Notesale: Turn your study into money

Document Preview

Extracts from the notes are below, to see the PDF you'll receive please use the links above

---

Germ layers
Categories of tissues

Covering Epithelia
Simple squamous
Simple cuboidal
Simple columnar
Stratified squamous
Stratified columnar
Pseudostratified columnar
Glandular Epithelia
Exocrine glands

Endocrine glands
Pancreas
Merocrine
Apocrine
Holocrine
Connective Tissues
Connective tissue
Collagen

MUST TO KNOW IN HISTOPATHOLOGIC TECHNIQUES
1
...
Mesoderm
3
...
Epithelial = 3 germ layers
2
...
Muscular = mesoderm
4
...
mt 04 |Page | 255

Dense CT

Dermis
Capsules
Tendons
Stroma of cornea

Special Connective Tissues
Cartilage
Hyaline = trachea
Fibrous = intervertebral discs
Elastic = ear, epiglottis
Bone
Cancellous/spongy/trabecular = epiphysis/ends of long bones
Compact/cortical = diaphysis/shaft
Others
Blood
Lymph
Hematopoietic tissues
Acid mucopolysaccharides
Fixative: Lead fixatives
Stain: Alcian blue
Osteogenesis imperfect
Brittle bone disease
Defective production of collagen
Deposits found in Connective Tissues (Eosinophilic)
Fibrin
Early: yellow
Old: blue
Stains:
Mallory’s PTAH
Lendrum’s MSB
Fibrinoid
Necrotizing vasculitis
Staining reactions identical to fibrin
Mixture of exudates & altered cytoplasmic constituents
Hyaline
Degenerated collagen
Hypertension, atheroma, diabetic kidney
Stain: PAS
Amyloid
TB, leprosy, osteomyelitis
Stains: “CoMT”
Congo Red
Metachromatic stain
Thioflavine
Muscle Tissues
Smooth
Involuntary
Intestines, blood vessels
Skeletal
Striated, voluntary
Skeletal muscles
Cardiac
Striated, involuntary
Heart
Nervous Tissues
CNS
Brain, spinal cord
PNS
Peripheral nerves
Special receptors
Ear, eye, nose
Inflammation
Inflammation
Latin word: Inflammare (to set afire)
5 Cardinal Signs of Inflammation
1
...
Calor
Heat

lec
...
Tumor

Swelling

4
...
Functio laesa

Retrogressive Changes = Organ/Tissue smaller than normal
Developmental defects: AAHA
Aplasia
Incomplete/defective development of a tissue/organ
Ex
...
)
Pathologic: Skin warts due to HPV
Compensatory hyperplasia Ex
...
Endometrial hyperplasia
Congenital hypertrophy
Phenytoin-induced
Degenerative Changes = Tissues have abnormalities
Metaplasia
Reversible
One adult cell type ↔ Another adult cell type
lec
...
Leukemia
Study of neoplasm
Tumors
1
...
Stroma = CT framework
1
...
mole)
- Malignant
2
...
mt 04 |Page | 258

N

M

Teratomas

Apoptosis
Necrobiosis
Necrosis
Types of Necrosis
Coagulation necrosis

Liquefaction/colliquative
necrosis
Caseous/caseation necrosis
Gangrenous necrosis
Fat necrosis
Fatty degeneration
1’ changes
2’ changes
Algor mortis (1st)

T1 = lesion <2 cm (T1a = <0
...
normal sloughing off of skin cells
Pathologic cell death
Most common
Tombstone formation
“MyLKS”
Myocardium
Lungs
Kidneys
Spleen
Pus formation
Brain & spinal cord
Yellow, cheesy, crumbly material
TB, syphilis, tularemia, lymphogranuloma inguinale
Sulfide gas production
a
...
Wet gangrene = venous occlusion
Chalky white precipitates
Pancreatic degeneration
Liver
Somatic death
During somatic death
“CRC”: circulatory, respiratory, CNS failure
After somatic death
“ARLP DPA”: Algor mortis, Rigor mortis, Livor mortis, Postmortem clotting,
Dessication, Putrefaction, Autolysis
Postmortem cooling
lec
...
Ecchymosis
Postmortem Lividity
Ecchymosis
Disappears on pressure (reappears when pressure is Opposite of postmortem lividity
released)
Oozing of blood (incision)
No oozing of blood (incision)
Postmortem Clot vs
...
Upper lateral third of the vagina
2
...
Endocervix
♫ 50% alcohol = All types
Fixation
50% alcohol = pleural & peritoneal
70% alcohol = sputum
95% alcohol = urine, bronchial & gastric
♫ Saccomanno’s fixative = 50% ETOH + 2% carbowax
Smear preparation
Fix immediately
2-3 slides/patient
a
...
spreading
lec
...
touch preparation/impression
d
...
carinii/P
...
George Papanicolau (1940)
Smears: prepared by rotary motion
1
...
Egg albumin = not recommended as adhesive agent (intensifies stain by Light
Green)
Pooled human serum/plasma
Celloidin ether alcohol
Leuconostoc culture
1
...
Ayer’s spatula = rotate 3600
3
...
vaginalis
Cells (Cervicovaginal Smears)
Parabasal | Intermediate | Superficial
↓ ----------Estrogen----------↑

