HISTOPATHOLOGY
FINAL NOTES
PPT
NAME BSMT 3A
STAINING TECHNIQUES
STAINING METHODS
Counter Staining
 Is the application of a different color or stain to
provide contrast and background to the staining of
the structural components to be demonstrated.
Metachromatic Staining
 Entails the used of specific dyes which differentiate
particular substances by staining them with a color
that is different from that of the stain itself.
Regressive Staining
 With this technique the tissue is first over stained to
obliterate the cellular details and the excess stain is
removed or decolorized from unwanted parts of the
tissue, until the desired color is obtained.
Progressive Staining
 Is the process whereby tissue elements are stained in
a definite sequence and the staining solution is
applied for specific periods of time or until the
desired intensity of coloring of the different tissue
elements is attained.
Decolorization
 Is the selective removal of excess stain from the
tissue during regressive staining in order that a
specific substance may be stained distinctly from the
surrounding tissues.
Direct Staining
 The process of giving color to the sections by using
aqueous or alcoholic dye solutions.
Indirect Staining
 The process where the action of dye is intensified by
adding another agent or mordant which serve as a
link or bridge between the tissue and the dye, to
make staining reaction possible.
Vital Staining
 Selective staining of living cell constituents,
demonstrating cytoplasmic structures by
phagocytosis of the dye particle.
Intravital Staining
 Intravital staining of living cells is done by injecting
the dye into any part of the animal body, producing
specific coloration of certain cells, particularly those
of the reticulo-endothelial system.
Supravital Staining
 Is used to stain living cells immediately after removal
from the living body.
DIVISIONS OF BIOLOGICAL STAINS PREPARED FROM DYES
 Natural Dyes- are those obtained from plants and
animals, previously utilized for dyeing of wool and
cotton.
 Examples: Hematoxylin, Cochineal dyes and its
derivatives, Orcein, Saffron
HEMATOXYLIN
 Derived by extraction from the core of the heartwood
of a Mexican tree known Hematoxylin
Campechianum
 The active coloring agent is hematin, formed by the
oxidation of hematoxylin.
 Strong oxidizing agents: Hydrogen peroxide, Mercuric
oxide, Potassium permanganate, Na perborate, Na
iodate
COCHINEAL DYES
 Extracted from the female cochineal bug (Coccus
Cacti), W/c is treated with alum to produce the dye
carmine.
 When combined with aluminum chloride (Bests’
Carmine stain) is used for demonstration of glycogen.
ORCEIN
 Vegetable dye extracted from certain lichens
 Used for staining elastic fibers
 Litmus is also obtained from lichens, treated with
lime and soda and exposed to ammonia and air.
SYNTHETIC DYES
 Known as “Coal Tar Dyes”
 Derived from the hydrocarbon benzene and are
collectively known as Aniline dyes
 Chromophores are substances with definite atomic
groupings and are capable of producing visible colors
 Benzene contains chromogens
CLASSIFICATION OF CHROMOPHORE DYES
 Acid Dyes- where the coloring substance is found in
the acid component.
 Basic Dyes- where the active coloring substance is
found in a basic component that combines with the
acid radical.
 Neutral Dyes- formed by combining aqueous
solutions of acid and basic dyes, capable of staining
cytoplasm and nucleus simultaneously and
differentially.
 Ex. Romanowsky dyes (hematology), Giemsa stain
and Irishman’s stain (Leucocyte differentiation)
COMMON STAINING SOLUTIONS USED
HEMATOXYLIN
a. Aluminum Hematoxylin Solutions
are recommended for progressive staining of tissues
and can also be used for regressive staining
b. Erlich’s Hematoxylin- generally used for regressive
staining
-Stained mucopolysaccharide
substances (cartilage & cement lines of bones)
c. Harris hematoxylin- a good regressive stain. Used
also for routine nuclear stain, exfoliative cytology and
for sex chromosomes
d. Cole’s Hematoxylin- recommended for routine
purposes, especially used in sequence with celestine
blue.
e. Mayer’s Hematoxylin- Alum hematoxylin ripened
with Na iodate. Used as regressive and progressive
stain.
