Staining of CARBOHYDRATES

Periodic Acid Schiff/PAS

PAS with Diastase
Best Carmine

Langhans
Iodine
method
(Carletons method)
Oldest stain, considered obsolete
Rapid stain but not a permanent
stain as it fades after a few
months
Fresh
Frozen
Azure
A
Metachromatic Stain
Alcian Blue Technique
Metachromatic
staining
Toluidine Blue Staining
Combined Alcian Blue PAS
Technique

For glycogen
For glycogen

For glycogen
Mast cell granules
Fibrin
Mucin
Not specific for glycogen
May also stain amyloid

For glycosaminoglycans
Most
popular
method
demonstration of acid mucins
For glycosaminoglycans

for

general

Demonstration of mucins
Separating acid mucins and neutral
mucins
MucicarmineStain
For mucus
Southgates
Mucicarmine For encapsulated fungi like Cryptococcus
Technique
neoformans
Hales Dialyzed (colloidal) Iron For acid mucins
Technique
Fluorescent Acridine Orange For acid mucopolysaccharides
Technique
Disadvantage
is
that
it
is
temporary and will only last for
about 2 hours once the section is
mounted
Staining of FATS or LIPIDS

Sudan Black
Sudan IV (Scharlach R)
Oil Red O method in Dextrin
Osmic Acid Stain
Not a dye but an unstable oxide
Used as fixative for electron
microscopy and in histochemistry
Nile Blue Sulfate

For lipids mainly triglycerides

For fats
Demonstration of unsaturated fats

For neutral fats

Cholestrin esters and cholestrin fatty acids
Cerebrosides
Fatty acids and soap

Toluidine
Method

Borohydride
(BHPS)

Blue

Acetone For sulfatide deposits

Periodic

Schiff

For gangliosides

Staining of PROTEINS

Alkaline Fast Green Method

Peracetic Acid Alcian Blue

Sakaguchis test

For basic proteins especially protamines and

histones
For cysteine and cysteine
For arginine

Staining of ENZYMES

Gomori Calcium method

Gomori Lead method
Lead
method
for
5nucleotidase
(Wachsstein
and Meisel)
Alpha
naphthyl
acetate
method
for
non-specific
esterases
Indoxyl acetate method for
non-specific esterases (Holt
and Withers)
Tetrazolium
method
for
monamine oxidase (Glenner
et. al)

For alkaline phosphatase

For acid phosphatase
For 5-nucleotidase

For non-specific esterases

For esterase activity

For monoamine oxidase activity

Staining of NUCLEIC ACID

Fuelgenstechinique for For DNA

nuclear DNA
Most reliable and specific
histochemical
staining
technique for DNA, best
known for chromatin and
nucleoproteins
Methyl
green-pyronin For RNA and DNA
method
Acridine
Orange For RNA and DNA
fluorescent staining
Most
commonly
used

fluorochrome
to
demonstrate DNA and RNA

Staining of CONNECTIVE TISSUE

Gomoris Silver Impregnation

Van Gieson
Massons Trichrome Stain
Weigerts Elastic Tissue Stain
Orcein (Taenzer-Unna-Orcein)
KrjiansTechinique
(employing
congo red)
Mallorys
Phosphotungstic
Acid
Hematoxylin (PTAH) method
Highmans Congo Red Technique
Method of choice in many laboratories
in demonstrating amyloid
Krajians
Amyloid
Staining
(Modified Bennhold Method)
Methyl Violet Crystal Violet
Method
Induced Fluorescent Staining with
Thioflavine T
Fluorescence may be imparted to
amyloid by staining with thioflavine T
and exposing the tissue to ultraviolet or
quartz iodine lamps

For reticulin fibers

For collagen
For collagen fibers
For elastic fibers
For elastic fibers
Repaid method of staining elastic fibers

Stain for both CNS material and

general tissue structures
For muscle striations
For amyloid

For amyloid
For amyloid
For amyloid

Staining of BONE MARROW and BLOOD ELEMENTS

Rapid

Toluidine

Eosin

Glycol methacrylate section

stain
for
glycol
methacrylate section
Wrights Giemsa Jenner
Stain
Peroxidase
Reaction
for
Myeloid cells