45-50μm
Pyknotic nucleus
True acidophilia
Folds/curls on edges
a
...
mt 04 |Page | 261

Doderlein bacillus

L
...
vaginalis infection
Clue cells
Wrinkled prune appearance
w/ perinuclear halo
HPV (LSIL)
Formation of salt crystals

C
...
vaginalis
Leptothrix
G
...
o
...
Doctor
2
...
File
Reports
Surgical pathology report
Cytopathology report
Autopsy report
Signatories
Request forms = patient’s doctor
Result forms = pathologist
Turnover of results
Surgical pathology & cytology = 24 hrs
Frozen section = 5-15 mins
Autopsy report = 1 week (Autopsy procedure: 24 hrs)
Storage
Specimen (tissue) = 1 month to 1 year
Tissue blocks (paraffin) = 3 to 10 years
Slides = indefinite
Suggested Guidelines for Record and Specimen Retention (Henry, 21st Ed
...
mt 04 |Page | 262

Specimens
Serum/other body fluids
Blood smears (routine)
Microbiology smears
BB donor/recipient
specimens
Pathology/BM slides
Pathology blocks
Cytogenetic slides
Cytogenetics diagnostic
images
Forensic Cases
Body fluids
Tissue for toxicology
Wet tissues
Paraffin blocks
Slides
Reports
Gross photographs/
negatives
Dried blood films
Frozen tissue for DNA
Autopsy

Types of autopsy

Preliminaries for PME

48 hours
7 days
7 days
7 days post-transfusion
10 years
10 years
3 years
20 years
1 year
1 year
3 years
Indefinitely
Indefinitely
Indefinitely
Indefinitely
Indefinitely
indefinitely
Autopsy (Postmortem Examination)
Gold standard for confirmation of a medical disease
Wherever scientific medicine of high quality is practiced, postmortem exams
are performed
Whenever a conscientious physician knows why he lost his patient, a
postmortem exam has been performed
Whenever criminal law is enforced
Whenever a death certificate shows accurately the causes of death & confirmed
medical diagnosis for the assembling of vital statistics, a postmortem has been
performed
Whenever there is medical research on the causes & nature of diseases such as
cancer, heart diseases & stroke, the investigative method is the postmortem
exam
An informed society requires a postmortem exam in human death for the good
of medical science, for the public’s health & for the future care of the living
patient
1
...
Partial autopsy
- Part of the anatomy
3
...
MI – heart)
1
...
Death certificate (Old: Blue form | New: Blue border/frame)
- Signed by:
a
...
Pathologist (back): will sign when PME has been performed
3
...
mt 04 |Page | 263

