HISTOPATHOLOGY PART 2

STAINING MECHANISM OF STAINING

 Process whereby tissue components are made visible
in microscopic sections by direct interaction with a dye
or staining solution
 A colored compound is used to produce a contrast
between different tissues and cellular components
based on their varying affinities for
most dyes and stains, so morphologic changes are
more easily identified, physical characteristics and
structural relationships of tissues and their cells can be
evaluated, and the presence or absence of disease can
be established
Two General Category:
 Pure Physical Stain: lipid stains
 Chemical Stain: tissue elements
 Nuclear Stain: nucleus
 acidic: generally anionic stain with basic dye
 positively charged, decalcified or contain only
basic nucleoproteins, stains with dyes-metal
mordant combination
HEMATOXYLIN

 ACCENTUATOR  deep blue-purple color

 accelerates the reaction  is a natural dye derived by extraction from the core or
the heartwood of a Mexican tree known as
 AUXOCHROME
"Hematoxylin Campechianum”
 links the dye to the tissue, ionizing group
 MORDANT
 Hematein
 serves as a link or bridge between the tissue
 oxidation product
and the dye, to make the staining reaction
 exposing the solution to air and sunlight (3-4
possible
months)
 CHROMOGEN  mixing oxidizer:
 compound containing chromophoric group.
 Sodium iodate
 simple benzene compounds
 Potassium Permanganate
 CHROMOPHORE
 Mercuric Oxide
 confers the property of color. Substances with
definite atomic groupings and are capable of  Sodium Perborate
producing visible colors.  Hydrogen Peroxide
 COUNTERSTAINING  Addition of acetic acid: retard
 LEUCO COMPOUNDS oxidation
 Overoxidation-Oxyhematin: brown

PREPARATION OF TISSUE FOR STAINING Variety depends on the choice of metal:

 Aluminum Hematoxylin
 Cole: artificially ripened using alcoholic iodine
REMOVAL OF PARAFFIN
 Slides in 60C oven for atleast 30 minutes to soften the
 Delafield: deep purple-red hue
wax
 Two changes of clean xylene-descending
 Erlich
concentrations of alcohol-distilled water
 Harris: used for routine H&E and PAS;
Mercuric Chloride as mordant
CLASSIFICATION OF DYES
 Mayer: preferred for IHC and Celestine Blue
 Natural Dyes Hemalum Method. Preservative: Citric acid &
 Hematoxylin chloral hydrate
 Cochineal Dyes
 Orcein  Gill: mucin in goblet cells; uses anhydrous
 Synthetic Dyes hematoxylin
 AKA “Coal Tar Dyes”
 Derived from the Hydrocarbon Benzene and  Iron Hematoxylin
are collectively known as Aniline dyes  Heidenhain’s: Mordant: ferric ammonium
 Affinity to Tissues sulfate; Violet-black/Blue black
 Basic Dyes: Cationic dyes: Positive charge
 Acidic Dyes: Anionic dyes: Negative charge  Phosphotungstic acid: Mordant: 1%
 Lipid stains: Nonionic: Neutral Phosphotungstic acid; Reddish brown-purple
 Chemical Composition
 Thiazine  Weigert’s: used when acidic staining is to be
 Azo-dyes applied; Mordant: ferric ammonium chloride;
 Rosalins Violet-black/Blue black

