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Culture Documents
The “N” score indicates the extent of lymph amorphous appearance in H&E stains
node involvement. called “fibrinoid” (fibrin-like)
The “M” score indicates whether distant
metastases are present SOMATIC DEATH
Secondary changes after death:
A – igor Mortis
TERATOMAS R – igor Mortis
“monstrous tumor” L – ivor Mortis
May contain hair, teeth, bones, P – ost Mortem Clot
eyeballs Dessication
Putrefaction
CELL DEATH Autolysis
Two Patterns of Cell Death:
1. Necrosis – (irreversible injury) LIVOR MORTIS ECCHYMOSIS
Changes produced by enzymatic Disappearance of Opposite of livor
digestion of dead cellular elements pressure <12 hours mortis
Protein denaturation Oozing of blood No oozing of blood
Nuclear changes : Karyolysis
(fading), pyknosis (shrinkage), Algor Mortis – cooling 7°F/hr
karyorhexis ( fragmentation) Rigor Mortis – stiffening of skeletal muscles
2. Apoptosis: Programmed cell death 2-3 hours (Head and Neck)
Vital process that helps eliminated Livor Mortis – post mortem lividity
unwanted cells Purplish discoloration
An internally programmed series of After 10-12 hours; does not blanch on pressure
events effected by dedicated gene or shift when the body is moved.
products. Occurs in the dependent parts of the body.
Unlike necrosis there’s no
inflammatory reaction POST MORTEM CLOT ANTEMORTEM CLOT
Patterns of Necrosis in Tissues or Organs Settling/ Separation Granular/friable
1. Coagulative – the architecture of dead of RBC
tissues is preserved for a span of at Chicken fat Not readily
least some days. The affected tissues appearance detachable from
exhibit a firm texture. -areas where RBCs vessels
are cleared
2. Liquefactive – digestion of the dead Currant Jelly- where
cells, resulting in transformation of the RBCs sediment
tissue into a liquid viscous mass.
3. Gangrenous – necrosis usually with Assumes vessel Seldom assumes the
superimposed infection shape vessel shape
4. Caseous – encountered in foci of rubbery
tuberculous infection “cheeselike
appearance” Dessication- drying and wrinkling of the cornea
5. Fat Necrosis – focal areas of fat and anterior chamber
destruction due to release of pancreatic Putrefaction – invasion of intestinal
lipases. “Chalky-white appearance” microorganisms
6. Fibrinoid – seen in immune reactions Autolysis – self-digestion of cells, Lysozyme
involving blood vessels. Bright pion and
HISTOPATHOLOGIC TECHNIQUES
Considerations:
FIXATION 1. Speed
2. Penetration – 1mm per hour for
First and most critical step formalin
Process of preserving cells and tissue 3. Volume: 10-20 times the volume of
constituents in a condition identical to that tissue to be fixed
existing during life 4. Duration of Fixation
HISTOPATHOLOGIC TECHNIQUES
-Absolute Alcohol
TYPES OF FIXATIVES ACCORDING TO -Newcomer’s
COMPOSITION -Acetone
1. Simple Fixatives – one compound
2. Compound Fixatives – two or more
Ex: Zenker’s- glacial acetic acid – ALDEHYDE FIXATIVES
swelling of tissues A. Formaldehyde (Formalin) – must never
Mercuric Chloride – shrinking of tissues be neutralized – violent explosions
Stock solution: 37%-40%
TYPES OF FIXATIVE ACCORDING TO ACTION Working solution: 10%
ACETONE XYLENE
-both fixative and dehydrating agent -AKA xylol
-for most urgent biopsies (dehydrates in half to -most commonly used in routine procedures
2 hours) but can cause considerable shrinkage -rapid clearing time: 15-30 minutes to 1 hour
and brittleness. -for tissue sections with a thickness of less than
5 mm
DIOXANE -not for nervous tissues and lymph nodes
-Diethylene Dioxide -it turns milky when tissues are incompletely
-both dehydrating and clearing agent dehydrated
Gaupner’s: Uses pure dioxane and paraffin
Weiseberger’s: Tissue is wrapped in gauze and TOLUENE
suspended in bottle containing dioxane and -Substitute for xylene or benzene
anhydrous calcium oxide
BENZENE
HISTOPATHOLOGIC TECHNIQUES
-Recommended for urgent biopsies and for Wax Impregnation/Infiltration: Replacing the
routine purposes clearing agent with the infiltrating medium to
-carcinogenic; toxic to man fill all tissue cavities
-can cause aplastic anemia Ratio: 25:1
B. CELLOIDIN
-AKA collodion
Other clearing agents -purified form of nitrocellulose
Oil of Bergamot: skin and smooth muscle -for spx with large hollow cavities w/c tend
Oil of Origanum: skin to collapse, hard and dense tissues
Oil of Wintergreen: artificial oil, for delicate -not requires heat during processing
tissues Wet celloidin: for bones, brain sections and
Carbon Disulfide: smooth muscle teeth.
