Histotechnique Flashcards
Cytology
the study of individual cells
Goal of histopathology
Preserve tissues in a life-like manner
–> abnormalities can be identified microscopically
Simplified workflow
obtain –> fix –> slice –> stain
Reason for fixative solution
tissues and cells die/degrade immediately after removal from body
fresh tissues must be transported immediately
placed in fixative solution for transportation
Sources of samples (2)
Surgical (biopsy with clean margins)
Post-mortem (autopsy)
Grossing
Macroscopic evaluation of specimen
Parameters (5)
Size Texture Number/Proportion Markings Locations
Fixation
Preservation go cells and tissues
Life-like as possible
STABILIZE PROTEIN so it is RESISTANT TO FURTHER CHANGES
Function of Fixatives (4)
- Prevents putrefaction (rotting) and autolysis (breakdown)
- Maintain proper relationships between cells and extracellular substances
- Bring out differences in refractive indexes and increase the visibility of/contrast between different tissue elements
- Secondary functions: enhance staining, limiting osmotic effects, prevent desiccation
Autolysis
Self-destruction after cells death
Caused by Intracellular enzymes
Temperature (warm=more damage)
Specialized cells - more rapid, worse damage
Nuclear changes: pyknosis –> karyorrhexis –> karyolysis
*condensed chromatin –> loose
Cytoplasm: granular and swollen
Putrefaction
decomposition by microorganisms
Modes of Action (fixing) (3)
Render - inactivate enzymes by stabilizing proteins
Kill - kill bacteria and molds (toxic)
Make - make tissues more receptive to dyes
Impact of Fixing on Tissues (5)
- Change in size (smaller)
- Change in texture (brittle, hard)
- Lost material (dissolve lipids)
- Chemical alterations (charges and properties may change)
- Fixation artefacts (deposits on and around tissues - affect microscopic imagine)
Volume ratio of Fixative/Tissue
20/1
T/F: dense tissue is faster to fix
FASLE - dense tissues required more time than porous tissues
Fixative Classification (4)
- Chemical action on proteins
- Effect on microscopic appearance of the tissues
- Number of fixing reagents in fixative solution
- Amount of time tissues can remain in fixative
Chemical action on Proteins:
Coagulant - tertiary structure, organelles, mesh
Non-coagulant - cross-linkages (impermeable), insoluble gel (hard to stain)
Additive - combines with protein
Non-additive - changes protein nature, structural configuration or activity
Microscopic appearance of tissue
- Microanatomical fixative - preserve microarchitecture
- Cytological fixative - preserve intracellular structures/inclusions (electron micro)
- Histochemical fixative - minimal changes
simple vs compound fixing sequence
Simple - 1 fixative
Compound - more than 1 fixative in solution
Tolerant vs intolerant fixing
Tolerant - time is flexible
Intolerant - timing is critical
Simple Fixatives (8)
- Formaldehyde and Formalin based fixatives
- Glutaraldehyde
- Osmium Tetroxide
- Potassium Dichromate
- Mercuric Chloride
- Picric Acid
- Ethanol
- Acetic Acid
Universal Fixative
Formaldehyde
Formaldehyde
Formalin-based fixatives
Glutaraldehyde
Osmium Tetroxide
Potassium Dichromate
Mercric Chloride
Picric Acid
Ethanol
Acetic Acid
Compound Fixative Formula
- 1 or more coagulating agent
- May contain a non-coagulating agent
- always in solution
4.
B-Plus Fixative
Bouin’s Fixative
Secondary Fixation
Formalin Pigment
How do you remove formalin pigment?
Mercuty Pigment
Chrome Pigment
Goal of Tissue Processing
- remove all water
- replace with an inert, stable material
3 steps to remove water
- Dehydration
- Clearing
- Infliltration
Incomplete dehydration
mushy & soft tissue
Prolonged dehydration
brittle & hard tissue
Dehydration
slow and gentle
increasing concentrations of alcohol (70%-100%)
Clearing
Remove dehydrating agent, replace with material miscible with infiltrating agent
Xylene
TOXIC agents
Improper: greasy feel and odor
Infiltration
Removal of clearing agent, replaced with intent, stable supportive material INTERNAL SUPPORT Long step (vacuum to speed up) Paraffin wax (with or without additives)
Processing Maintenance
Replace all single solutions with new
“dirtiest” repeat is replaced and shifted to end position
Occurs after every run
Processing Outcomes
Tissue shrinkage
Brittle
Lipids dissolved (tightly bound myelin stays)
Microwave processing
FAST
finicky
1 sample at a time