For myeloid cells except basophils

Staining of MUSCLE and BONE

Modified
Gomoris
Trichrome Stain
Mallorys Phosphotungstic
Acid Hematoxylin (PTAH)
Heidenhains
Iron
Hematoxylin
Lissamine
Fast
Red
Tartrazine
SchmorlsPicroThionin
Method

For muscle fibers and collagen

For muscle, neuroglia, myelin, collagen
Muscle striations, mitochondria,
chromatin
For muscles and bones

myelin,

and

For bones (lacunae, canaliculi, and bone matrix)

Staining of CENTRAL NERVOUS SYSTEM

Bielschowsky Technique
Bodians Stain

Sevier Munger Technique

Cresyl Fast Violet (Nissl) Stain for

paraffin sections
Weigert Pal Technique of staining
Normal Myelin Sheath
Kluver and Barrera Luxol Fast Blue
Stain
for
myelin
with
Nissl
Counterstain
Luxol Fast Blue H&E Stain

For neurons, axons, and neurofibrils

For nerve fibers and nerve endings

For demonstrating neuritic plaques
and neurofibrillary tangles for the
diagnosis of Alzheimers disease
Foe neural tissues
Commonly used for demonstrating
neuritic plaques and neurofibrillary
tangles for the diagnosis of
Alzheimers disease
For missle substance, neurons
For myelin sheath
For myelin

For myelin

Luxol Fast Blue PAS Hematoxylin

Stain
Weils Method
Cajals Gold Sublimate
Modified
holzers
astrocytic processes

Method

For myelin
For myelin sheath
For astrocytes, nerve cells and nerve
fibers

for

Staining of TISSUE PIGMENTS and DEPOSITS

Perls Prussian Blue

Gomoris Prussian Blue

Turnbulls Blue Reaction for
ferrous iron (hemosiderin)
Benzidine Nitroprusside Stain
Modified Fouchets Technique
Schmorls
Ferric
Ferricyanide
method for reducing substances

Gomoris Aldehyde Fuchsin

Mallorys Fuchsin Stain
Masson Fontane Technique
Widely
used
for
melanin
demonstration

Calcium Dye lake Reaction

Von
Kossas
Silver
Nitrate
Method
Lindquists Modified Rhodamine
Technique

For hemosiderin the iron containing

pigment of hemoglobin, seen as yellow to
brown granules normally found inside the
cell. It is the most common hemoglobin
derivative
Stain for iron pigments
For hemosiderin
For hemoglobin and oxidase granules
For liver bile pigments

For argentaffin cells, chromaffin

For thyroid colloid For bile, melanin,
lipofuscins yellow brown to reddish
brown pigment produced by slow
oxidation of lipids and lipoproteins, it
can be found in hepatocytes, cardiac
muscle cells, adrenal cortex, and other
organs
For lipofuscin
For hemofuscin pigment
For argentaffin granules and melanin an
autogenous pigment (brown or black)
normally found in the skin and eyes
pathological deposition of melanin occurs in
benign lesions such as nevus or mole or in
melanoma
For staining skeletal system in embryos and
fetuses
For calcium demonstration
For staining copper

Staining of MICROORGANISMS

Gram Twort Stain

Brown and Brenn

For bacteria
For bacteria, Nocardia, and Actinomyces

ZiehlNeelsen Method
Wade Fite Technique

Auramine Rhodamine Fluorescent

Method
Toluidine Blue Stain for Helicobacter
Cresyl Violet Acetate Method for
Helicobacter
Dieterie Method
Levaditis
Wathin-starry Method
Modified
Steiner
and
Stainer
Technique for Spirochetes

GrocottMethamine Silver (GMS)

LendrumsPhloxine-Tartrazine Method
Rapid Giemsa

For AFB
For leprosy bacilli
Nocardia
For Mycobacteria

(M.

leprae)

and

For Helicobacter
For Helicobacter
For Legionella pneumophilia
For spirochetes
For spirochetes
For spirochetes
Donovan bodies
Fungi and bacteria
For fungi
For viral inclusions
For HBsAg

For blood and bone marrow

parasites (Leishmania, Malaria,
and Trypanosomes)
Inclusion conjunctivitis
Toxoplasma
Spirochetes and other bacteria

EXFOLIATIVE CYTOLOGY

Microscopy study of cells that have been shed, desquamated/ scrapped off from lining
epithelium and mucosal surfaces.

