LECTURE (FINALS)

STAINING ➢ The use of specific dyes which differentiate particular

➢ Process of applying dyes on the sections to see and study substances by staining them with a color that is different
the architectural pattern of the tissue and physical from that of the stain itself
characteristics of the cells COUNTERSTAINING
➢ Acidic part of the cells or tissue have greater affinity for ➢ Application of different color or stain to provide contrast
basic dyes while basic constituents take more of the acid and background to the stain of the structural components
stains to be demonstrated
STAINING OF TISSUE CAN BE CLASSIFIED INTO 3 MAJOR METALLIC IMPREGNATION
GROUPS ➢ Specific tissue elements are demonstrated not by stains
HISTOLOGICAL STAINING but by colorless solutions of the metallic slats which are
➢ Process whereby the tissue constituents are thereby reduced by the tissue producing an opaque,
demonstrated in sections by direct interaction with a dye usually black deposit on the surface of the tissue or
or staining solution, producing coloration of the active bacteria
tissue component VITAL STAINING
HISTOCHEMICAL STAINING ➢ Selective staining of living cell constituents,
➢ Process whereby various constituents of tissues are demonstrating cytoplasmic structures by phagocytosis of
studied through chemical reactions that will permit the dye particle (cytoplasmic phagocytosis)
microscopic localization of a specific tissue substance ➢ Nucleus of a living cell is resistant to vital stains
IMMUNOHISTOCHEMICAL STAINING INTRAVITAL STAINING
➢ Allows phenotypic markers to be detected by using a wide ➢ Staining of living cells is done by injecting the dye into any
range of antibodies (Ab) part of the animal body
➢ Combination of immunologic and histochemical staining SUPRAVITAL STAINING
METHOD OF STAINING ➢ Used to stain living cells immediately after removal from
DIRECT STAINING the living body
➢ The process of giving color to the sections by using ➢ Biological stains or coloring substances are prepared
aqueous or alcoholic dye solutions from dyes which may generally be divided into two
o Example: methylene blue and eosin categories:
INDIRECT STAINING o Natural dyes
➢ Process whereby the action of the dye is intensified by o Synthetic dyes
adding another agent or mordant – which serves as a link NATURAL DYES
or bridge between the tissue and the dye to make staining ➢ Those obtained from plants and animals
reaction possible HEMATOXYLIN
➢ Mordant combines with the dye to form a colored “lake” ➢ Derived from the core or heartwood of a Mexican tree
➢ Lake combines with the tissue to form “tissue mordant- known as “Hematoxylin Campechiamun”
dye-complex” ➢ Hematoxylin itself is not a stain. The active coloring agent
➢ Accentuator: accelerates or hastens the speed of the is hematin, which is formed by the oxidation of
staining reaction hematoxylin process known as “ripening”
PROGRESSIVE STAINING 1. Allum Hematoxylin
➢ Tissue elements are stained in a definite sequence ➢ Used in routine H&E
➢ No differentiation or decolorization is involved ➢ Mordant: Potassium Aluminum Sulfate
REGRESSIVE STAINING ➢ Produce good nuclear stain (red)
➢ Tissue is first overstained to obliterate the cellular details 2. Iron Hematoxylin
and the excess stain is removed or decolorized ➢ Weigert’s
METACHROMATIC STAINING ➢ Mordant: Ferric Chloride
3. Heidenhain’s
➢ Mordant: Ferric Ammonium Chloride
Made by: AGANG
LECTURE (FINALS)

