Week 1

Question 1

what is the flash point

Answer 1

lowest temperature where the vapours can ignite . The higher the FP the less flammable something is

Question 2

what is fixation

Answer 2

-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

Question 3

how does fixation prevent post mortem activities

Answer 3

• Autolysis
  -Putrefaction
  -Maintains relationship between cells and extracellular substances - collagen, reticulin, Elastin

Question 4

what is the difference between Autolysis and Putrefaction

Answer 4

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

Question 5

how does fixation bring out the differences in RI

Answer 5

-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

Question 6

how does fixation make cell parts insoluble

Answer 6

-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

Question 7

how to stabilize proteins using fixatives : Heat
Desiccation, chemical reagents

Answer 7

-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

Question 8

how does the nucleus act when reacting with fixatives

Answer 8

dna and rna entrapped by fixed or stabilized nuclear proteins

Question 8

how do protiens act when reacting with fixatives

Answer 8

-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

Question 8

how do lipids and carbs act when reacting with fixatives

Answer 8

lipids - preservation by osmium tetroxide and chromic acid
carbs like glycogen are preserved by entrapment actions of fixed proteins

Question 9

how to remove fixation pigments

Answer 9

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

Question 10

what can occur if fixation is delayed - autolysis and how to solve

Answer 10

-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

Question 11

what problems can incomplete fixation cause and how can it be solved

Answer 11

-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

Question 12

what is accessioning and what to look for

Answer 12

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

Question 13

when grossing what do you look for

Answer 13

-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)

Question 14

what is tissue marking

Answer 14

using marking dyes, india ink or meurochrome
notching the tissue

-helps give the orientation

Question 15

what is embedding

Answer 15

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

Question 16

QA for wax reservoir

Answer 16

-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

Question 17

what are the three steps in tissue processing and what do they help with

Answer 17

dehydration, clearing and infiltration

aid in hardening tissue

Question 18

describe the work flow in histotechnology

Answer 18

accessioning - grossing - fixation - processing - enbedding- microtomy- staining

Question 19

what types of processors are used

Answer 19

rotatory tissue transfer processor - open system
fluid exchange processor - closed system

Question 20

what is dehydration
what happens if there is too much or not enough

Answer 20

• 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

Question 21

how is water removed by the dehydrant?

Answer 21

hydrophilic dehydrants - attract tissue water

diluting dehydrants - dilute tissue fluids

Question 22

what reagents are used as dehydrants

Answer 22

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

Question 23

why is ethanol commonly used

Answer 23

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

Question 24

what is the clearing step and what is used for it

Answer 24

• 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

Question 25

what is the infiltration step

Answer 25

-removal of xylene and replacing with support medium like paraffin
-needs a vacuum to speed up reagent exchange

Question 26

what are the different types of support mediums

Answer 26

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

Question 27

what factors affect paraffin infiltration

Answer 27

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

Question 28

what steps are taken in microwave oven processing

Answer 28

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

Question 29

how does temperature affect fixation

Answer 29

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

Question 30

how does tissue size affect fixation

Answer 30

• 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

Question 31

how does volume ration affect fixation

Answer 31

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

Question 32

how does time affect fixation

Answer 32

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

Question 33

how does penetration affect fixation

Answer 33

rate of penetration is affected by heat not concentration with formaldyhe being fastest and picric acid the slowest

Question 34

how does tissue storage affect fixation

Answer 34

-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

Question 35

how does pH affect fixation

Answer 35

-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

Question 36

how does osmolality affect fixation

Answer 36

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