HISTOPATHOLOGY Flashcards
Process
RECEIVING > NUMBERING > LOGBOOK
NAME & AGE OF PATIENT
ATTENDING PHYSICIAN
INITIAL DIAGNOSIS
TYPE OF SPECIMEN
> PATHOLOGIST: GROSSS EXAM (COLOR, CONSISTENCY, GROSS WEIGHT) > DISSECTION > TISSUE CASETTES
MEASURE > CUTTING > MEDTECH: PROCESS
Occurs once fixative penetrates the tissues
FIXATION
Primary Goal of Fixation:
TO PRESERVE TISSUES AS CLOSE TO THE ORIGINAL AS POSSIBLE
Secondary Goals of Fixation:
- TO HARDEN SPECIMEN TO FACILITATE EASY CUTTING INTO THIN SLICES
- TO PROTECT TISSUE FROM TRAUMA OF FURTHER HANDLING
Factors Involves in Fixation:
- THICKNESS/SIZE
- pH OF FIXATIVE
- OSMOLALITY
- CONCENTRATION OF FIXATIVE
- CORRECT VOLUME
- TIME AND DURATION OF FIXATION
- TEMPERATURE
Thicker = ___ fixation time
LONGER
Required size of tissue in fixation
3 x 2 cm
Required thickness of tissue in fixation
3-5 mm
Thickness of lungs tissue in fixation
1-2 cm
Ideal pH of fixative
6-8
Osmolality: by the book
SLIGHTLY HYPERTONIC
Osmolality: in practice
ISOTONIC
Osmolality: cell swelling
HYPOTONIC
Osmolality: cell shrinkage
HYPERTONIC
Concentrated solution = ___
DAMAGING
-We dilute to produce 10% solution
10% FORMALIN
Need to dilute
GLUTARALDEHYDE
Penetration rate of formalin
1 mm/hr
Maximum effective fixation
20x THE VOLUME OF SPECIMEN
Osmium tetroxide
5-10x THE VOLUME OF SPECIMEN
Ratio of fixative to specimen
15-20:1
Usual time and duration of fixation
24-48 HOURS
Temperature: EM & histochemistry
0-4 degC
Temperature: Autotechnicon
40 degC
Temperature: Manual
ROOM TEMPERATURE
Factors to be Considered when choosing the Right Fixative:
- URGENCY OF THE CASE / NEED FOR IMMEDIATE EXAM
- TYPE OF TISSUE TO BE PROCESSED
- TISSUE STRUCTURE TO BE STUDIED
- STAINING TECHNIQUE TO BE APPLIED
- TYPE OF SECTION TO BE MADE
Needs to use fixative that is fast-acting
URGENT BIOPSY
Commonly used bc rapid in action
FORMALIN
Brain tissues for diagnosis of rabies
CARNOY’S & ACETONE
Do not use for kidneys
BOUIN’S
Best for preserving Glycogen
PICRIC ACID FIXATIVES LIKE BRASIL’S FLUID
For enzymes (lipases & phosphatases)
Do not use for fats bc it dissolves fats
ACETONE
Routine staining
Compatible with many fixatives
H&E STAINING
Inhibit hematoxylin
OSMIUM TETROXIDE
Type of Section to be Made:
SERIAL / INDIVIDUAL
Factors that Prolong Fixation Time:
- COLD TEMPERATURE
- PRESENCE OF BLOOD & MUCUS IN THE SPECIMEN
- SIZE & THICKNESS OF SPECIMEN
- PRESENCE OF FATS
Remedy for Presence of blood & mucus in the specimen
WASH THE TISSUE WITH NSS
Remedy for Presence of fats
CUT THEM THINLY TO ENSURE RAPID ENTRY OF FIXATIVE
Factors that Accelerate Fixation:
- SIZE & THICKNESS OF TISSUE
- HEAT APPLICATION
- AGITATION
Smaller = ___ fixation time
SHORTER
Continuous agitation → ___
RAPID ENTRY OF FIXATIVE TO TISSUE
Faster fixation time than manual method
AUTOTECHNICON
Problems in Fixation:
Loss of Substance Soluble in Fixing Agent - ___
WRONG CHOICE OF FIXATIVE
Problems in Fixation:
Too hard/brittle - ___
OVERFIXATION
Problems in Fixation:
Too soft - ___
INADEQUATE FIXATION
Problems in Fixation:
Presence of artifacts - ___
INADEQUATE WASHING
Problems in Fixation:
___ - Wrong choice of fixative
LOSS OF SUBSTANCE SOLUBLE IN FIXING AGENT
Problems in Fixation:
___ - Overfixation
TOO HARD/BRITTLE
Problems in Fixation:
___ - Inadequate fixation
TOO SOFT
Problems in Fixation:
___ - Inadequate washing
PRESENCE OF ARTIFACTS