PURPOSES

Assess malignant/cancerous condition Detect asymptomatic cancer in women

assess femal hormonal
activity (sterility, endocrine disorder) determine genetic sex and presence of
infection

ACCURATE CYTOLOGIC INTERPRETATION OF CELLULAR MATERIALS DEPENDS ON

Method of specimen collection

fixation, fixatives
PRSV of fluid specimen prior to processing
Prep of materials for microscopic exam
Staining and mounting of cell sample

COLLECTION OF SPECIMEN
Vaginal scraping = scraping/swab
Endocervical & endometrial scrapings = swab, curettage
prostatic secretion = prostatic massage
bronchial aspirate/ sputum = deep cough/ bronchoscopy
serous fluid = pleural (thoracentesis), pericardial (pericardiocentesis), peritoneal
(paracentesis)
gastric & duodenal = nasogastric tube aspiration
nipple discharge
CSF fluid = lumbar puncture
Bone marrow aspirate = BM tap
urinary sediment = urine collection, catheterization
PAP SMEAR/ ROUTINE CERVICAL/ ENDOCERVICAL

Endocervical mucus = prevent air-drying during collection of the subsequent cervical

component.
extended tip spatula = scrape material from the whole circumference of the cervix
slide = spread evenly to the frosted end
fixation = immediately, drop slide to fixative or spray (8-12 inches)

FIXATION & FIXATIVES

decompose rapidly
cellular & nuclear details are destroyed before fixation
poorly preserved specimen
marked distortion on cell constituents
smear = before staining (1 hour)

COMMONLY USED FIXATIVES

Alcohol - ether Best fixative for cytological smear

95% ethyl alcohol
most widely used

universal fixative

doesnt contain volative & flammable ether

used for vaginal, cervical, prostatic, breast, biopsy smears
used for final fixation of all smears in lab

Carnoys fluid
for bloody spn
hemolyze RBC
Stain in hematoxylin (reduce overstaining)
3-5 minutes

Coating fixatives
hairspray with high OH content
has lanolin/oil as fixative
dual action (fix cells when dry, form a thin coating)
not recommended for fluid smear
not recommended for blood (clumps RBC)

High mucus content (sputum, bronchial, mucocoele) = PRSV 12-24 hr if ref

High protein content (pleural, peritoneal, pericardial) = PRSV 24-48 hr if ref
Low mucus content (urine, CSF) = 1-2hr Low pH (gastric) = collect on ice

TRANSPORTATION/MAILING OF SPECIMEN

Air dry after fixation = 2 hr

Place in wooden cardboard/ plastic slide containers = avoid spillage of fixative
Glycerin technique = fix smears for 30 min, cover with glycerin

FIXATION OF FLUID SPECIMEN

Place in alcohol, and ref

Pleural 50%, Peritoneal 50%, Sputum 70%, Urine 95%, bronchial & gastric 95%

PREP OF MATERIAL FOR MICROSCOPIC EXAM

Smearing
Streaking
Spreading
Pull apart
touch/imprint

CONCENTRATING CELLS FROM FLUIDS

(Cell in fluids: CSF, coelomic fluid, urine, cell suspension)
cell pellet with swing out centrifuge
direct concentration (cytocentrifuge) (spray fixative for papaniculao)
filter techniques (millipore, nucleopore)

ADHESION AND ADHESIVES

Secretion : viscid, directly immersed, fixed with ether alcohol

REQUIRE ADDITION OF AN ADHESIVE AGENT

Urinary sediments
bronchial lavage specimen
specimen with proteolytic enzyme during processing (trypsinfrom GIT)

CHARACTERISTIC OF A GOOD ADHESIVE AGENT

permeable to both fixative and stain

retain the stain

EXAMPLES OF GOOD ADHESIVE AGENTS FOR CYTOLOGIC METHOD

Pooled human serum or plasma

Celloidin ether alcohol
Leuconostoc culture

STAINING AND MOUNTING OF CELL SAMPLE COMMONLY USED STAINING TECHNIQUES

Pap smear (papnicolau)

H&E
Modified staining (wrights)

PAPS STAIN: Hematoxylin, Orange G6, Eosin 36

Advantages
Disadvantages
Transparent
blue
staining
of
Lengthy procedure
Complicated procedure
cytoplasm
Excellent nuclear staining