 Used as nuclear counterstain  Examples: Carbol-Fuchsin, Coleman’s
F. Iron hematoxylin- Used for differential or Feulgen Reagent, Schiff’s reagent, Mallory’s Fuchsin
regressive staining stain, Aldehyde Fuchsin
Examples: Weigert’s solution- using ferric ammonium
chloride and EXFOLIATIVE CYTOLOGY
Heidenhein’s solution using ferric ammoniun sulfate
as mordants. Demonstrates nuclear and cytoplasmic
inclusions, voluntary muscle straitions and myelin
G. Phosphotungstic Acid Hematoxylin (PTAH)
-It is a progressive stain
- Demonstrates structures in paraffin,
celloidin and frozen sections.
EOSIN
 Used for differentially staining connective tissues and
cytoplasm
 Used as counterstain after hematoxylin and before
methylene blue.
3 FORMS OF EOSIN
1. Yellowish (Eosin Y)- shows a green yellow
fluorescence especially in alcoholic medium.
2. Eosin B, Erythrosin B (Bluish)- deeper red color
3. Ethyl eosin- Eosin S, Eosin alcohol-soluble
OTHER STAINS USED
 Van Gieson’s Stain (Acid Fuchsin-Picric Acid)- is a
mixture of picric acid and acid fuchsin for
demonstration of connective tissues.
 Acridine Orange- is a basic acridine fluorochrome
which permits discrimination between dead and
living cells, giving green fluorescence for DNA and a
red fluorescence for RNA
 Acridine Red 3B- demonstrates deposits of Calcium
salts and possible sites of phosphatase activities.
 Alcian Blue- is for specific for connective tissue and
epithelial mucin.
 Aniline Blue- is a cytoplasmic stain used for
counterstaining of epithelial sections
 Basic Fuchsin- is a plasma stain used for deep staining
of acid-fast organisms, for mitochondria, for
differentiation of smooth muscles with the use of
picric acid.
 INTERMEDIATE FILAMENT MARKERS
1. ACTIN- IDENTIFY TUMORS FROM SMOOTH,
SKELETAL, AND CARDIAC MUSCLE
2. VIMENTIN-IS 57kD intermediate filament that is
present in normal mesenchymal cells
3. DESMIN
4. GLIAL FIBRILLARY ACIDIC PROTEIN (GFAP)
5. NEUROFILAMENT (NF)
6. S-100
NEUROENDOCRINE MARKERS
1. NEURON-SPECIFIC ENOLASE (NSE)
2. CHROMOGRANIN
3. SYNAPTOPHYSIN
GERM CELL TUMOR MARKERS
1. HCG-CHORIOCARCINOMA
EPITHELIAL TUMOR MARKERS 2. APP ( ALPHA-FETOPROTEIN)-
I. KERATIN-highly sensitive for marker for epithelial cells and Endodermal Sinus Tumors Showung Yolk Sac
non-epithelial (mesotheliomas and non-seminomatous germ Differentiation
cell tumors) 3. PLAP (PLACETA-
 1. CK7 (CYTOKERATIN 7)-more frequently found in Like Alkaline Phosphatase)-Germ Cell Tumors
carcinomas of the lung, breast, uterus, and ovaries (Germinomas), Embryonal Carcinomas,
(serous tumors). Choriocarcinomas And Endo-Dermal Sinus Tumors
 Negative for CK20 MESENCHYMAL TUMOR MARKERS
 2. CK20 (CYTOKERATIN 20) 1. MYOGENIC TUMORS-
 3. transitional cell carcinomas of the bladder and MUSCLE-SPECIFIC ACTIN AND DESMMIN, myo-D1,
mucinous ovarian tumors are usually positive for myoglobin and myogenin
both CK7 and CK20 2. FIBROHISTIOCYTIC TUMORS-
 4. Renal cell carcinomas, hepatocellular carcinomas, CD68 or FAM 56, combined with more nonspecific
prostatic adenocarcinomas, thyroid carcinomas and proteolytic enzymes such as alpha-1-antitrypsin and
squamous cell carcinomas (skin, lung and alpha-1-antichymotrypsin to diagnose malignant
esophagus)are negative for CK7 and CK20. fibrohistiocytic sarcomas
II. EMA (epithelial membrane antigen)-adenocarcinomas of 3. vascular tumors