HISTOPATHOLOGY PART 2 GARSY

 CYTOPLASMIC STAIN
 Brought about by proteins or charged groups on the
amino acid sidechains or proteins
 Proteins are amphoteric
 pH 6: isoelectric point of proteins
 below 6: (+) affinity for anionic sye (eosin)
 above 6: (-) affinity for cationic dye
 If pH is too low: Eosin is uncharged
and stains tissue nonspecifically.
 If above pH 6, proteins will have a
net negative charge and will no
longer attract eosin.
 Cytoplasmic dyes:
 Anionic dyes
 Eosin counterstain: Eosin Y; pH 4.6-
5; green fluorescence
 Eosin-Phloxin B Counterstain: more
vivid
 Eosin B
 Ethyl Eosin
 Nuclear Fast Red
H&E METHOD
 Tissue specimen
 Hematoxylin: dark blue to purple
 Eosin: shades of pink
 NUCLEI: dark blue to purple (basic dye)
 EXTRACELLULAR MATRIX AND
CYTOPLASM: different shades of pink
(acidic affinity)
METHODS OF STAINING
 Direct staining
 is the process of giving color to the sections
by using aqueous or alcoholic dye solutions.
 Ex: Methylene Blue, Eosin, Iodine, Gentian
Violet
 Indirect staining
 is the process whereby the action of the dye
is intensified by adding another agent or a
MORDANT
 Uses a mordant or accentuator
 Progressive staining
 staining follows a definite sequence. No
washing out
 without differentiator
 Regressive Staining
 With this technique, the tissue is first
overstained to obliterate the cellular details,
and the excess stain is removed or
decolorized from unwanted parts of the
tissue, until the desired intensity of color is
obtained
HISTOPATHOLOGY PART 2
 Metachromatic Staining
 use of specific dyes which differentiate
particular substances by staining them with a
color that is different from that of the stain
itself
 Ex: Methyl Violet, Cresyl Blue, Thionine,
Toluidine blue, Crystal Violet
 Metallic Impregnation
 is a process where specific tissue elements
are demonstrated, not by stains, but by
colorless solutions of metallic salts which are
thereby reduced by the tissue, producing an
opaque, usually black deposit on the
surface of the tissue or bacteria
 Ex: Gold (Gold chloride) and Silver (Silver
nitrate)
 Vital staining
 is the selective staining of living cell
constituents
 Ex: RES staining by the MYPON blue
 Intravital Staining
 injecting the dye into any part of the animal
body (either intravenous, intraperitoneal or
subcutaneous), producing specific
coloration of certain cells, particularly those of
the reticulo-endothelial system
 Common dyes used are lithium, carmine and
India ink
 Supravital Staining
 method of staining used in microscopy to
examine living cells that have been removed
from an organism
 Ex: Neutral Red, Janus Green, Trypan Blue,
Nile Blue, Nile Blue and Thionine Blue
PERIODIC ACID SCHIFF REACTION
 Primarily stains carbohydrates
 0.5-1% aqueous solution for 2-10 mins (ave. of 5 mins)
at room temperature
 Osmic, chromate and permanganate fixative should be
avoided
 Schiff agent:
 Basic Fuchsin Rosanilline, Pararosaniline and
Magenta II
 Preparation:
 Barger and Delamater Method: Uses thionyl
Chloride
 De Tomasi-Coleman Method: Uses
sodium/potassium metabisulfide
 Itikawa and Iguru Method: Uses sulfur dioxide
gas
 Counterstain: Hematoxylin (+) Magenta Red
GARSY
 STAINING OF GLYCOGEN
 Histochemical Methods:
 BEST CARMINE METHOD  Free Fatty Acids
 Carmine/Carminic acid  Cholesterol
 Counterstain: Erlich Hematoxylin  Cerebrosides
 Added to remove background stain:  Gangliosides
Potassium Carbonate and Potassium  Borohydride-periodic Schiff (BHPS) Method
Chloride for gangliosides
 (+) Bright Red Granules

 LANGHAN’S IODINE METHOD (CARLETON’S STAINING OF CONNECTIVE TISSUE

MODIFICATION)
 Oldest Stain; Not specific  Reticulum and Collage Fibers stain selectively with
 (+) Mahogany Brown Acid Aniline Dyes:
 Aniline Blue
 PAS WITH DIASTASE METHOD  Basic Fuchsin
 Method of choice  Methyl Blue
 (+) RED/ Magenta Red  Indigo carmine
 Azocarmine Stain: amyloid
connective tissue, mucus colloid (+)
STAINING OF MUCIN blue