➢ For mitochondria muscle striations, BASIC FUCHSIN

chromatin and myelin 1. Carbol Fuchsin
COCHINEAL DYES 2. Coleman’s Feulgen
➢ Old histologic dye extracted from the female cochineal 3. Schiff’s
bug (Coccus cacti), which is treated with alum to produce 4. Mallory’s Fuchsin
the dye, carmine 5. Aldehyde Fuchsin (Gomon’s)
ORCEIN ➢ A plasma stain utilized also for deep staining of acid-fast
➢ Vegetable dye extracted from certain lichens which are organisms
normally colorless, but which is treated with ammonia and ➢ Mitochondria and differentiation of smooth muscles with
exposed to air the use of picnic acid
➢ Produce blue or violet colors BENZIDINE
SYNTHETIC DYES ➢ Used for staining hemoglobin
➢ Sometimes known as “Coal Tar Dyes” since they were BISMARCK BROWN
originally manufactured from substances that have been ➢ Use for staining diptheria
taken from coal tar ➢ Used as a contrast stain for grams technique acid fast
➢ They are derived from the hydrocarbon benzene (CH) an and papanicolau method
are collectively known as Aniline dyes CARMINE
EFFECTS OF SYNTHETIC DYES ➢ Used as a chromatin stain for fresh materials in smear
1. Chromophore preparations
• responsible for the coloring property ➢ Best carmine combined with aluminum chloride to stain
2. Auxochrome glycogen
• responsible for the dyeing property MAYER’S CARMALUM SOLUTION
3. Lysochrome Dyes ➢ A mordanted dye acting as a basic dye and staining acid
substances
• oil soluble dyes
CELESTINE BLUE
4. Amphoteric Dyes
➢ Recommended for routine staining of fixed sections
• dyes with a neutral pH CONGO RED
STAINING ➢ Best known as indicator
VAN GIESON’S (ACID FUCHSIN PICNIC ACID) ➢ May be utilized as a stain for axis cylinders in embryos
➢ Mixture of picnic acid and acid fuchsin for the ➢ Used for staining elastic tissues amyloid and myelin
demonstration of connective tissues (Krajan’s Method)
➢ Also, mucin and elastic tissue CRYSTAL VIOLET
ACRIDINE ORANGE ➢ A nuclear or chromatin stain used for staining amyloid in
➢ A basic acridine fluorochrome which permits frozen sections and platelets
discrimination between dead and living cells ➢ Gentian Violet = mixture of crystal violet, methyl violet and
➢ DNA – Green fluorescence
dextrin
➢ RNA – Red fluorescence
GIEMSA
ACRIDINE RED 3B ➢ Used for staining blood to differentiate leukocytes or WBC
➢ For calcium salts and phosphatase activity GOLD SUBLIMATE
ALCIAN BLUE ➢ Used for metallic impregnation made up of gold chloride
➢ Stain acid mucopolysaccharides and mercuric chloride
➢ More specific for connective tissue and epithelial mucin IODINE
ANILINE BLUE ➢ The oldest of all stains
➢ A cytoplasmic stain used for counterstaining of Epithelial ➢ Used to remove mercuric fixative artifacts pigments
sections ➢ Used reagent to other crystal and methylene violet which
may retain certain bacteria and fungi
Made by: AGANG
LECTURE (FINALS)

a. Gram’s – microorganism and fibrins in SUDAN BLACK B

tissue section ➢ Most sensitive of the oil soluble dye
b. Lugol’s – test for glycogen amyloid and ➢ Stain triglyceride or neutral fat in the body
corpora amylacea ➢ Stains black or blue
JANUS GREEN B (STAR) SUDAN 4 (SHARLACH R)
➢ Used for demonstrating mitochondria during supra vital ➢ Most commonly used and has no secondary amino group
staining ➢ Color red
MALACHITE GREEEN (STAR) SUDAN 3
➢ Used as a counter or decolorizer stain for ascharis eggs ➢ first Sudan dye and stain fats in CNS Tissue
and erythrocytes OTHER STAIN FOR FATS
METHYLENE BLUE OIL RED O (ORO)
➢ Plasma cells, cytological test for Fresh sputum for ➢ stain neutral fats and LIPIDS
malignant evaluation and differentiation of bacteria cells OSMIC ACID
➢ Diphtheria diagnosis and Nervous tissue vital staining ➢ unsaturated fats in frozen sections
NEUTRAL RED (STAR) SOLVENTS USE FOR STAININGS
➢ Best Vital Dye 1. Water
➢ For demonstration cell granules and vacuoles of 2. Alcohol
phagocytic cell a. Ethyl alcohol – dehydration
ORCEIN b. Methyl alcohol – wet and dry smear
➢ Excellent stain for Elastic Fibers and most delicate fibers 3. Aniline water – 10 ml per 1 liter of hot distilled water
in skin CARBOHYDRATES STAIN
OSMIUM TETROXIDE ➢ Glycogen is the storage form of carbohydrates in human
➢ used to stain fats PERIODIC ACID SCHIFF STAIN
PRUSSIAN BLUE ➢ Stain GBM hyaline cast and glycogen,
➢ Used as Contrast stain ➢ PAS COLOR: MAGENTA
➢ Intravital staining for circulatory system colored salt PERIODIC ACID SCHIFF W/ DIASTASE CONTROL
Ferric ➢ Methd of choice for glycogen
➢ Manufactured of Paints BEST CARMINE
RHODAMINE B ➢ Glycogen- bright red
➢ Used with osmic acid to fix and stain blood and glandular LANGHAN’S
tissues ➢ Oldest stain for glycogen color is: MAHOGANY BROWN
SILVER NITRATE (STAR) STAINS FOR MUCIN
➢ Used in identification of Spirochetes, reticulum and other
ALCIAN
fiber stains
➢ Most popular method for acid mucin
TOLUIDINE BLUE (EXAM)
➢ Color: blue
➢ Recommended for staining of Nissi granules of
AZURE A
chromophilic bodies Nuclear stan for fixed tissues
➢ most useful metachromatic dye
➢ Used as a substitute for thionine in fresh tissue sections
➢ Color: red violet or crimsons
VICTORIA BLUE
➢ For demonstration of neuroglia in frozen sections URANYL ACECATE AZURE A
STAINS USED FOR FATS ➢ Metchromatic stain same color w/ azure a
OIL SOLUBLE DYES (LYSOCHROMES) TOLUIDINE BLUE
➢ Gives color to lipids because they’re more soluble in lipid ➢ Red Purple
medium of the tissue than the 70% alcohol
Made by: AGANG
LECTURE (FINALS)