breast, lung, kidneys 4. melanomas
5. Lymphomas
III. CEA (Carcinoembryonic antigen)-carcinomas of GIT,
CELL PROLIFERATION MARKERS
pancreas, lung, breast, ovary, uterus and cervix

IV. TTF-1 (Thyroid transcription factor-1)- pos itive for thyroid, CANCER-ASSOCIATED GENES
lung and neuroendocrine tumors
INFECTIOUS AGENT MARKERS
V. PSA (Prostate specific antigen)-prostatic adenocarcinoma,
pancreatic and salivary gland tumors
 EXFOLIATIVE CYTOLOGY
 -study of cells that are separated from superficial or
deep serosal or mucosal surfaces
 - cervico-vaginal smears, bronchial
washing/brushing;bronchoalveolar lavage, sputum,
urine sediment, bladder washing/brushing;
peritoneal, pericardial, pleural, cerebrospinal and
synovial fluids, gastric washing/brushing;prostatic
secretions/fluids, discharges cytology and scrape
cytology
 -detects malignancy
 -cells are shed into the body fluids and secretions w/c
are used for evaluation
 -if malignancy is suggested then biopsies can be used
for confirmatory
IMPRINT/ABRADED CYTOLOGY-
cells are directly taken from the surfaces of
excised/incised specimens by touching them with a clean
glass slide
 Also known as impression cytology
 Indicated in cases of hematolymphoid malignancy
 5 techniques; smear (malignancy; chromosomal sex;
type of infection;abnormalities); crush (mucoid and
viscous fluids);technique;pull-push technique (other
types of fluid)
 ALL SAMPLES SHOULD BE PRESERVE WELL-EQUAL
PARTS OF THANOL (95% ETHYL ALCOHOL) AND
ETHER IS THE IDEAL FIXATIVE
CELL BLOCK TECHNIQUE-
is a paraffin-embedded specimen derived from different fluids
and aspirated materials
 Mainly used with smears as an adjunct for
establishing a more definitive cytopathologic
diagnosis
 For malignancy det.
 Also known as microbiopsy
 For architectural evaluation (histologic pattern of
tumor)
 Categorization of tumors not seen in smears
 Special stains and immunohistochemistry
 Immunophenotyping, molecular studies  -cells are isolated thru series of centrifugation steps
 Archival material for future studies to concentrate the cells into a small Suspension
LIQUID-BASED CYTOLOGY
- enable cells to be spread in a monolayer
 -improve sensitivity of screening of cervico-vaginal
smears; breast and thyroid samples
 -METHODS :
Thin Prep (Cytyc Corp.) and SurePap (Tripath Imaging,
Inc)
• ADVANTAGES:
• improved cell representation;
specificity,sensitivity; abnormal cells are
clearly seen and easily identified; remaining
or residual cell suspension can be used to
make further cytological preparations or
other tests like detection of human
papilloma virus (HPV) DNA and
immunocytochemistry.
ASPIRATION CYTOLOGY-
study cells that do not shed spontaneously
 : palpable lesion (fine needle aspirate-accuracy is
dependent on size of lesion: smaller size means less
sensitive diagnosis and cant distinguish between in
situ and invasive lesion) and deep seated/ non
palpable lesions (image-guided biopsy)
MEMBRANE FILTER METHOD-
using filter paper with a specified pore size
 -made up of polycarbonate and cellulose esters
 -cytodiagnosis in urine, spinal fluid, bronchial washing
and substances with low cellular
content, sex chromatin determination on amniotic
fluid
 -simple; small amount samples, availability, excellent
preservation of cellular samples it needs
CONCENTRATION TECHNIQUE- USES CYTOSPIN AND
SEDIMENTATION PREPARATIONS