4
...
physical injury
Other Uses of Death
Certificate

PME is permitted w/o
consent in the following
circumstances

If pathologist is not
available
The coroner has authority
in the following cases

Somatic death
Criteria for the
pronouncement of death

Burial & cremation purposes
Transport of body from hospital  funeral  cemetery
Medical insurance claiming
- If suicide: (-) insurance
- Acts of God (lightning, flood): (-) insurance
- Civil war: (-) insurance
1
...
When it is necessary to complete the death certificate
3
...
Deceased military personnel who dies in active services/training duty or
military services
The medico-legal examiner or the coroner has jurisdiction in medico-legal cases
& may authorize the pathologist to proceed w/ an autopsy
1
...
Newborns in the 1st 24 hrs of life
3
...
All deaths due to unknown cases
5
...
All abortion, whether self-induced or otherwise
7
...
All accidental deaths
9
...
All cases w/o medical attendance w/in 36 hrs prior to the hour of death
11
...
All deaths due to shooting, stab wounds, burns, electricity, lightning,
tetanus, etc
...
Homicides
14
...
All poisoning
16
...
Premature death
Death of an organism
Cessation of circulation & respiration (1960’s)
1
...
Advance life-sustaining equipment capable of maintaining cardiovascular &
respiratory functions despite severe brain injury
3
...
Brain death: cannot be revived anymore [National institute of neurological
diseases & stroke in the US (1977)]
- Clinically dead & dead are the same
lec
...
Coma (patient will not respond) & cerebral unresponsiveness
b
...
Absent cephalic (brainstem) reflexes
d
...
irreversible cessation of circulation & respiratory functions
2
...
Coma
2
...
Algor mortis
- 1st demonstrable change after death is cooling of the body
- At room temp: 2’-2
...
5-2’F/hr (next 12 hrs)
- 1’F/hr (next 12-18 hrs)
- As a rule, the body cools at 1
...
Rigor mortis
- Rigidity of the body due to hardening of the skeletal muscles caused by a
series of physiochemical events after death
- ( formation of locking-chemical bodies
between actin & myosin
- This interlocking is fixed & produces rigor mortis w/o shortening of the
muscles
- Sets w/in 2 hrs after death (head & neck)
- Complete w/ 12 hrs
- Persists about 3-4 days
3
...
Postmortem clotting of blood
5
...
Putrefaction
7
...
mt 04 |Page | 265

Technique of Rokitansky

Technique of Ghon
Technique of Letulle

Autopsy: Larynx  Rectum

Teasing/Dissociation
Crushing/squash
preparation
Smear preparation
Frozen section
Freezing of unfixed tissue
Freezing of fixed tissue
Formal (formol) calcium
Commonly used methods of
freezing
Staining methods
(frozen sections)

H&E

Freeze-drying
Freeze-substitution
Cold knife procedure

In-situ dissection in part combined w/ en bloc technique
♫ En bloc:
- By cavity
- Interrelated to each other
- Systemic dissection
- Ex
...
2 slides/coverslip  Vital stain
Spread lightly over a slide (wireloop/applicator)
Frozen Section
(-) Fixative
Best frozen section
To localize hydrolytic enzymes & other antigens
Derivative of formaldehyde
Fix at 4’C for 18 hrs
Liquid nitrogen = most rapid
Isopentane cooled by liquid nitrogen
CO2 gas
Aerosol sprays
“PATH”
Polychrome methylene blue
Alcoholic pinacyanol
Thionine
H & E = progressive, no decolorizer
a
...
Regressive
- w/ decolorizer (acid-alcohol)
- for routine histology staining
w/o use of any chemical fixative
♫ Quenching: rapid freezing (-160’C)
♫ Sublimation: removal of H2O in the form of ice (-40’C) – vacuum
Similar to freeze drying but:
Frozen tissue  Rossman’s formula/1% acetone
Dehydrated in absolute alcohol
Any microtome
Uses CO2
Knife: -40 to -60’C
Tissue: 5 to -10’C
Environment: 0 to -10’C
lec
...
C
...
(Optimal Cutting
Temperature)

Steps

Fixation

pH
Temperature
Microanatomical fixatives

Cytological Fixatives

Nuclear fixatives

Cytoplasmic fixatives

Temperature: -18 to -20’C
Cryostat: refrigerated cabinet w/ rotary microtome
Best mounting media for cryostat sections

Tissue Processing
“FDCIETS SMoL”
1
...
Dehydration
3
...
Impregnation/Infiltration
5
...
Trimming
7
...
Staining
9
...
Labeling (slides)
Fixation
st and most critical step
1
1’ aim: preserve cell (life-like)
2’ aim: harden & protect tissues
Most important: stabilization of proteins
6
...
0
Room temp = Surgical specimen
0 to 4’C = EM and Histochem
...
10% Formol saline
b
...
Heidenhain’s SuSa
d
...
Zenker’s solution
f
...
Bouin’s solution
h
...
Nuclear fixatives: w/ glacial acetic acid – destroys mitochondria & golgi
bodies (pH ≤4
...
Cytoplasmic fixatives: w/o glacial acetic acid
c
...
mt 04 |Page | 267

Histochemical fixatives

Aldehyde Fixatives
Formaldehyde

10% Formol saline
10% NBF

Formol-Corrosive
(formol sublimate)
Glutaraldehyde
Karnovsky’s
paraformaldehydeglutaraldehyde
Acrolein
Formol-calcium
Mercuric Chloride

