydrates of bacteria and fungi in tissue sections with special reference to capsules of cryptococcus neoformans and staphylococci. Am. J. Path., 32: 628. Pearse, A. G. E. 1960 Periodic acid-Schiff technique (after McMannus). I n : Histochemistry, Theoretical and Applied. J. A. Churchill Ltd. edit. London, 2nd. edition, p. 832. Pioch, W. 1957 Uber die Darstellung saurer Mukopolysaccharide mit dem Kupferphtalocyaninfarbstoff Astrablau. Virchow’s Arch. path. Anat., 330: 337-346. Schauer, A., and M. Eder 1962 Die Entwicklung von Mukopolysacchariden und Bildung histochemisch nachweisbarer Enzyme wahrend der Mastzellreifung. Virchow’s Arch. path. Anat., 335: 72-83. Spicer, S . S. 1960 Siderosis associated with increased lipofucsins and mast cells in aging mice. Am. J. Path., 37: 457-475. 1963 Histochemical properties of mucopolysaccharide and basic protein in mast cells. Ann. N. Y. Acad. Sc., 103: 322-332. Steedman, H. F. 1950 Alcian blue 8 GS: a new stain for mucin. Quart. J. micr. Sc., 91: 477479. Walton, K. W., and C. R. Ricketts 1954 Investigation of the histochemical basis of metachromasia. Brit. J. exp. Path., 35: 227-240.