mikro-graf

Volume
Spring 2 0 1 2

41
Issue

02 O ff ic ial Publ ication of the Mich ig an S o c ie t y of Hi stotech nolo g i sts

of Histo
iety te
oc
MAST CELLS

ch
S
Michigan

nolo s
gist
Peggy A. Wenk, HTL(ASCP)SLS
William Beaumont Hospital, Royal Oak
Tech Points
Unusually prominent mast cells in an H&E.
photo by Ed Uthman, Flickr
AhhhSpring is here!
Beautiful flowers!................ Sneezing!

this issue Trees in leaf!........................ Runny eyes!

Presidents Message
Editors Note
Walks in the fields!............. Hives!
2
Blooming gardens!.............. Asthma!
2
Pollenating insects!............. Anaphylactic shock!

Inbox
Test Your Knowledge
Springtime in Michigan
MSH News & Events
Student Spotlight:
Decalcification Comparison 12
Signals: Helicobacter pylori
Around The Region
NSH Update
Officers And Chairpersons
W
hy is spring a wonderful time of the year for some people, while
3
allergies make it so miserable for others? Mast cells are one of the
culprits. Besides being involved in allergies, mast cells are also
found in some tumors, skin diseases, and inflammation of various organs.
3 All of these conditions might involve a biopsy or specimen being sent to the
histology laboratory for diagnosis. So what are mast cells, what do they do,
and how do we stain for them?
8
Mast cells were first described by Paul Ehrlich in his 1878 doctoral thesis,
which described their granules and staining characteristics. Erlich named the
9 cells "mastzellen", from the German word for breast, as he thought that the
granules fed or nourished the surrounding cells. His paper emphasized impor-
tant histological chemical reactions, particularly of aniline dyes. He used the
mast cell to discuss the application of these dyes to tissue. He also empha-
sized the need to diagnose mast cells based on their staining characteristics,
not just by their shape and size.
Mast cells are immune cells that stay in the body for a long time. They appear
15 species-wide in all organs and connective tissue that have blood circulation.
Mast cells are not found in avascular tissues such as the cornea, cartilage and
mineralized bone. They usually appear in the tissue close to blood vessels,
16 and also in any surfaces that are the boundary between the outside and the
body, such as skin, gastro-intestinal tract and the lungs. There are also more
mast cells in the areas of the body that are more likely to be injured, such as
17 the feet, the nose and the mouth. Therefore, one of the roles of mast cells
is to be among the first cells to defend against invasion by outside (foreign)
agents.
19