Heidenhain’s SuSa
HgCl2
G
...
Paraformaldehyde
- White crystalline precipitates
- Due to prolonged standing
- Removed by: 10% METOH/filtration
b
...
B5 = for BM biopsies
b
...
Schaudinn’s
d
...
Heidenhain’s SuSa = (-) black pigments
f
...
Zenker-formol (Helly’s) = pituitary gland, BM, & blood containing organs
Su = sublimat (HgCl2)
Sa = saure (acid)
Shrinks tissues
Swells tissues, counteracts HgCl2
Removal of mercuric deposits
H2O  I2  H2O  Sodium thiosulfate  H2O
“ROCK”
a
...
Orth’s = for Rickettsia, tissue necrosis
lec
...
Chromic acid = preserves CHO
d
...
(H2O soluble)  Add 70% ETOH  Insoluble
Never wash in H2O before dehydration
For glycogen (excellent)
a
...
Brasil’s alcoholic picroformol = less messy than Bouin’s, glycogen (excellent)
Solidifies at 17’C
Fixes & precipitates nucleoproteins, chromosomes, & chromatin material
Most commonly combined w/ other fixatives
Disadvantage: polarization (glycogen granules  poles/ends of the cells)
“MEICAN”
a
...
Ethanol = preserves but does not fix glycogen (Disadv: polarization)
c
...
Carnoy’s = most rapid (1-3 hrs) | for chromosomes | Dx: rabies (acetone)
e
...
Newcomer’s = for MPS | nuclear & histochemical fixative
Inhibits hematoxylin
Produce black precipitate crystals (osmium oxide)
For lipids
a
...
Flemming’s w/o acetic acid = for mitochondria
Precipitates proteins
Swelling effect  counteract shrinkage by other fixatives
Weak decalcifying agent (softening effect)
Recommended for H2O-diffusible enzymes (phosphatases, lipases)
Rabies
Bacteriologic smears
Microwave: 45-55’C
Underheating: poor sectioning
Overheating (>65’C): vacuolation, overstained cytoplasm
Placing an already fixed tissue in a 2nd fixative
Primarily fixed tissue  2
...
Tap H2O = remove excess chromates, formalin, osmic acid (NOT Bouin’s)
b
...
Alcoholic I2 = remove excess mercuric fixatives
Glutaraldehyde
PtCl3
PtCl3 – formalin (Zamboni’s)
AuCl
Osmium tetroxide
10% NBF = acceptable but not recommended
“PUL”
lec
...
PTA = 1st general stain
2
...
Lead
Factors that Affect Fixation of Tissues
Retarded by:
Size & thickness
(+) Mucus

Prevents complete penetration of fixative
Wash w/ NSS
Fatty tissues: cut in thin sections, fixed longer
Flush out w/ NSS  fix
Inactivates enzymes

(+) Fat
(+) Blood
Cold temperature
Enhanced by:
Size & thickness
Agitation
Automatic/mechanical tissue processing
Moderate heat
37-56’C
Principles and Precautions in Handling and Fixation of Specimens in General
Autopsy materials
Fixed ASAP
If not possible  mortuary refrigerator (4’C) or arterial embalming
Surgical specimens
Fixed ASAP
If not possible  refrigerate
If placed in NSS during
Autolysis may occur before fixation
operation
If tissues are refrigerated
Avoid slow freezing (ice crystal formation)
Repeated freezing & thawing  destroy organelles, release enzymes…
Not more than 5mm thick
Size of tissues
Except lung edema: 1-2 cm thick
20:1
Ratio of fixative to tissue
Except osmium tetroxide (expensive) = ratio is 5-10:1
50-100:1
Ratio of fixative to tissue in prolonged fixation (ex
...
mt 04 |Page | 270

Removal of substances soluble in fixing agent
Wrong choice of fixative
Presence of artifact pigments on tissue sections
Incomplete washing of fixative
Tissues are soft & feather-like in consistency
Incomplete fixation
Loss/inactivation of enzymes needed for study
Wrong choice of fixative
Shrinkage & swelling of cells & tissue structure
Overfixation
Tissue blocks are brittle & hard
Prolonged fixation
♫ An incompletely fixed tissue may lead to improper & incomplete clearing & impregnation, and may later
prove to be a hindrance to normal sectioning & staining of specimen
Pigment
Acid formaldehyde hematin

Mercuric chloride pigment
Chromate pigment
Osmium tetroxide pigment
Crush artifact
20:1
37’C
55’C
RT (18-30’C)
24-48 hrs
Decalcifying agents

HNO3

5% Formic acid
HCl (Von Ebner’s)
EDTA
Ion exchange resins
Electrophoresis
Measuring extent of
decalcification
Post-Decalcification
Tissue softeners