 ACID MUCOPOLYSACCHARIDES  Gomori’s Silver Impregnation

 Primarily made up of hyaluronic acid, heparan Stain for Reticulin: black
sulfate and chondroitin sulfate
 Alcian Blue: most popular (will stain  Masson’s Trichrome Stain:
blue) collagen and mucus (blue)
 Combined Alcian Blue-PAS
Technique for Acid and Neutral  Mallory’s Aniline Blue: collagen
Mucins: 2nd stain (PAS) (red) elastic fibers (pink and yellow)
 Combined Alcian Blue-Aldehyde
Fuchsin: purple (carboxylated:  PAS Method for Basement
blue) Membrane
 Colloidal (Dialyzed) Iron
Technique: toluidine blue  Van Gieoson’s stain for collagen:
 Fresh Frozen Azure: red purple simplest; PA and BF; (+) pink or
 Fluorescent Acridine Orange deep red
Technique: black
 Mucicarmine Stain: red
 Uranyl-Acetate-Azure A: red violet ELASTIC FIBERS
or crimson
 Weigert’s
 NEUTRAL POLYSACCHARIDES  Stain: Basic Fuchsin, resorcin and ferric
 Mucoproteins and glycolipids chloride
 PAS (+); Alcian Blue (-)  Differentiator: Acid Alcohol
 Counterstain: Neutral Red, H&E or
hematoxylin
STAINING OF FATS OR LIPIDS  Elastic fibers: dark blue or blue black

 Best demonstrated by frozen sections  Verhoeff’s

 Potassium Chromate and chromic acid fixatives  Differentiator: 2% aqueous frric chloride
 Formol Calcium is the fixative of choice for lipid  Elastic fiber: black
histochemistry  Collagen: red

 Sudan Dyes:  Orcein (Tanzer-Unna Orcein Method)

 Nile Blue Sulfate Method  Vegetable dye
 Oil Red O Method in Dextrin  Differentiator: Acid alcohol
 Osmic acid/Tetroxide  Counterstain: Methylene blue or alum
 Sudan Black B hematoxylin
 Sudan III  Elastic fiber: dark brown
 Sudan IV/ Sharlach R
 Krajan’s Technique
 Congo Red
 Elastic Fiber: bright red