Types of Fixatives as to Mechanism of Action:
ADDITIVE
NON-ADDITIVE
Binds to tissue, becomes part of the tissue
ADDITIVE
ALL EXCEPT ___ are Additives
ACETONE & ALCOHOL
Does not bind / become part of the tissue
NON-ADDITIVE
Types of Fixative as to Action / Purpose:
- HISTOCHEMICAL
- CYTOLOGICAL
- MICROANATOMICAL
Preserve chemical components like enzymes (phosphatase, lipase)
HISTOCHEMICAL
Acid mucopolysaccharides, acid mucins
HISTOCHEMICAL
Preserve all parts of the cell (nucleus / cytoplasm)
CYTOLOGICAL
Nuclear Fixatives:
CARNOY’S
BOUIN’S
NEWCOMER’S
HEIDENHAIN’S SUSA
FLEMMING’S W/ HAc
Cytoplasmic Fixatives:
REGAUD’S / MOLLER’S
ORTH’S
FORMALIN W/ POST CHROMING
FLEMMING’S W/O HAc
General microscopic study of tissue structures w/o altering structural pattern & normal intercellular relationship of tissues
Preserve tissues & cells as a whole
MICROANATOMICAL
Prepare 1 liter of 10% Formalin
100 mL of 37-40% FORMALIN + 900 mL DISTILLED WATER
Types of Fixatives as to Active Component Substance:
- FORMALDEHYDE / FORMALIN
- GLUTARALDEHYDE
- MERCURIC CHLORIDE
- CHROMATE
- LEAD
- PICRIC ACID
CNS & post mortem tissues
NSS used to dilute
10% FORMAL SALINE
Same but sodium phosphate is added as buffer to preserve tissues w/ iron pigments
10% NEUTRAL BUFFERED FORMALIN
Sputum specimens & microincineration technique
Ethanol + Glacial acetic acid + Formaldehyde
ALCOHOLIC FORMALIN / GENDRES
Lipids & neutral phospholipids
Formalin + Mercuric chloride
FORMOL CORROSIVE / FORMOL SUBLIMATE
For EM
GLUTARALDEHYDE
Small tissue fragments / needle biopsies
2.5% SOLUTION
Larger tissues, >4 mm thick
4% SOLUTION
Most common metallic
Trichrome staining & tissue photography
MERCURIC CHLORIDE FIXATIVE
Disadvantage: Leave black mercury deposits
MERCURIC CHLORIDE FIXATIVE
Liver spleen, CT & nuclei
ZENKER’S FLUID
Bone marrow
B5
Tumor skin biopsies
HEIDENHAIN’S SUSA
Pituitary gland, BM, spleen, liver
HELLY’S FLUID / ZENKER FORMOL / FORMOL ZENKER
3 Types of Metallic Fixative:
- MERCURIC CHLORIDE
- LEAD
- CHROMIC
Carbohydrates
1-2% CHROMIC ACID
Lipids, mitochondria
POTASSIUM DICHROMATE 3%
Mitochondria, RBC, & colloid containing tissues
REGAUD’S FLUID / MOLLER
Rickettsia & other bacteria, tissue necrosis
ORTH’S FLUID
Acid mucopolysaccharide
LEAD
Excellent for glycogen determination
PICRIC ACID FIXATIVES
Versatile but impart a yellow color
PICRIC ACID
Under picric acid as main ingredient
Embryos, pituitary biopsies & endometrial curettings
Not for kidney structures
Not compatible w/ Feulgen’s
BOUIN’S
Touch preparations
95% ISOPROPYL ALCOHOL
Solidifies at 17 degC
Contraindicated for cytoplasmic fixation
Combination w/ other fixatives to produce a compound fixative
GLACIAL ACETIC ACID
Ice cold temperature
ACETONE
Most rapid fixative
Chromosomes & lymph glands
CARNOY’S
Mucopolysaccharides & nuclear protein
NEWCOMER’S
Alcohol Fixatives:
ETHANOL
METHANOL
IPA / ISOPROPYL
CARNOY’S
NEWCOMER’S
Picric Acid Fixatives:
BOUIN’S
BRASIL’S
HOLLANDE’S
For Electron Microscopy:
OSMIUM TETROXIDE
GLUTARALDEHYDE
PARAFORMALDEHYDE
Fixation Methods:
- PHYSICAL METHOD
HEAT FIXATION
MICROWAVE TECHNIQUE - CHEMICAL METHOD
Thermal coagulation of proteins
HEAT FIXATION
Typical, immerse in chemical fixatives
CHEMICAL METHOD
Fixative & dehydrating
Phosphatases & lipases
ACETONE
Disadvantage: Evaporates easily & dissolves fats
ACETONE