Week 1 Flashcards
what is the flash point
lowest temperature where the vapours can ignite . The higher the FP the less flammable something is
what is fixation
-Means to stabilize protein through chemical and physical means so it is resistant to changes through all the other stages of tissue processing
-The process turns soluble cell contents to insoluble contents
-Stops autolysis and putrefaction
-Preserves cells and tissue
-Hardens tissues
how does fixation prevent post mortem activities
- Autolysis
-Putrefaction
-Maintains relationship between cells and extracellular substances - collagen, reticulin, Elastin
what is the difference between Autolysis and Putrefaction
Autolysis - enzyme activity
-self destruction
-independent of bacterial action
-slowed by cold temps and increased with high over 30 but inhibited by 50
-can cause desquamation where the epithelium separates from its basement membrane
Putrefaction- bacterial action
-when proteins liquify
-produce ammonia and hydrogen sulfide
-resembles autolysis
-sample will have spaces due to gas production
how does fixation bring out the differences in RI
-Fixation increases visibility or contrast between tissue
-if air and the tissue had the same RI (velocity of light in air: velocity of light in liquid or solid) then the tissue would be invisible
how does fixation make cell parts insoluble
-Fixation uses tissue proteins for stabilization
-some fixatives have mordants that link dye to tissue for better staining
-fixatives mask AG sites for IHC staining, preserve lipids and glycogen in liver
-fixatives make tissues firmer and enhance staining
how to stabilize proteins using fixatives : Heat
Desiccation, chemical reagents
-microwave the specimen in saline heat to - 50-60 or 45-55
-if the above 65 C tissue will become pyknotic, lose enzyme activity . lyse RBC
-if under 45 there will be poor fixation
Desiccation - air dry
Chemicals - primary fixation method.
Additive - binding to tissue to change it (positive to anionic carboxyl and negative to amino cationic)
Non additive-organic reagents acetone/alcohols that dont link to the tissue
Coagulant-allows solution to penetrate tissue
Non coagulant - creates a gel barrier making it harder for solutions further in the reaction to penetrate
how does the nucleus act when reacting with fixatives
dna and rna entrapped by fixed or stabilized nuclear proteins
how do protiens act when reacting with fixatives
-additive fixative changes the 3D shape of a protein by changing the charge at the attachment site
-non additive fixative alters the tertiary structure of proteins making them insoluble
how do lipids and carbs act when reacting with fixatives
lipids - preservation by osmium tetroxide and chromic acid
carbs like glycogen are preserved by entrapment actions of fixed proteins
how to remove fixation pigments
formaldehyde- 37-40% - unfiltered and can form formalin pigment
the working solution is 10% neutral buffered formalin = 3.7-4.0 formaldehyde
if pH is under 4 formalin reacts with heme in hemoglobin to form acid formalin hematin
Formalin Pigments must be removed before staining use picric acid OR 3% NH4OH’
Mercuric precipitate can also be formed. It can be removed with alcoholic iodine/sodium thiosulphate. Mercuric chloride can render tissues with high calcium to be radioopaque
what can occur if fixation is delayed - autolysis and how to solve
-cells can dissapear
-loss of chromatin
-cell shrinks
-lose AG
troubleshoot by:
-putting fresh specimen in fixative 30X volume
-open up breast, uterine or GI specimen
-bread loaf large organs and bisect lymph nodes, kidneys before putting them in fixative
what problems can incomplete fixation cause and how can it be solved
-tissue components seperate
-poor tissue morph
-fuzzy nuclei with no chromatin
-nuclear bubbling
-tissue center is more eosinophilic than periphery
troublshoot:
-leave in fixative longer
-change the fixative and include formalin alcohol in processing
-use thinner sections
-have enough formalin
-dont pack cassettes tightly
-use agitation during processing
-reprocess the tissue
breast tissue needs to be fixed for min 48 hours
what is accessioning and what to look for
lab processing before grossing
-priority for sample -Bone marrow, STAT, urgent, routine, autopsies
-labels and requisition match
-assign a lab number
S – Surgical specimen
1234 – Laboratory number
23 - Current year
A1 – Indicator for tissue type submitted
-all containers need a lab number and indicator for tissue type
-cassettes must be labelled
-separate pt with same last name or same tissue type
when grossing what do you look for
-Macroscopic examination
-note fixative used, specimen type, color, texture, measurements -3D, weight
-dont let tissues dry out
in breast tissue fixation time is very important - need collection time, the time sample was placed in formalin after marking and grossing, and then need ischemic time
Ischemic time = (Time tissue is placed in formalin) – (Collection time)
what is tissue marking
using marking dyes, india ink or meurochrome
notching the tissue
-helps give the orientation
what is embedding
casting/ blocking - putting tissue in an embedding medium and letting it harden for microtomy
the embedding medium should have a high MP to support hard tissues
proper orientation of the tissue is the most important step
QA for wax reservoir
-record daily temps. They must be 2-3 degrees above melting point
-dont let the thermometer touch the metal parts otherwise the reading will be inaccurate
-record in log
-embed flat side of tissue face down use light pressure against base of the mold
what are the three steps in tissue processing and what do they help with
dehydration, clearing and infiltration
aid in hardening tissue
describe the work flow in histotechnology
accessioning - grossing - fixation - processing - enbedding- microtomy- staining
what types of processors are used
rotatory tissue transfer processor - open system
fluid exchange processor - closed system
what is dehydration
what happens if there is too much or not enough
- removal of free water to prepare the tissue for embedding in non aqueous medium
if too much - molecular bound water is removed too causing hard brittle tissue
too little - free water still remaining causing soft mushy tissues
how is water removed by the dehydrant?