Doesnt give accurate acidophile

Color range is predictable and great
index
value in cells ID
STAINING RESULTS
Bacteria
Cytoplasm
Mycelia
Nucleus (pyknotic)
Nucleus (vesicular)
Trichomonas vaginalis

Dark blue
Bright red/ greenish blue
Violet
Dark blue to black
Blue
Pale greenish blue blob cytoplasm

INTERPRETATION OF PAP SMEAR

CLASS I
CLASS II
CLASS III
CLASS IV
CLASS V

Normal, Negative (no atypical/ abnormal

cell)
Cytology
atypical,
Negative
(no
malignancy)
Suspicious,
Cytology
suggestive
of
malignancy (not conclusive)
Positive,
strongly
suggestive
of
malignancy
Positive, Malignancy conclusive

NON EPITHELIAL CELLS

RBC

PMN
Histiocyte

menstrual flow
scraping of friable lesion
round/oval, eosinophilic, anucleate
Cell size, darkly staining poly lobate
nucleus
phagocytosis
small, eccentric nucleus, bean shape

Spermatozoa
EPITHELIAL CELLS

Basal Cell

Endocervical Cell

Endometrial Cell
Intermediate SC

Mature superficial SC

small, round, large nuclei

basophilic
Columnar, group appearance
Picket fences, palisade, honeycomb
pattern
Vesicular
nucleus
with
coarse
chromatin pattern
Cilia, plaque
cylindrical, less basophilic
Medium, polyhedral, elongated cell
Cytoplasm staining: basophilic
Nucleus:
large,
vesicular,
fine
chromatin network
a) Navicular cell: boat shaped, tendency to
fold/curl on edges. Has combined E-P effect
(androgen stimulation)
b) Pregnancy cell: round, oval, boat shaped.
Translucent basophilic cytoplasm. Double
walled boundary. Deeper blue cytoplasm
Large, flat, polygonal, translucent
cytoplasm
Cytoplasm
staining:
acidophilic,

Parabasal SC

Superficial

eosinophilic
Dark and keratohyaline granules
Nucleus:
small,
dense,
homogenous, pyknotic nucleus
a) True Acidophlia = cells under estrogen
influence
b) Pseudoacidophilia = drying of cells
during fixation, prolapse, infection, chemical
Thick, oval, fried eggs sunny side
up
Cytoplasm staining: basophilic
Nucleus: large vesicular
2 weeks of age to puberty
polyhedral,
flat,
acidophilic/basophilic, small dark
pyknotic nuclei.
-smear conta from vulva
-epidermization of vagina from prolapse
-leukopakia of cervix
-ruptured membrane in pregnant woman
-hyper estrinism

QUANTITATION IN VAGINAL CYTOLOGY

Acidophilic/ Eosinophilic index AI/EI

Crowded cell index CCI

Femininity index - FI
Folded cell index FCI
Karyopyknotic index KPI

Maturation Index MI

Eos/Acido Cyanophilic
Mature SC in cluster 4 mature SC in
single/cluster 4
Superficial parabasal intermediate
Folded mature SC Flat mature SC
Mature
Superficial
Cell

Mature
Intermediate cells
Parabasal intermediate superficial

SMEAR WITH ONLY INTERMEDIATE CELLS:

Sheehans dse
women without ovaries (castrated, child bearing age)
massive cortisone therapy
estratrophy
pure progresterone, without estrogen
androgen
LACTOBACILLUS ACIDOPHILUS (DORDERLEINS BACILLUS)
most important of all organism growing in vagina
low pH 3.8-4.3
stimulate vaginal epithelium
increase = corpus lutheum phase

FERNING
Mucus on drying exchibits a fern/ palm leaf pattern (arborization)
formation of salt crystals in high NaCl concentration
under estrogen influence
inhibited by progresterone
indicates basis of early pregnancy

IMMUNOHISTOCHEMICAL TECHNIQUES

IMMUNOHISTOCHEMISTRY

or IHC is the localization of antigens in tissue sections by the use of labeled antibody as
specific reagents through antigen-antibody interactions that are visualized by a marker
such as fluorescent dye, enzyme, radioactive element or colloidal gold.