STAINS FOR PROTEINS, ENZYMES AND NUCLEIC ACID STAINS FOR PATHOLOGIC STAGES AND DEPOSITS FOUND IN
ALKALINE FAST GREEN CONNECTIVE TISSUE
➢ Histones and protamines FIBRIN
➢ Color: green ➢ insoluble febrile protein seen after tissue damage, blood
PERACETIC ACID clots and acute inflammatory reaction
➢ Alcian blue- cystine and cysteine (amino acid) FIBRINOID
➢ Color: blue green ➢ eosinophilic material, identical staining reaction to fibrin
SAKAGUCHI’S but it is seen collagen disease hypersensitivity and SLE.
➢ Stain arginine color: orange red HYALIN
ENZYME HISTOCHEMISTRY ➢ wide variety of pathologic exudates and deposit, for
GOMORI CALCIUM periodic acid Schiff but not specific.
➢ Alkaline phosphatase AMYLOID
➢ Color: brownish black ➢ Semi translucent ground glass, chondroitin sulfuric acid
GOMORI LEAD protein complex deposited in the kidney, spleen and
➢ Acid phosphatase CONGO RED
➢ color: black ➢ stains amyloid color: red
LEAD METHOD THIOFLAVIN
➢ Stains 5- nucleosidase ➢ used for amyloid
➢ Color: blackish brown ➢ Yellow fluorescence (star)
ALPHA-NAPHTHYL ACETATE STAINS FOR MUSCLE AND BONES
➢ Nonspecific esterase SCHMORL’S PICRO- THIONIN
➢ Color: reddish brown ➢ Lacunae and canaliculi
INDOXYL ACETATE ➢ DARK BROWN
➢ nonspecific esterase ➢ Bone matrix- YELLOW/ BROWNISH YELLOW
➢ Color: blue ➢ Cells- ReD
FEULGEN TECHNIQUE (STAR) STAINING OF BM AND BLOOD ELEMENTS
➢ Stains nuclear DNA ZENKERS SOLUTION
➢ Color: red purple ➢ Recommended
➢ Most reliable and specific histochemical staining ➢ fixative for BM preparation
technique for DNA ROMANOWSKY STAINS
CONNECTIVE TISSUE STAINING o WRIGHT STAIN
MASSON’S TRICHROME STAIN o LEISHMAN STAIN
➢ use for collagen o GIEMSA STAIN
➢ Color: blue o WRIGHT STAIN/ JENNER-GIEMSA
VAN’S GIESON’S STAIN o MAY-GRUNWALD-GIEMSA
➢ Collagen PEROXIDASE
➢ color: deep pink or red (SIMPLEST) ➢ Myeloid cell
GOMORI SILVER IMPREGNATION (STAR) ➢ color: green to dark blue. All myeloid cells are positive
➢ reticulin except basophils, lymphocytes and erythroblast are
➢ color: black negative.
Methyl green pyronin
➢ used to demo plasma cell

Made by: AGANG

LECTURE (FINALS)