[continued on page 4]
 Mikro-Graf
[continued from page 1]
Mast cells were originally thought to be related to basophils.
While they both probably originate from the same hemato-
poetic stem cell, they diverge almost immediately after that.
Basophils are entirely mature when they leave the bone
marrow, whereas mast cells do not mature before leaving
the bone marrow. Mast cells disperse, settling into various
tissue types. Their final location, the proteins (cytokines,
interleukins) and other tissue factors (fibroblasts, endothe-
lial cells, etc.) encountered will determine the type of mast
cells and characteristics (type of granules) into which they
will mature. There are two main types of mast cells:
MCT (mast cell-tryptase), are immune cell-associ-
ated, contain tryptase and are found in the mucosa
of the intestine and the respiratory system, where
they associate with T-cells.
MCTC (mast cell-tryptase-chymase) cells contain
both tryptase and chymase, along with other pro-
teases, and are found in connective tissue in places
such as skin, submucosa of stomach and intestine,
breast, heart, lymph nodes, conjunctiva of the eye,
and synovium of joints.
A third type of mast cells containing chymase but not
tryptase have been identified.
MCC (mast cell-chymase) seem to be found mostly
in the submucosa and/or mucosa of the stomach,
small intestine, and colon.
Because mast cell characteristics are based on the tissue
type where they are located, mast cells in different animals
will also have different characteristics.
CELL MORPHOLOGY AND GRANULES
Human mast cells are round to oval shaped, about 8 to 20
m in diameter. The nucleus is also round to oval shaped.
The cytoplasm has lots of secretory granules, about 1.5 m
in diameter. The secretory granules contain dozens of dif-
ferent types of proteins, which they subsequently release.
These proteins allow the mast cells to move about, change
their environment, cause the itching/swelling/allergy symp-
toms, attract other white blood cells (WBC), promote blood
vessel growth, and repair injured tissues. Table 1 contains
a partial list of mast cell proteins.
INFLAMMATION AND GRANULAR DEGRADATION
Acute inflammation caused by mast cells is actually a good
thing. It can isolate the area that is damaged, call other
inflammatory cells (and their proteins) to the area, and start
to repair the damaged tissue.
Chronic inflammation, on the other hand, is not only longer
lasting, but in all likelihood, is too much of a good thing.
After being exposed to an allergen, i.e., pollen or a cat, the
body does not really need to over-react by causing a runny
nose, asthma, or even anaphylactic response. Autoimmune
diseases are also a type of chronic inflammation. Mast cells
are implicated in most of these conditions.
4 www.mihisto.org
Mast cell membranes bind to the Fc portion of IgE antibod-
ies. IgE is produced in small amounts by plasma cells.
IgE is supposed to protect the body from parasites, but is
found in abundance in allergic reactions to all other types of
allergens. Exposure to the allergens will cause the specific
antigen to bridge IgE molecules on the surface of the mast
cell. This will cause the mast cell to release many of the
proteins found in the granules, particularly the histamine
and some of the proteases. This is known as degranulation.
There are two different pathways: Exocytosis and Piece-
meal degranulation.
Exocytosis is also known as anaphylactic degranu-
lation. Large amounts of granules will very quickly
bind to each other and to the mast cell membrane.
This will open up channels in the mast cell, allowing
the quick release of the granule contents. This is the
cause of the symptoms in very severe acute allergic
reactions and anaphylactic shock.
Piecemeal Degranulation is much slower, with only
some of the granules being released slowly and with-
out the granules binding together or making channels
to the mast cells exterior. This is more often seen in
chronic inflammation or with cancer.
Table 1: Mast Cell Proteins
Proteases that degrade neuropep-
Tryptase, Chy-
tides and interleukins, break apart
mase
collagen.
Dilates blood vessels, increases blood
vessel permeability allowing some
WBC and proteins to pass through,
causes edema (swelling) in the imme-
diate area (runny nose, watery eyes,
wheal from an insect bite), warmth,
and redness.
Histamine Contracts smooth muscles, such as in
lung bronchi (asthma) and the GI tract
(diarrhea).
Irritates nerves, causing pain and
itching.
Increases mucus production (such as
in asthma).
Acidic sulfated glycosaminoglycans.
Acts as anticoagulant, involved in
Heparin
angiogenesis (growth of new blood
vessels in an area).
Attract all other inflammatory cells to
Cytokines the area, such as monocytes, T and B
lymphocytes and neutrophils.
Involved in tissue remodeling involv-
Urokinase
ing fibroblasts and endothelial cells.
Blood vessel growth factors involved
FGF, VEGF
in angiogenesis.
MG41.02
 Official Publication of the Michigan Society of Histotechnologists
DISEASES INVOLVING MAST CELLS
Allergies: Obviously, mast cells play a large role in all
allergies, whether in the skin (itching, eczema) or in the