ANTIGEN

a substance that stimulates antibody response. It has many antigenic sites on its surface
called epitopes which have the capacity of binding to the antibody.

ANTIBODY

defined as an immunoglobulin (Ig) capable of specific combination with the antigen. It is

produced in response to the invasion of an antigen in the body. It consists of two heavy
chains and two light chains, and named according to its heavy chains; e.g. IgG has
gamma type heavy chains and IgA has alpha light chains.

ANTIBODY TYPES
1. MONOCLONAL ANTIBODY

Binds to specific antigen. Monoclonal antibodies are generally considered to

exhibit greater specificity than polyclonal antibodies.
2. POLYCLONAL ANTIBODIES - are a heterogeneous mix of antibodies that recognize
several epitopes of one antigen. They are made by injecting animals with peptide
antigens, and then after a secondary immune response is stimulated, isolating
antibodies, against different epitopes of the antigen, from whole serum.

TECHNIQUES OF IHC STAINING

DIRECT IHC STAINING

o one-step staining method, and involves a labeled antibody reacting directly with
the antigen in tissue sections. This technique utilizes only one antibody and the
procedure is therefore simple and rapid.
INDIRECT IHC STAINING
o
involves an unlabeled primary antibody (first layer) which reacts with tissue
antigen, and a labeled secondary antibody (second layer) which reacts with the
primary antibody.

TECHNICAL ASPECTS OF IHC STAINING

1) FIXATION AND SECTIONING
A. To ensure the preservation of tissue architecture and cell morphology, prompt and
adequate fixation is essential.

2) TISSUE ADHESIVES
A. In case of IHC, the tissue section is liable to fall off due to frequent rinsing of the
slides, use proteolytic enzymes or microwave. This problem can be avoided by using
positively charged slides or coating the slides with adhesives e.g. poly-L-lysin,
albumin
3) ANTIGEN RETRIEVAL
A. involved the application of heat for varying lengths of time to formalin-fixed, paraffinembedded tissue sections in an aqueous solution (commonly referred to as the
retrieval solution).
4) BLOCKING
A. Inadequate or delayed fixation may give rise to false positive results due to the
passive uptake of serum protein and diffusion of the antigen. Such false positives are
common in the center of large tissue blocks or throughout tissues in which fixation
was delayed.
5) STAINING
A. PAP Method (peroxidase anti-peroxidase method)
is one development of the indirect technique and it involves a third layer
which is a rabbit antibody to peroxidase, coupled with peroxidase to make a
very stable peroxidase anti-peroxidase complex. The complex, composed of
rabbit gaba-globulin and peroxidase, acts as a third layer antigen and becomes
bound to the un-conjugated goat anti-rabbit gabaglobulin of the second layer.
B. Avidin-Biotin Complex (ABC) Method
ABC method is standard IHC method and one of widely used technique for
immunohistochemical staining. This technique involves three layers. The first
layer is unlabeled primary antibody. The second layer is biotinylated secondary
antibody. The third layer is a complex of avidin-biotin peroxidase. The
peroxidase is then developed by the DAB or other substrate to produce
different colorimetric end products.
C. Labeled Strept-Avidin Biotin (LSAB) Method
The first layer is unlabeled primary antibody. The second layer is biotinylated
secondary antibody. The third layer is enzyme-strept-avidin conjugates to

replace the complex of avidin-biotin peroxidase. The enzyme is then visualized

by application of the substrate chromogen solutions to produce different
colorimetric end products.

CONTROLS OF STAINING
1) Negative control: stained sections following all steps of IHC except for the step of antibody
application which is replaced by non-immune serum or even PBS. It is done in every run to
avoid interpretation of any non-specific staining (seen also in the negative control) as specific.
2) Positive control: a stained section known to be positive for the antigen following all steps
of IHC, exactly as the specimen. It is done in every run to verify the validity of the technique
and the quality of the reagents.

NUCLEAR IMMUNOSTAINING
ER nuclear staining
A- benign adenosis
B- B- Ductal carcinoma

P63 nuclear immunostaining

CYTOPLASMIC IMMUNOSTAINING

Cytoplasmic immunostaining of metalloproteinase in prostatic carcinoma

MEMBRANOUS IMMUNOSTAINING

Her-2 immunostaining in breast cancer