HISTOPATHOLOGY PART 2 GARSY

 FIBRIN
 Martius, Scarlet, Blue (MSB)
 Martius yellow: early fibrin
 Brilliant crystal scarlet: fibrin
 Methylene Blue: old fibrin
 Fibrin: red
 Early Fibrin: yellow
 Very old fibrin: blue
 Mallory’s Phosphotungstic Acid Hematoxylin
 Fibrin: dark blue
 Collagen and elastic fibers: deep brown
AMYLOID
 Congo Red
Modifications:
 Hignman’s congo red
 Alkaline congo red
 High pH congo red technique
 Gram’s iodine: oldest staining method
 Induced fluorescent Staining with Thioflavin T
 Krajan’s Amyloid Stain: red
 Metachromatic staining: pinkish red (+)
MUSCLE
 ATPase
 Heidenhain’s Iron Hematoxylin Method
 Muscle Striations: gray black
 Lissamine Fast Red Tartrazine Method
 Muscles: red
 Collagen: yellow
 Mallory’s Phosphotungstic Acid Hematoxylin
 Muscle: blue
 Collagen: reddish
 Coarse Elastic Fibers: bluish
 Modified Gomori’s Trichrome Stain
 Muscle Fibers: red
 Collagen: green
 Rapid Gomori Trichrome Stain for Frozen Muscle
Tissue
 Myofibrils: green
 Intermyofibrillar material: bright red
BONE
 Schmorl’s Picro-Thionine Method
 Lacunae and canaliculi: dark brown to black
 Bone Matrix: yellow or brownish yellow
 Cells: red
HISTOPATHOLOGY PART 2
BONE MARROW AND BLOOD ELEMENTS
 RAPID TOLUIDINE-EOSIN FOR GLYCOL
METHACRYLATE SECTIONS
 Nuclei: blue
 Cytoplasm: blue or pink
 Eosinophilic Granules: red
 Basophils and Mast Cells granules: blue
 ROMANOWSKY STAINS
 Consists of methylene blue/azure B and eosin
dissolved in acetone free alcohol
 Jenner, Giemsa, May-Grunwald, Leishman
and Wright
 Wright Stain: Methylene blue is polychromed
by heating with sodium bicarbonate
 Giemsa Stain: Azurophilic granules: red
 Wright-Giemsa or Jenner-Giemsa:
 Nuclei: purple or blue
 Cytoplasm: pink or blue
 Eosinophils: pink or red
 PEROXIDASE REACTION FOR MYELOID CELLS
CENTRAL NERVOUS SYSTEM
 Bielschowsky’s Nervous Tissue
 Neurofibril, axons and dendrites: black on
gray background
 Bodian’s Stain for Nerve Fibers and Nerve Endings
 Demonstrating neurotic plaques and
neurofibrillary tangles for dx of: Alzheimer’s
Disease
 Sevier-Munger technique for staining Neural
Tissues
 Axons: black
 Myelin sheath: light brown
 Neurotic plaques and tangles: black
 Argentaffin granules: black
 Nissl Bodies
 Cresyl Fast Violet
 Nissl Substance: purple to dark blue
 Neurons: pale purple blue
 Cell Nuclei: purple blue
GARSY
 Gmelin Bile and Hematodin Green
 Myelin Sheath Gomori’s Aldehyde Lipofuchsin Purple
 Weigert-Pal technique of staining Normal Fuchsin
Myelin Sheaths: blue black Gomori’s Prussian Iron Blue
 Kluver & Barrera Luxol Fast Blue Stain: blue Blue
or green Lindquist’s Copper Red
 Luxol Fast Blue-H&E stain: blue or green Modified Orange
 Luxol Fast Blue-PAS-Hematoxylin Stain: blue Rhodamine
or green Mallory’s Fuchsin Hemofuchsin Red
 Weil’s Method: black stain pigment
Masson Fontana Melanin and Black
 Astrocytes Argentaffin
 Cajal’s Gold sublimate: black on a light Modified Fouchet’s Liver Bile Pigments Emerald to
brownish background blue green
 Modified PTAH Stain for Reactive Astrocytes Perl’s Prussian Hemosiderin Blue
 Modified Holzer’s Method for Astrocyte Blue
Processes: blue
Schmorl’s Ferric Reducing Black
Ferricyanide Substances
Stein’s Iodine Bile Pigments Black
 Oligodendrocytes
Tumbull’s Blue Ferrous Iron Blue
 IHC method using antibodies to
Reaction
galactocerebroside, myelin basic protein or
carbonic anhydrase-C (no reaction) Von Kossa’s Silver Calcium Black
Nitrate
 Microglial cells
 Rod shaped nuclei on H&E
 CD68 and CD45 positive (LCA)
MICROORGANISMS

TISSUE PIGMENTS AND DEPOSITS  Auramine-Rhodamine Stain for Mycobacteria:

golden yellow
 Anthracosis: black coloration of lungs due to carbon
 Hematogenous pigments  Brown and Brenn
 Hemosiderin: iron containing pigment of  Nocardia and Actinomyces
hemoglobin  Gram Positive: blue
 Hematoidin: iron-free pigment of hemoglobin  Gram Negative: red
 Hematin: hemoglobin minus the globin
molecule  Dieterle Method for Legionella Pneumophilia: dark
 Hemozoin: black granule formed by malarial brown to black
parasite living in RBCs 
 Hemofuchsin: iron-free brownish yellow  Grams Stain, Gram-Twort Stain
pigment that occurs with  Gram Positive: blue black
hemosiderin in hemochromatosis  Gram Negative: pink red

 Helicobacter pylori
 Warthin-Starry
 Steiner
 Toluidine Blue: dark blue
 Cresyl Violet: violet

 Wade-fite technique for Leprosy, Bacilli and

Nocardia: red

 Ziel-Neelsen’s Method of Staining AFB: red

HISTOPATHOLOGY PART 2 GARSY

Spirochetes ADHESIVES
 T. pallidum
 Dierterle  Mayer’s Egg Albumin
 Levaditi: black on yellowish background for all  most commonly used
spirochetes  easy to make, inexpensive
 Warthin-Starry: black  filter and add thymol crystals to prevent the
 Modified Steiner: black growth of molds