STAINS FOR CNS PTF1

BODAIN’S STAIN ➢ Thyroid transcription factor 1
➢ NERVE FIBERS AND ENDINGS ➢ Present in thyroid gland neoplasms
➢ Demonstrate ... diagnostic of Alzheimer’s ➢ Distinguishes lung adenocarcinoma from mesothelioma
Sevier-Munger Technique PSA (PROSTATE SPECIFIC ANTIGEN)
➢ Prostate or prostatic adenocarcinoma
➢ neurotic plaque and tangles.
➢ Pancreatic and salivary tumors
Bielschosky’s
ERPR (ESTROGEN RECEPTOR AND PROGESTERONE RECEPTOR):
➢ neurons, axons and neurofibrils.
FOUND IN BREAST EPITHELIAL CELLS
Schlei??? ➢ Breast specific
➢ Negri bodies appear magenta in color diagnosis for ➢ IHC stain for prognosis and treatment of breast
RABIES. carcinoma
STAINS FOR TISSUE PIGMENTS ➢ Identification of metastatic breast carcinoma
➢ Pigmentation- occurs if the substances produces color INTERMEDIATE FILAMENT MARKERS
➢ Normal due normal metabolism of the body ACTIN: SENSITIVE MARKER FOR MUSCLE DIFFERENTIATION
ABNORMAL ➢ Tumors from smooth skeletal and cardiac muscles
ENDOGENOUS VIMENTIN: NORMALLY PRESENT IN MESENCHYMAL AND
➢ Haematogenous NEOPLASTIC CELLS SUCH AS SARCOMAS, LYMPHOMAS,
o Hemosiderin is iron containing pigment of hgb LEUKEMIAS, SEMINOMAS AND NEURAL TUMORS
➢ Melanomas and schwannomas: always stain positive in
o Hematoidin:iron free pigment
Vimentin
o Hematin: hgb without globin molecule
DESMIN
o Hemofuscin: iron free brownish yellow pigment
➢ Highly specific for myogenic tumors
NONHEMATOGENOUS PIGMENTS GFAP
➢ Melanin ➢ Glial fibrillary acidic protein
EXOGENOUS PIGMENTS ➢ For diagnosis of astrocytoma
➢ Foreign materials like asbestos, silica, iron, silver and o From astrocyte cells (type of neuroglial cell)
tattoos S100 PROTEIN
EPITHELIAL TUMOR MARKERS (CK: CYTOKERATIN) ➢ Calcium binding protein
KERATIN: MARKER OF EPITHELIAL CELLS ➢ Also positive for melanomas
➢ CK7: indicated positive carcinoma of lungs, breast uterus NUEROENDOCRINE MARKERS
and ovarian cancers NSE (NEURON SPECIFIC ENOLASE)
➢ CK20: colon and stomach cancers ➢ (+) Neural endocrine differentiation
➢ If both 7 & 20 are positive: indicates transitional cell CHROMOGRANIN
carcinomas and mucinous ovarian tumors ➢ Positive neuro endocrine differentiation and keratin and
➢ If both negative: thyroid and squamous cell carcinoma, chromogranin
liver carcinoma, renal cell carcinoma ➢ For neuroendocrine carcinomas
EPITHELIAL MEMBRANE ANTIGEN: DETERMINES THE SITE OF SYNAPTOPHYSIM
TUMOR ➢ Present in synaptic vesicles of neurons, normal neurons
➢ (+) breast, adenocarcinoma, lungs and kidney carcinomas and neuroendocrine cells
CEA (CARCINOEMBRYONIC ANTIGEN): ONCOFETAL AG GERM CELL TUMOR MARKERS
➢ Present in carcinomas of GI tract (place where we find HCG (HUMAN CHORIONIC GONADOTROPIN
colon cancers), pancreas, lung, breast, ovary, uterus, ➢ Synthesized by placental cytotrophoblasts and liver
cervix ➢ Indicated choriocarcinoma and germ cell tumor
➢ Differentiates adenocarcinoma from mesothelioma

Made by: AGANG

LECTURE (FINALS)