Color
Brown/black granules

Removed by:
“SAKaL”
a
...
Alcoholic KOH
c
...
Lillie’s method
Black granules
Alcoholic iodine
Fine, yellow brown
Acid-alcohol
Black precipitate crystals
Cold H2O
Intense eosinophilic staining at the center of the tissue (H & E)
Due to partial coagulation of partially fixed protein
Decalcification
Ratio of decalcifying agent to tissue
Impaired nuclear stain by Van Gieson’s stain
Tissue  Digestion (24-48 hrs)
Optimum temperature
Time
Acids
Chelating agents (EDTA/versene)
Ion exchange resins
Elec
...
Perenyi’s = tissue softener & decalcifying agent
b
...
mt 04 |Page | 271

Dehydration
10:1
Ethanol
Methanol
Butanol
Denatured alcohol
Acetone
Dioxane
(Diethylene dioxide)
Tetrahydrofuran (THF)
Graupner’s method
Weiseberger’s method
Cellosolve
Triethyl phosphate
Additives to dehydrating
agents
Methods of determining
incomplete dehydration
Xylene (Xylol)
Toluene

Chloroform
Benzene
Methyl salicylate
Methyl benzoate
Cedarwood oil
Clove oil
CCl4
Aniline oil
Glycerin
Gum syrup
Others

1% HCl in 70% alcohol
Dehydration
Aim: To remove fixative & H2O
Ascending grades of alcohol (Start: 65%)
Embryonic & animal tissues: 30% ETOH
Ratio of dehydrating agent to tissue
Best dehydrating agent
Blood & tissue films
Plants & animals
Ethanol + methanol
Both fixative & dehydrating agent
Both dehydrating & clearing agents
Dehydration w/ dioxane
Ethylene glycol monoethyl ether
Combustible and toxic
-a
...
Anhydrous CuSO4 (Last ETOH bath)
- both dehydrating agent & indicator of H2O content of 100% ETOH
- (+) H2O = White  Blue
1
...
Xylene  Milky
Clearing
Most commonly used
Clearing time: ½ to 1 hr
Block size: <5mm
Substitute for xylene/benzene
Clearing time: 1-2 hrs
Not carcinogenic
Toxic fumes
Toxic to liver
For clearing tough tissues
Does not make tissue translucent but removes alcohol
For urgent biopsies
Minimum shrinkage
Aplastic anemia
For double embedding techniques
For CNS, smooth muscles, skin
Minimum shrinkage
Has tendency to become adulterated
Similar to chloroform but is cheaper
Disadvantage: similar to chloroform
For delicate tissues, embryos and insects
No dealcoholization but make the tissues clearer
Citrus fruits oil
Trichloroethane & petrol
Impregnation
lec
...
Green’s no
...
mt 04 |Page | 273

Blocking-out Molds
Leuckhart’s embedding
mold
Compound embedding unit
Plastic embedding rings &
basse mold
Tissue Tek
Disposable embedding
molds
Celloidin/Nitrocellulose
method
Double embedding method

Cold knife procedure
Cryostat (Cold microtome)
Fixation
Freeze-drying
Algor mortis
Formol calcium fixation
Impregnation
Embedding
Flotation water bath
Trimming
Routine histopath
...
Peel-away: perfect even block w/o trimming
2
...
Paper boats: cheap & easy to make
Bones, teeth, eyes
Bell jars: control evaporation
1st: celloidin
2nd: paraffin
Brain
Recall Temperatures
Knife = -40 to -60’C
Tissue = 5 to -10’C
Environment = 0 to -10’C
-18 to -20’C
Surgical specimen: room temp
HC & EM: 0-4’C
Quenching: -160’C
Sublimation: -40’C
7’F/hr (3
...
5μm
Trefall
Simplest
Minot
Most commonly used for paraffin embedded tissues
Adams
Most dangerous (movable knife)
a
...
Standard sliding
- Block: stationary
- Knife: moving
lec
...
Belgium yellow: Best
b
...
Fine carborundum: for badly nicked knives
Removal of gross burrs
Toe to heel (Edge last)
Paddle strop (horseleather)
- Mineral oil = not recommended
- Vegetable oil (castor oil) = applied into the back of the strop, not the surface
Staining
From plants & animals
a
...
Cochineal dyes: female Coccus cacti
c
...
Saffron
A
...
a
...
Quinoid ring: Basic fuchsin
b
...
Xanthene: Eosin
d
...
Ethyl group
b
...
Sulphonic acid
Alum hematoxylin
Tissue stained w/ fat or oil-soluble dyes
H & E Staining
Hematoxylin capechianum/ Hematoxylon campechianum
Nuclear/basic/1’ stain
Waldeyer: 1st to use hematoxylin
lec
...
Weigert’s
- Mordant: FeCl3
- Weigert’s + Van Gieson’s = CT & E
...
Heidenhain’s
- Mordant: Ferric ammonium sulfate
a
...
Eosin Y (Yellowish) = most commonly used
b
...
Ethyl eosin/Eosin S/Eosin alcohol soluble
Holds 5-9 slides
Holds 5-19 slides
Holds 10-30 slides
1
...
Descending grade of alcohol = rehydration
3
...
Remove fixative artifact pigments after rehydration & before staining
5
...
H2O
7
...
Ammonia water (blueing agent): Nucleus = blue
- NH4OH
- LiCO3
- Scott’s tap H2O
9
...
Stain: Eosin Y
11
...
Xylene = dealcoholization/clearing
13
...
mt 04 |Page | 276