 L. interrogans (Levaditi, Warthin Starry, Modified  Dried Albumin (NaCl + crystals)

Steiner)
 Fungi and Actinomycetes
 Grocott Methamine Silver: sharply outlined on
black
 Viruses
 Lendrum’s Phloxine-Tartrazine Method for
Viral Inclusions: bright red
 Orcein Method for HBsAg: brown black
PROTEINS, ENZYMES AND NUCLEIC ACID
 Alkaline Fast Green Method for Basic Proteins
 Histones and Protamines (found in nuclei)
 Alpha-napthyl Acetate Method for Non-Specific
Esterase: reddish brown
 Gelatin
 Starch Paste
 Plasma: not recommended
 Poly-l-lysine: used for immunohistochemistry
 Apes/3-aminopropyl-triethoxysilane: cytology
CHARACTERISTICS OF A GOOD MOUNTING MEDIUM
 Refractive Index should be as near as possible to that
of glass slide which is 1.518 and it should be as near
as possible to that of the tissue which is 1.53-1.54
 Should not dry quickly
 Should not dissolve out or fade tissue sections
 Should not cause shrinkage or distortion.
 Should set hard-producing permanent mounting of
sections

 ATPase TWO MAIN GROUP OF MOUNTING MEDIA

 Type 1 Fibers: light brown
 Type 2 A, B, C Fibers: dark brown
 AQUEOUS MEDIA
 Feulgen Technique for DNA: red to purple  For water miscible preparations
 Fluorescent Staining  Water
 Acridine Orange  Glycerin Jelly (contains phenol as an
 DNA: yellow green fluorescence antiseptic): moist sections and fats
 RNA: brick orange red  Gum Arabic/Farrant’s Medium: fat sections
(1.43)
 Gomori Calcium for ALP: brownish black  Apathy’s Gum Syrup: fluorescent
microscopy (1.52)
 Gomori Lead for ACP: black  Brun’s Fluid- frozen sections
 Highman’s Medium: metachromatic dye
 Indoxyl Acetate Method for Non-Specific Esterase: (methyl violet)
blue
 RESINOUS MEDIA
 Lead Method for 5-Nucleotidase: blackish brown  Used for preparations that have been
dehydrated and cleaned in xylene or
 Methyl Green Pyronin Method toluene
 DNA (Chromatin): green or blue green  Natural Resin
 RNA (Nucleoli): rose red  Canada Balsam: extracted from Canadian
tree, Abusbalsamea (1.54)
 Peracetic Acid-Alcian Blue  Synthetic Resin
 Cystine and Cysteine:  Harden quickly than natural resins,
neutral reaction, uses Xylene as
 Sakaguchi’s Test solvent
 Arginine:orange red
Clarite 1.54 Histoclad 1.54
 Staining for Acetylcholinesterase (Felipe and
Lake): brown to black Coverbond 1.53 Permount 1.54
DPX 1.53 Pro-Texx 1.49
 Succinic Dehydrogenase: black or bluish black
H.S.R 1.54 XAM 1.52
 Tetrazolium Method for Monoamine Oxidase: bluish
black