nose, eyes and lungs (runny nose, runny eyes, asthma).
These are examples of localized mast cell degranulation.
Anaphylaxis involves systemic mast cell degranulation,
which can lead to such severe blood vessel dilation (and
the side-effects) that death is pos-
sible. Taking an anti-histamine will
block the action of the released
histamine, but does not prevent
the release of histamines. Some of
the newer anti-allergy medications
work by stabilizing the mast cells,
stopping the channel formation, and
thus stopping the degranulation.
Autoimmune diseases: Mast cells
are involved in recruiting additional
inflammatory cells in autoimmune
diseases, such as rheumatoid arthri-
tis (synovial tissue and joint fluids),
bullous pemphigoid (subepidermal
blisters of the skin), and multiple
sclerosis (demyelination of nerves).
Interstitial cystitis: In this condition
an increased number of mast cells
in the bladder, particularly around
the nerves, cause the pain.
Implicated in: Cardiomyopathy,
atheroscerlosis, endometriosis.
Mast cell tumors: Mastocytosis
is a rare disease caused by hav-
ing too many mast cells and CD34
mast cell precursors. Most of these
tumors are found in the skin (cuta-
neous mastocytosis), with the most
common being urticarial pigmen-
tosa. Brown or red spots are found The role of mast cells in the development of allergy.
on the skin, and hives are often
seen in the area involved. Other
mast cell degranulation symptoms can also occur.
Involvement of any other organ is known as systemic
mastocytosis. In every case, the bone marrow is involved.
Sometimes, even organs that normally do not contain large
numbers of mast cells can be affected. Examples include
liver, spleen, lymph nodes, and the GI tract. People with
either type of mastocytosis live with severe allergic reac-
tions every day, and something as simple as rubbing the
skin can cause a massive release of the granules proteins,
leading to itching, hives and, sometimes, anaphylactic
response.
Other cancers: Other types of mast cell cancers include
mast cell leukemia and mast cell sarcoma.
Spring 2012
MAST CELL DEMONSTRATION IN TISSUE SECTIONS
Many types of traditional histology stains and several IHC
CD markers will demonstrate mast cells. If the mast cells
have been degranulated, positive staining will not be seen.
Tissues must also be quickly fixed, as the granules will
deteriorate with time.
Giemsa: The Romanowsky-type stains (Giemsa, Jenner-
Giemsa, May-Grunwald, etc.) are
polychromatic. They are comprised
of two distinct dyes, such as meth-
ylene blue and eosin, in a water,
alcohol and glycerin solution. Meth-
ylene blue is an impure thiazine dye
composed of numerous basic/cat-
ionic dyes (thionins, azures, methyl
violet). Because the composition
varies by manufacturer and the thi-
onin dyes will oxidize into the other
azure dyes as the dye powder and
stain solutions age, there can be
up to seven different blue dyes in
the Giemsa-type stains. Thus, tis-
sue components can vary from light
blue to violet. The second dye in the
staining solution is one (or more) of
the xanthene or eosin dyes, such as
eosin Y and/or eosin B.
The Giemsa-type stains are used
regressively. The tissue is over-
stained in the methylene blue/eosin
mixture, then the methylene blue is
differentiated with an acidic solution,
while the eosin is differentiated with
graded alcohols. The differentiation
steps are critical, as too much blue
in the background makes it difficult to
see the purple mast cells. However,
over-differentiation can remove the
purple color.
Giemsa-style stains result in baby-
blue nuclei, purple mast cells and
basophil granules, with a pink background.
Toluidine blue acid: Although staining results look the
same with the Toluidine blue-acid method as the Giemsa-
style stains (blue nuclei, purple mast cell granules, pink
background), the staining mechanism is different, being
metachromatic. Toluidine blue is a dye of the thiazine family
(basic/cationic), but is made up of only one dye toluidine
blue, which is a linear dye molecule.
A very dilute aqueous solution (<0.1%) of toluidine blue is
made at pH 4.0 with dilute acetic acid or hydrochloric acid.
Slides are stained for 1 hour, rinsed quickly in water, dipped
once in eosin, differentiated in 70% alcohol until very little
pink color remains in the tissue, then dehydrated, cleared
[continued on page 6]
www.mihisto.org 5
 Mikro-Graf
[continued from page 5]
and coverslipped. There is no differentiation step for the
toluidine blue, but it is critical to have a very pale eosin
(short staining time, long alcohol differentiation), so that
the pink of the background does not overwhelm the purple
mast cells.
The nuclei pick
up the toluidine
blue in a random
arrangement, and
are the baby blue
color. This back-
ground color is
called orthochro-
matic staining. In
Mast Cells Intestine, 40x.Toluidine blue. some areas, such
as granules of
mast cells, baso-
phils, and Nissl, the linear dye molecules bind to the tissue
close to each other in an almost parallel arrangement. The
parallel dyes then bind with each other via hydrogen bonds