hydrophilic dehydrants - attract tissue water
diluting dehydrants - dilute tissue fluids
what reagents are used as dehydrants
Alcohol - etoh, meoh, isoh, buoh
Acetone - rarely used - can be used as dehydrant and clearing agent
Universal solvents - dioxane, tertiary butanol - not good for delicate tissue
use hydrometers to check if there is contamination in alcohols between processing steps
why is ethanol commonly used
hydrophilic
clear colorless flammable
governments controlled
must be denatured
MUST BE USED IN ASCENDING GRADE - so slowly adding high concentrations to not shrink the tissue must start at 60% after fixation with buffered formalin. If you start at over 70 then it will form salt precipitate - artifacts
butanol is 2nd best for plant and animal tissues
what is the clearing step and what is used for it
- slow removal of dehydrating agent- alcohol
-the high RI of xylene makes tissue transparent
-clearing must be done properly because alcohol and paraffin don’t mix.
if there is improper dehydration, clearing , infiltration - causing soft and mushy tissues
if prolonged exposure - hard and brittle tissues especially fibrous, muscular CS and cartilaginous tissues
if the xylene is contaminated with water it will turn cloudy
xylene is flammable cannot be poured down the sink, it is a neurotoxin and defatting meaning it dissolves lipids on skin - GLOVES
what is the infiltration step
-removal of xylene and replacing with support medium like paraffin
-needs a vacuum to speed up reagent exchange
what are the different types of support mediums
beeswax - lowers crystal size and increases stickiness /adhesion
rubber - reduces brittleness, increases stickness which is good for ribboning
plastic - increase hardness and support
paraffin - variable MP (55-58) - high MP therefore becomes harder providing more tissue support allowing for thinner sections and low MP soft, difficult to get thin sections but easy to ribbon
-Crystallization
-staining can use low MP paraffin to preserve AG
what factors affect paraffin infiltration
time - extra exposure causes hardening/shrinkage
temp- 2-4 over MP
-if the paraffin is overheated it can overharden the tissues
-dont process large or fatty tissues in the same cycle
-over processing causes artifacts, brittle tissues
ensure there are three changes of paraffin with the last bath being the cleanest/anhydrous
what steps are taken in microwave oven processing
takes about 45 mins, fix for 30 and ensure the casettes are rinsed in water so there is no salt formation
ethanal, isopropanol - boiled off , and paraffin (60C) are used -
paraffin is molten at 60 - 65 and then 84 C
how does temperature affect fixation
increase temp - increases fixation, autolysis, diffusion of cellular elements
-even if temps go to 45C tissue morp is not affected
Cold fixation only in electron microscopy
room temp fixation with formalin is best
how does tissue size affect fixation
- bisection or loafing must be done so fixative can penetrate all parts of tissue
-tubular specimen - esophagus, intestines have to be cut so all layers are exposed to fixation
-tissue sections should touch the bottom or top of cassette
how does volume ration affect fixation
fixative must be 30X the tissue volume
-if there is not enough fixative volume the additive effect will cause the molecules to bind to tissue components causing decrease of fixative molecules and tissue salts
-poor volume also affects staining
how does time affect fixation
fixation time dependant on tissue size
-small biopsies need 2-6 hours
-large tissues need overnight
-whole organs must sliced first
-fatty tissues need more time as well
how does penetration affect fixation
rate of penetration is affected by heat not concentration with formaldyhe being fastest and picric acid the slowest
how does tissue storage affect fixation
-dont let tissues dry out out back in original container
-storage in 10% neutral buffered formalin is forever
however tissues can be transfered into 70% alcohol to stop protein cross linking
how does pH affect fixation
-always use high quality fixative with known pH
-most are acidic - if the pH is below 4 pigments are produced - formalin reacts with heme therefore youd find pigments in blood vessels
formalin must be buffered to 7.2-7.4
how does osmolality affect fixation
physiological saline 0.3 Osm is used to hold specimen that cannot be fixed right away
-hyper or hypotonic solutions cannot be used as tissue and cellular distortion can happen