Pap Smear staining

Benzidine
Acridine orange
Gentian violet
Congo red
Iodine
Malachite green
Janus green
Night blue
Victoria blue
Lysochromes

Adhesives

Mounting media

Stains on skin
Restaining of old sections

Broken slides

Hematoxylin = nuclear stain
OG-6 = cytoplasmic stain (mature superficial cells)
EA (Eosin Azure) = cytoplasmic stain (immature cells: parabasal/intermediate)
EA 65 = for body fluids
EA 36/50 = for gynecologic smears
Other Stains
Hgb
RNA (red fluorescence)
DNA (green fluorescence)
Crystal violet + methyl violet + dextrin
Elastic tissue, amyloid, myelin
Oldest stain
Ascaris eggs
Mitochondria (intravital stain)
Substitute for carbolfuchsin
Neuroglia
Oil soluble dyes
a
...
Sudan III = orange
c
...
Mayer’s egg albumin = add thymol crystals (inhibit mold growth)
b
...
Gelatin
d
...
Starch paste
f
...
Poly-L-lysine = IHC
h
...
518 = refractive index of glass
1
...
DPX = 1
...
XAM = 1
...
Canada balsam (Abus balsamea) = 1
...
Clarite = 1
...
Aqueous media = for lipids (no xylene)
- Water = temporary mounting, low refractive index
- Glycerin jelly = 1
...
43
- Apathy’s medium = 1
...
5% acid alcohol  tap water
Slide  Xylene (24hrs) or gently heat until mounting medium begins to bubble
 Remove coverslip  Section: Xylene (30mins)  H2O  0
...
Mount the broken slide to another clean xylene-moist slide w/ drop of
lec
...
If replacement not possible, the section (if intact) may be transferred to
another slide:
Broken slide  Xylene (rem
...
mountant)  6 parts
butyl acetate + 1 part durofix  incubate (mixture  film)  Cut the film
around the section  Cold H2O until the film & section float off  Film w/
section  mount on a clean slide  incubate  butyl acetate  xylene 
mount

Ringing

Enzyme histochemistry
IgG
Polyclonal
Monoclonal
Epithelial Tumor Markers
(+) CK 7
(-) CK 20

Shortcut: “Xi6B1DiCuCoFSMiBXM”
Broken slide  Xylene  Incubate  6 Butyl acetate + 1 Durofix  Incubate 
Cut film  Cold H2O to float film & section  Film w/ section  mount 
incubate  butyl acetate  xylene  mount
Sealing the margins of the coverslip
Prevent escape/evaporation of fluid
Immobilize the coverslip
Prevent sticking of slides
a
...
Durofix (cellulose adhesives)
Immunohistochemistry
Trypsin & protease = most commonly used
Most commonly used antibody
Rabbits (1’) > Goat (2’) > Pig (3’) > Sheep (4’) > Horse (5’) > Guinea pig (6’)
Mice

“LUBO” = paired
Lung
Uterus
Breast
Ovary
(+) CK 20
Stomach
(-) CK 7
Colon
(+) CK 7
Transitional cell carcinoma of the bladder
(+) CK 20
Mucinous ovarian tumor
(-) CK 7
HCC
(-) CK 20
RCC
SCC
Thyroid carcinoma
Prostatic adenocarcinoma
EMA (Epithelial membrane (+) carcinoma “BuLK” = paired
antigen)
Breast
Lungs
Kidney
CEA
Oncofetal antigen
GI carcinoma
Differentiates adenocarcinoma (+) & mesothelioma (-)
TTF-1 (Thyroid
Differentiates lung adenocarcinoma & mesothelioma
Transcription Factor)
(+): Thyroid, lung, neuroendocrine tumors
PSA
Prostate cancer
Intermediate Filament Markers
Actin
Smooth muscle
Skeletal muscle
lec
...
k
...
“built-in control”
Contains the target antigen
Other Topics
Faults During Tissue Processing
Brittle/hard tissue