from nearby water molecules. This bonding typically length-
ens the wavelength, which alters the color being seen. In
the case of toluidine blue, this color shift changes the ortho-
chromatic blue to a metachromatic purple/violet.
Stains for sulfated acid mucopolysaccharides (AMP):
Mast cells contain large quantities of sulfated acid muco-
polysaccharides. Therefore, they will stain brown with
aldehyde fuchsin (also used to stain elastin violet) and
baby blue with Alcian blue. However, it's difficult to see the
pale blue mast cells against the collagen, which also has
AMP that stains with the Alcian blue. As a result, neither
of these stains is recommended for the demonstration of
mast cells.
Leder Chloracetate esterase: Unlike most enzyme stains,
the chloroacetate esterase can be done on fixed, paraffin
embedded tissue. The slides are incubated in a solution
containing the substrate naphthol AS-D chloroacetate.
Then the esterase contained in the neutrophils and mast
cells binds with the chloroacetate. This releases the naph-
thol group, which binds to the diazonium dye pararosanalin
(basic fuchsin), another component of the incubating solu-
tion. Pararosanilin or basic fuchsin gives a deep pink-red
color to the granules, while hematoxylin counterstains the
nuclei blue.
When compared with
the Giemsa and Tolu-
idine blue-acid meth-
ods, the Leder stain
involves more tech
time, as all solutions
must be made at the
time of the stain. This
increases the cost
significantly over the
other two stains. Mast Cells Intestine, 40x.Esterase.
6 www.mihisto.org
IHC: Cell surface markers for the various types of mast
cells include: CD2 (early T cell marker), CD23 (low affinity
receptor for IgE), CD25 (cutaneous mast cells), CD68 (mast
cells, macrophages, melanomas), CD88 (mast cells, neu-
trophils, monocytes, macrophages, hepatocytes), CD117
(c-kit mutation found in systemic mastocytosis), CD203c
(mast cells and basophils, and their precursors). In addi-
tion, IHC for tryptase and chymase can be used.
Positive Controls
Tissue sections with a high number of mast cells, such as
urticarial pigmentosa, bladder interstitial cystitis, or skin
inflammations can be used.
Normal tissues that have a fair number of mast cells, like
small intestine or fallopian tubes, can also be used.
Our understanding of mast cells and of the special stains
used to demonstrate specific structures, began with a doc-
toral thesis presented 134 years ago on June 17, 1878, by
a 24 year old medical student named Paul Ehrlich, Thank
you, Dr. Ehrlich.
REFERENCES:
Crivellato E, Beltrami CA, Mallardi, F, Ribatti D: The Mast Cell: An
Active Participant or an Innocent Bystander? Histology and Histopa-
thology (2004) 19:259-270.
Crivellato E, Beltrami CA, Mallardi F, Ribatti D: Historical Review:
Paul Ehrlichs Doctoral Thesis: A Mile Stone in the Study of Mast
Cells. British Journal of Haematology. 2003. 123, 19-21.
Metcalf DD, Baram D, Mekori YA: Mast Cells. Physiological
Reviews (Oct. 1997) 77(4):1033-1079.
Wong E, Morgan EW, MacDonald: The Chloroacetate Esterase
Reaction for Mast Cells in Dermatopathology: A Comparison with
Metachromatic Staining Methods. Acta Dermatovener (Stockholm).
1982. 431-434.
Bancroft JA, Gamble M: Theory and Practice of Histological Tech-
niques, 6th edition. 2007. Churchill Livingstone.
Brown JA, Smoller BR: Special Stains in Dermatopathology. Chap-
ter in Connections 14 H&E and Special Stains.2010. Dako. www.
dako.com/28829_connection_14.pdf
Carson FL, Hladik C: Histotechnology: A Self-Instructional Text, 3rd
edition. 2009. ASCP Press.
Kiernan JA: Histological and Histochemical Methods: Theory and
Practice, 4th edition. 2008. Scion Publishing.
Stevens A, Lowe J: Human Histology 3rd edition. 2004. Mosby.
http://en.wikipedia.org/wiki/File:Mast_cells.jpg
MG41.02
 Earn 0.5 contact hours of continuing education by reading articles in the
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^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^
DATE OF ARTICLE: Spring 2012
TITLE: Mast Cells
AUTHOR: Peggy A. Wenk, HTL(ASCP)SLS

DIRECTIONS:
1. Answer the following questions by circling the one (1) BEST answer for each question.
2. Complete the information required at the bottom of the page.
3. Submit questions & check made out to MSH(in US funds) to: Sarah Bajer, 35669 Impala Dr, Sterling Heights, MI, 48312

To earn Continuing Education credit from MSH, completed form must be submitted within
twelve (12) months of original date of the article.

1. All of the following organs/tissues will have mast cells in them EXCEPT:
A. Bone
B. Liver
C. Lung
D. Skin

2. Which of the following components found in mast cell granules is most involved in the itching, runny nose and
watery eyes found in most allergies?
A. cytokines
B. heparin
C. histamine
D. tryptase

3. All of the following demonstrate mast cells in dark, easy to see granules against a light background, EXCEPT:
A. Alcian blue
B. Chloroacetate esterase
C. Jenner-Giemsa
D. Toluidine blue-acid

4. TRUE or FALSE (circle one): CD 117 marker will demonstrate a genetic defect seen in systemic mastocytosis.

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