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Histochemical studies on developing mast cells.

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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.
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