Clearing agent  Milky

Incomplete dehydration
lec
...
mt 04 |Page | 280

holder when cut
Frozen tissue chips into
fragments

Tissue is frozen too hard

lec
...

neutral lipids)
-Blue oxazone (reacts w/
PL and FFA)

Fast green stains basic
groups in tissues

Blue-green
Orange-red
Brownish-black
Black
Blackish brown deposits
Dark brownish-black ppt
Cobalt phosphate ppt
Reddish brown

Uses Milton reagent
Substrate: β-glyceroPO4
For skel
...
muscle biopsies
lec
...
mt 04 |Page | 283

Stain
Schmorl’s Picro-Thionin
method

Substance Stained
Lacunae & canaliculi
Bone matrix

Bielschowsky’s technique

Neurofibril, axons &
dendrites
Neuroglia & collagen
Bodian’s stain
Nerve fibers & nerve
endings
Sevier-Munger technique
Peripheral neuritis
Axons
Myelin sheath
Neuritic plaques &
tangles
Argentaffin granules
Toluidine blue
Nissl granules & nucleoli
Polychrome methylene blue Nissl granules & nucleoli
Thionine
Nissl granuls & nucleoli
Cresyl fast violet
Nissl substance
Neurons
Weigert-Pal technique
Myelin sheath
Luxol fast blue
Myelin
Weil’s method
Myelin
Cajal’s gold sublimate
Astrocytes
method
Perl’s Prussian blue
Hemosiderin
Gomori’s Prussian blue
Iron pigments
Turnbull’s blue
Ferrous iron
(Hemosiderin)
Benzidine-nitroprusside
Hemoglobin & oxidase
stain
granules
Mod
...
k
...

Tigroid substances

Dark blue
Emerald to blue green
Blue-purple then green
Depend on the oxidation
of the pigment to green
biliverdin by iodine

Bile, lipofuscins,
melanin, argentaffin
cells, chromaffin, thyroid
colloid
Lipofuscin
Hemofuscin
Melanin
Argentaffin cell granules

Dark blue

Calcium

Black

Copper

Red to orange-red

Purple
Red
= Black
= Black

Argentaffin reaction:
melanin reduces
ammoniacal silver
solutions w/o use of a
reducer

lec
...
leprae
H
...
pylori
L
...
1% urea + 5% NaSO4
Metastasis
Degree of localization
Dunn-Thompson
K2MnO4
H2 O 2
Helly’s
Formalin ammonium
bromide
Alcohol as 1’ fixative
Glutaraldehyde
Carnoy’s
Orth’s
Zenker’s
Formaldehyde
Ethanol

Mucin & glycogen
Mycelia & hyphae
RBCs
Viral inclusions

(+) Color/Result
= Blue-black
= Pink-red
= Green
= Black
= Blue
= Red
Golden yellow
Dark blue against blue
BG
Blue-violet
Dark brown to black

Comments

Black on a yellowish BG
Black
Black
= Sharply outlined I
black
= Gray-black
= Old rose
= Yellow
Bright red

HBsAg
Brown-black
Bacteria
= Blue
Recommended for blood
Mast cell granules
= Deep blue
and BM parasites,
Eosinophilic granules
= Red
inclusion conjunctivitis,
Nuclei
= Blue
Toxoplasma, spirochetes
Cytoplasm
= Pink
& other bacteria
Most sensitive technique for identifying DNA
DNA amplification
Pseudogout
Pigment removal
70% ETOH + 28% NH3 water
Remove yellow color of HNO3
Most definitive of malignancy
Most reliable indicator of prognosis of malignant tumors
Hgb = emerald green
Removes excess melanin
Contains formalin, K2CrO4 and HAc
Fixative for CNS (gold/silver stain)
Increased tissue shrinkage
Not satisfactory for PAS
Nonaqueous fixative
Pheochromocytoma
PTAH for cross-striations
Wash tissue in water after fixation in Zenker’s
Combines w/ amino group
Nonadditive fixative
lec
...
0 (formalin)
Universal solvents
Soft paraffin
Weigert’s hematoxylin
Natural resins (mounting)
Formalin-alcohol
Churukian-Schenk
technique
Masson-Fontana
Muscle biopsies
Paraffin sections
Zamboni’s PAF
Glutaraldehyde
10% NBF
Paraformaldehyde
Warthin-Starry stain
Iris diaphragm
Substage condenser
Pathology
Cornelius Celsus
Littoral cells
Hoffbauer cells
Cancer
Biohazards
Mercuric chloride
According to the presence
of a mordant
According to the presence
of differentiator
According to the resultant
color
Vital staining