HISTOPATHOLOGY PART 2 GARSY

 EXFOLIATIVE CYTOLOGY MATURE SUPERFICIAL CELLS

 Study cells that are separated from superficial or deep  SIZE: 45-50um
serosal or mucosal surfaces.  SHAPE: polyglonal squamous cells
 Used in the following:  NUCLEUS: dark pyknotic (less than 6um)
 Staging of Cancer  CYTOPLASM: pale pink
 Pap Smear  REMARKS: true acidophilia
 Assesment of Hormonal status in case of
sterility and endocrine disorders. INTERMEDIATE CELLS
 Determination of Genetic Sex
 Detection of Infectious agents  SIZE: medium sized
 SHAPE: polyhedral or elongated cellls
 Specimen Preparation Techniques  CYTOPLASM: basophilic vacuolated
 Smear Technique
 Screening Test
PARABASAL
Types (Based on the viscosity of the sample)
 SIZE: 15-30um
 Crush Technique: Viscous /Mucoid  SHAPE: round to oval
 NUCLEUS: large vesicular granule than intermediate
 Pull-push Technique: Other types of fluid cell
 Fixative: Equal parts of 95% ethanol and ether  CYTOPLASM: small dense basophilic
 95% ethanol- most commonly used  REMARKS: normally found in 2 weeks of age to
 100% methanol- substitute puberty, after childbirth, abortions and after
 Cytospray (1ft distance) menopause
 For Bloody Specimens: Carnoy’s fixative; 3
units/mL of heparin for bloody specimens
NAVICULAR CELLS
 If delay is anticipated: 50% alcohol;
Saccomano preservative
 SIZE: medium sized
 SHAPE: boat shaped
 Air-drying: Stained with Diff-Quik, Giemsa, or May
 REMARKS:
Grunwald-Giemsa; Hematolymphoid tissue
 folded/curl edges
 Alcoholic Fixation: 95% alcohol for 15 minutes then  combine estrogen-progesterone effect
stained with Harris Hematoxylin for nuclear stain and found during:
OG-6 and EA-36 for cytoplasmic stain  later half of menstrual cycle
 during pregnancy
 menopause
CELL BLOCK TECHNIQUE
PREGNANCY CELLS
Methods of Cell Block Preparation:
 SHAPE: round to oval or boat shaped
 Centrifuge Method: 2000 RPM for 2 mins  CYTOPLASM: translucent basophilic (glycogen
 Membrane Filter Method accumulation)
 Concentration Technique  REMARKS: deeper blue cytoplasm at the periphery
 Liquid Based Cytology
 Sampling in the T-zone ENDOMETRIAL CELLS
 Collection:
 Endocervical brush: Endocervical  SIZE: small occurring in 3 or more
canal  SHAPE: slightly cylindrical
 Four Quadrant Vaginal Scrape:  REMARKS: found 1-10 days after menstruation
Vaginal Adenosis
 Lateral Vaginal Scrape: Hormonal
Evaluation ENDOCERVICAL CELLS
 Vaginal Scrape: Patients with
Hysterectomy  SHAPE: honeycomb appearance
 Vulvar Scrape: Herpetic Lesions/  NUCLEUS: with finely granular chromatin
Carcinoma  CYTOPLASM: pale blue/gray, finely vacuolated
 REMARKS: occur in large groups or small sheets

HISTOPATHOLOGY PART 2 GARSY

 AUTOPSY/POST MORTEM EXAMINATION POST MORTEM CHANGES

 Pathologist/Coroner/Medico-Legal Officer  ALGOR MORTIS: cooling

 RIGOR MORTIS: stiffening
 Prosecutor: cuts the body  LIVOR MORTIS: post mortem clotting
 POST MORTEM CLOTTING OF BLOOD
 Diener/Morgue Attendant: cleans the body  DISCOLORATION OF TISSUE
 PUTREFACTION
 Medical Laboratory Scientists: prepares the  DESSICATION
materials needed

 Attending Physician/Referring Authority (Judge)

TYPES OF AUTOPSY

 MEDICOLEGAL
 Manner of death
 Institution of Justice
 Deals Gross Examination like Presence of
tattoo, past procedures

 MEDICAL
 Cause of Death
 Education of the medical personnel and
surviving relative
 Deals with microscopic and ancillary studies

HOW TO PERFORM AUTOPSY

 Preliminary investigation
 External exam/Gross
 Internal

 Cranial
 coronal incision from back of the ear to
the outer back of the ear then scalp flap
 make a wedge cut using a saw. Remove
the skull cap and get the brain

 Trunkal
 Y-shape incision: tip of the shoulder
(coracoacromial joint) straight toward the
tip of the sternum (xyphoid process)
down to the middle of the pubic bone
 Thoracic-U: preservation of breast
 Certical- throat down to pubic; young
cadavers
 T-shaped adult need not to put in coffin

TECHNIQUES OF EVISCERATION

 Virchow: one by one, most widely used, father of

modern pathology
 Rokitansky: in situ
 Ghon: “en bloc”, organs closely associated to each
other are removed
together
 Letulle: “en masse” most common

HISTOPATHOLOGY PART 2 GARSY

HISTOPATHOLOGY PART 2 GARSY