AFP • Clinical abstract in coordination of Attending

➢ Synthesizes by normal liver physician and medical records
➢ Indicates hepatocellular carcinomas or hepatoma ➢ 2nd: External Inspection
PLAP (PLACENTAL-LIKE ALKALINE ANTIGEN) • Scrutinization of anterior and posterior
➢ Late pregnancy detection surfaces
➢ Indication of embryonal carcinoma and choriocarcinoma • To observe signs of violence
and seminoma • Lacerations
MESENCHYMAL TUMOR MARKERS • Identifying marks
MYOGENIC TUMORS • Edema
➢ Positive for desmin and actin, myoglobin and myogenic or • Distention
myogenic tumors • Hemorrhage
FIBROHISTOCYTIO • Jaundice
➢ Positive for alpha1 antitrypsin, alpha 1chymotrypsin and ➢ 3rd: Internal Inspection
cd68 • Abdomen and thorax
VASCULAR TUMORS • Through Y-incision from right shoulder to
➢ Positive for CD 31, FACTOR RELATED ANTIGEN, ulex middle sternum to left shoulder
europaeus, 1. To examine the heart, kidneys, lungs
LYMPHOMA and other organs
➢ Leukocyte common antigen (CD45) 2. To analyze fluids: acetic fluid, pleural
CELL PROLIFERATION MARKERS fluid, pericardial fluid
➢ KI-67 3. For culture, cell cytology and cell block
➢ Proliferating Cell Nuclear Antigen and chemical analysis
➢ Use to asses tumor proliferation, greater aggressiveness • Dissection of organs
or probability of metastasis • Analysis of tissues
➢ Immunoglobulins are produced by plasma cells which are ➢ No consent conditions
in turn synthesized by b-cells or b lymphocytes • Ordered by police or NBI
o THESE ARE Y-SHAPED composed of heavy and • To complete death certification
light chains • When deceased person gave permission before
o Structure is a 4-chain polypeptide: 2 identical death
heavy and light chains ▪ You can call this advanced directive
▪ 2 heavy chains ▪ Ex: donation of organs, transplantation
• IgM, G, E, A, D • Solider or officer who died from active services
▪ Light chains TYPES OF AUTOPSY DONE IN THE PHILIPPINES
• Kappa and lambda ACCORDING TO ITS PURPOSE
AUTOPSY ➢ For routine hospital autopsy: ex cause of death
➢ Systemic study of a cadaver to determine the cause of ➢ Medico legal autopsy: ex NBI or gov't prosecution
death ACCORDING TO COMPLETENESS
➢ Pathologists conduct the study ➢ Partial: SMALL SECTIONS LIKE organs
➢ Purpose: to determine cause of death and pathogenesis, ➢ WHOLE BODY: HEAD TO FOOT
preservation of dead tissues for further research all for ACCORDING TO THE MANNER OF INCISION OR OPENING OF
the sake of increasing standard of living CADAVER
HOW IS IT PERFORMED? ➢ Y-shape incision: for adults and females
➢ 1st: Preparatory Measures ➢ Straight cut incisions: infants and children cut from the
• Consent midline
• Documents

Made by: AGANG

LECTURE (FINALS)

TECHNIQUES OF AUTOPSY MUST KNOW:

TECHNIQUE OF VIRCHOW 1. Domains regions /sections of
➢ Organs removed or directed individually from the body ➢ Ab Variable Amino terminal. First 110 AA OF Ab
TECHNIQUE OF ROKITANSKY molecule
➢ in situ dissection in part combined with unblock technique ➢ Contstant/Carboxy Terminal
• Organs removed according to/by cavity or systemic 2. Hinge Region
TECHNIQUE OF GHON ➢ Flexible portion of the Ab
➢ unblock technique by cavity only ➢ Located between CH1 and CH2
TECHNIQUE OF LETULLE ➢ Rich in AA (PROLINE)
➢ in masse technique ➢ H & L chains are held together by noncovalent
REMOVAL OR ALL PARTS OF THE BODY forces and disulfide interchain bridges
Clinical pathology lab report 2 years
Autopsy forensic reports Indefinite
Surgical pathology report (biopsy) 10 years
Cytogenetic reports 20 years
Specimen of pathology tissue blocks 10 years
Slides Indefinite
IMMUNOHISTOCHEMISTRY
➢ used to determine
• origin
• prognosis
• treatment of a tumor
➢ General Immunology exerts its control through HUMORAL ANTIBODY VARIATONS
(antibody) and cellular comportments. ISOTYPE BASED ON HEAVY CHAIN TYPE
• ANTIBODY ➢ 1g6 gamma
➢ Host protein (immunoglobulin) produced in response to ➢ IgA- alpha
the presence of Foreign molecules, organisms or other ➢ IgD delta
agents in e body. ➢ Ig M- mu
• “immunoglobulins” ➢ IgE- epsilon
• produced by B lymphocytes in response to antigenic ALOTYPE
stimulation. ➢ genetic var based on constant region
• FOUR-CHAIN POLY PEPTIDE IDIOTYPE
• 2 identital Heavy chains (H) - 1gs subclass ➢ genetic var. based on variable region
• 2 identical Light chains (L)-Kappa Lambda ADDITIONAL NOTES
STRUCTURE OF IMMUNOGLOBULIN ➢ Ab Fragmentation
➢ Y-shaped protein molecule that is composed of both • 1g M- Dithiotreitol, 2-mercapto ethanol (destroys J
heavy and light chains. chain)
• EPITOPE ➢ 1g6 -PAPAIN
o upper arms of Y region bind to short arms of ➢ ANTIGEN
specipic antigen (Ag)- site which Ab attaches to • made up of proteins, CHO, or other polymers and is
tissue. capable of producing an immune response in animals
or cell cultures for production of antibodies

Made by: AGANG