Neurons

Prevent brown microcrystalline deposits in H & E stain
Both dehydrates & clears
For thick sections
Not easily decolorized w/ acidic solutions
Inherently acidic
Microincineration
Substance that can bind silver but need a chemical reducer
Substance that can both bind & reduce silver
Isopentane at -150’C
If isopentane is low, dust muscle w/ talc then freeze in liquid nitrogen
Naphthol AS-D chloroacetate esterase
Specimens may remain indefinitely
Specimens may be removed after 2-4 hrs
pH 7
...
4
Pure polymer of formaldehyde
Calibrate pH meter to 7
...
Direct staining = w/o mordant
b
...
Progressive = w/o differentiator/decolorizer
b
...
Orthochromatic = “ortho”: correct/same | same color = dye & tissue
b
...
Intravital stain = injection of dye  animal body
- Ex
...
Supravital stain = staining of cells immediately after removal from the animal
body
...
Dendrites (Greek: “Tree”) = conduct impulses to the cell body
lec
...
Axons = conduct impulses away from the cell body
Criteria for the diagnosis of Marked progesterone effect
normal pregnancy
At least 50% of intermediate cells in clusters
At least some typical pregnancy cells present
<30% of matured superficial cells
Doderlein-filled dirty BG
Staining solutions used in
a
...
OG-6 = orange w/ a hint of green
(Macroscopic)
c
...
Eosin Y
b
...
Light green SF
# in EA designates the proportion of SF
♫ Affinity:
OG-6
Matured superficial cells
Keratinizing malignant cells
♫ Cytoplasmic:
Bright orange to yellow orange
♫ Affinity:
EA 36/50
Immature vaginal cells (parabasal, intermediate)
♫ Cytoplasm color:
Transparent blue to gray to brown hue
Presently, the Bethesda system divides squamous cell abnormalities into 4 categories:
ASCUS
Atypical cells of undetermined significance
L-SIL
Low-grade squamous intraepithelial lesion
H-SIL
High-grade squamous intraepithelial lesion
SCC
Squamous cell carcinoma
Description
Bethesda 2001
Papanicolau
Normal
(-) for intraepithelial lesion/malignancy
Class I
Atypical
ASCUS
Class II
HPV
L-SIL
Class II
Mild dysplasia
L-SIL
Class II
Moderate dysplasia
H-SIL
Class III
Severe dysplasia
H-SIL
Class III
Carcinoma in situ
H-SIL
Class IV
Invasive carcinoma
Invasive carcinoma
Class V
Microtome
Usual
Description
Tissues embedding in
Microtome
Knives
Length
Plane concave
25 mm One side flat, other Less concave: celloidin-impregnated tissues Sliding
is concave
More concave: paraffin embedded tissues

Biconcave

120 mm

Plane wedge

100 mm

Carmine
Best Carmine
Mucicarmine

Both sides are
Paraffin
concave
Both sides are
Frozen sections
straight
Hard, tough tissue specimen (paraffin)
Chromatin stain
Carmine + Aluminum chloride = For glycogen
Carmine + Aluminum hydroxide = For C
...
mt 04 |Page | 287

Picrocarmine
Carmine + Picric acid = for neuropathological studies
Duke’s staging for neoplasia One of the most frequently applied for staging individual tumors
of the rectum
Biopsy
Biopsy
Excision and exam (living subject)
Preferred: perform the biopsy at the periphery of the tumor (advancing tumor
margin)
Types of Biopsy
Exfoliative cytology
Desquamated cells
Sex hormonal status in females
Sex chromatin phenotype
Excisional biopsy
Complete removal of a lesion
Most reliable
Incisional biopsy
Removal of part of a lesion/small piece of tumor directly incising the tumor
capsule
Preferred for large tumors that can’t be excised completely
Needle biopsy
Aspiration of fluid
Bite biopsy
Small pcs of tumor are removed w/ special forceps
Cutaneous biopsy
Skin fragments
Punch biopsy
For specimens >2mm  embed in a single paraffin block
Shave biopsy
Curettage specimens
Wedge biopsy
Specimen is subdivided w/ a razor blade
Marginal excision
Shell-out end

lec

---