Carbon xylene: friable tissues Dry Celloidin: for processing whole eye
Terpineol: delicate material like eyes sections, uses Gilson’s mixture for storage
Phenol: smooth muscles Nitrocellulose Method (Low Viscosity
High Test Aviation Lead Free Gasoline: excellent Nitrocellulose): soluble in equal
clearing agent concentration of ether and alcohol
-preferred since it produces a harder tissue
block and thinner sections
WAX IMPREGNATION AND -add plasticizer (castor oil) to prevent tissue
cracking
EMBEDDING
C. GELATIN
HISTOPATHOLOGIC TECHNIQUES
Red Oxazone – dissolves neutral lipids -adsorbed into mucin at low pH,
Blue Oxazone – basic and reacts with subsequently staining with Prussian blue
phospholipids and free fatty acids -greater sensitivity than alcian blue but
more complex and time consuming
Solvents used for staining
1. Water STAINS FOR PROTEIN, ENZYMES AND NUCLEIC
2. Alcohol ACIDS
3. Aniline Water Histochemical Identification of Proteins
4. Phenol -detection of amino acids molecules
o Alkaline Fast Green Method-
CARBOHYDRATE STAINS Basic Proteins (Protamines and
Periodic Acid Schiff Histones)
-Intensity of PAS proportional to sugar content o Peracetic Acid Alcian Blue-
Schiff Reagent Cystine and cysteine
-Basic Fuchsin (rosanilin, pararosanilin, magenta
III) Enzyme Histochemistry
Methods of Preparation: -tissue requirement= -70C
Barger and de lamater: thionyle chloride o Gomori Calcium- ALP
De Tomasi-coleman: potassium metabisulfite o Gomori Lead – ACP
Itikawa and Oguru: sulfur dioxide gas o Lead method – 5-nucleotidase
o Metal Precipitation for ATPase
MUCOPROTEIN most common PAS positive o Alpha Naphtyl Acetate- Non
substance specific esterase
PAS with Diastase o Indoxyl Acetate Method – non
-Diastase can digest glycogen specific esterase
Best Carmine o Acetylthiocholine –
-selective and highly specific for glycogen acetylcholinesterase
o Tetrazolium – monoamine
ACID MUCOPOLYSACCHARIDES oxidase
Alcian blue
-most popular method for acid mucins Nucleic Acid Stains
Metachromatic Staining o FEULGEN TECHNIQUE for nuclear
Azure A – most useful DNA
Uranyl Acetate – excellent results for CT -most reliable and specific
mucins histochemical technique
Combined Alcian blue PAS Technique for o Methyl Green Pyronin Method for
Acid and Neutral Mucins DNA and RNA
-demonstration of any mucins o Fluorescent stain for DNA and RNA
Gomori Aldehyde Fuchsin Stain for sulfated -Fluorescein-widely used
mucin fluorochrome
Mucicarmine -Rhodamine
-carmine with aluminum hydroxide to -Auramine- most common
improve mucin staining -Acriflavin
-useful for staining encapsulated fungi In situ hybridization – “on site”
(Southgate’s mucicarmine technique) -most sensitive technique for DNA
Acridine Orange Idenfication
Colloidal iron PCR
HISTOPATHOLOGIC TECHNIQUES
TUMOR MARKERS
Copper – Lindquist Modified Rhodamine
I. Epithelial Tumor Markers
Technique
1. Keratin (CK 7, CK20, CK 5/6)
Urates and Phosphates – birefringence, use of
2. EMA
lithium carbonate
3. CEA
Carbon – most common exogenous pigments
4. TTF-1
5. PSA
HISTOPATHOLOGIC TECHNIQUES
Mounting – use of a medium and a coverslip to Specimens that require adhesive agent
facilitate the handling, storage, protection of 1. Urinary Sediment
the tissue section 2. Bronchial lavage
Fixation
-exfoliated cells decompose rapidly which may
destroy cellular and nuclear details, in turn will HORMONAL CYTOLOGY
give inadequate results for diagnosis. - Based on the specific response of the
vaginal epithelium to steroid hormones
1. Equal parts of 95% eTOH and ether ESTROGEN: produced by the
2. 95% eTOH proliferating granulose theca cells of
3. Carnoy’s fluid the ovarian follicles. It acts upon the
4. Equal parts of tertiary butyl alcohol and SUPERFICIAL CELLS
1 part 95% EtOH PROGESTERONE: produced by corpus
5. SCHAUDINN’S FLUID – sat. Aq, hgCl2, luteum formed after ovulation. It acts
absolute acetic acid upon the INTERMEDIATE CELLS
6. Methanol – dried films
7. Saccomano’s – 50% alcohol and 2% Hormonal changes are best mirrored in the
carbowax upper third of the vagina.
CELLS
PAPANICOLAU SMEAR AND STAIN 1. Superficial cells
- Nicolas Papanicolau -cytoplasm: may be acidophilic or
- Screening test for cervical cancer basophilic
Anatomic Sites for Cytologic Samples -presence of small dark pyknotic nuclei
(Gynecological) (less than 6u)
1. Upper (Proximal) third of the vaginal -true acidophilia (estrogen influence)
wall 2. Intermediate Cells
-ideal for studying the hormonal status -cytoplasm: basophilic with vacuoles
of, evaluation of inflammatory -vesicular nuclei (6-9u)
conditions
2. Ectocervix Navicular cells – boat shaped
-most common for cancer screening intermediate basal cells with a
- use of Ayre’ s spatula strong tendency to fold and curl
T zone where most malignancies arise their edges.
3. Endocervix -expression of the combined
-for detection of endocervical lesions , estrogen-progesterone effect
intrauterine lesions Pregnancy cells – translucent
Histology: simple columnar epithelium basophilic cytoplasm (glycogen
accumulation)
PAPS STAIN -cytoplasm stains deep blue or
1. Harris Hematoxylin – nuclear stain green + cell membrane = a
2. OG-6 – strong affinity for mature cells double cell wall appearance
3. EA 50 – strong affinity for 3. Parabasal Cells
_____________ -round to oval cells
HISTOPATHOLOGIC TECHNIQUES
Bethesda System
Report Format
-Specimen Adequacy
-General Categorization
-Descriptive Diagnosis
Adequacy
- Satisfactory
- Limited
- Unsatisfactory
Descriptive
- Normal
- Benign
- Epithelial cell abnormality
- Atypical squamous cells of unknown
significance ( ASCUS)
- Low grade squamous intraepithelial
lesion (LGSIL)
- High grade squamous intraepithelial
lesions (HGSIL)