Histopathological Techniques Flashcards
What is neuropatholgy?
CNS & PNS disease. Skeletal muscle disease, ophthalmic pathology, & temporal arteritis investigation
What is cytopathology?
investigation of diseased cells, serious effusions, CSF, urine, FNAs, bronchial lavages, biliary brushings.
What is immunohistochemistry?
exploiting the principle of antibodies specific to antigens.
What is electron microscopy?
renal tissue disease, neuromuscular biopsies
What is the core diagnostic laboratory?
biopsies from endoscopy, breast clinic, skin – larger samples from surgery e.g. breast, lung, colon – perinatal pathology e.g. placenta, POCs.
What is molecular pathology?
melanoma, lung, colorectal muscle – determine the suitability of targeted therapies.
What type of pathology is carried out on a patient posthumously?
Postmortem pathology
What are samples received in?
10% buffered formalin
What is every sample given before testing?
Accession no. - allowed to be tracked
What is minimum acceptance criteria for a sample?
patient name, MRN, DOB, nature of specimen & site, relevant clinical information (suspected problem), if an urgent report is needed
What are types of large specimens?
colon, breast mastectomy/lumpectomy, kidney, ovary & uterus, & prostate
What are small specimen types?
core needle biopsies (lung, breast, liver, lymph node, prostate), stereotactic biopsies (breast), skin biopsies (punch, shaves, excisional, incisional, curetting, wide local excisions)
Describe the histology workflow?
fixation of tissue → processing of tissue → embedding of tissue → microtomy → staining → diagnosis/result
Describe cell break down.
lysosomes release enzymes in cytoplasm – break down proteins, nucleic acid, & lipids – self-digestion & autolysis.
What is putrefaction?
bacteria & fungal spores grow on cell – microbial spoilage.
What is fixation and its purpose?
preserve tissue in life like conditions – prevent autolysis & putrefaction - allow the preparation of thin stained sections – coagulate tissue – crosslink proteins - facilitate downstream staining with dyes & reagent.
What are features of fixatives?
kill bacteria & enzymes quickly with minimum distortion to tissue (life-like condition) – penetrate tissue quickly & evenly – good optical differentiation – inhibit autolysis & putrefaction – harden tissue, insensitive to further treatment – permit the application of numerous staining procedures, allow cell constituents to be seen easier – pH 6-8, room temp. (0-4˚ EM samples), larger tissue=longer fixation, 2-24 hours fixation size-sample & downstream technique.
what is the difference between a simple & compound fixative?
simple = 1 chemical, compound = >1 chemicals
Write a note on aldehydes as fixatives?
create covalent chemical bonds between protein & tissue – protect secondary & tertiary structure – strength & rigidity.
What are2 type of aldehyde fixatives?
Formaldehyde & glutaraldehyde
Write a note on formaldehyde.
commonly used – mixed with other reagents
Write a note on glutaraldehyde.
Electron microscopy (small renal biopsies) – similar chemical properties to formaldehyde – inactivates all enzymes – unsuitable for downstream staining techniques.
Give an example of an oxidising agent as a fixative.
Osmium Tetroxide (OsO4) – joins with side chain of protein molecule – formation of cross link between proteins – stabilize tissue structure – expensive, hazardous, irritation to the eyes, nose & throat – permanently fixes fat – poor penetration power (small biopsies only) – buffered fixative in EM.
What are 2 examples of denaturing agent?
acetic acid & ethanol
Write a note on acetic acid.
counter the shrinkage effect of fixatives – preserve DNA – many compound fixatives.
Write a note on ethanol as a fixative.
cytology – preserves proteins without denaturation – increase penetration by other fixatives.
Write anote on Mercuric chloride as a fixative
in many compound fixatives – precipitates proteins – penetrates & hardens tissue rapidly – leave a black precipitate needed to be removed.
Write a note on picric acid as a fixative.
binds with histones & basic proteins – form crystalline picrates with AA & precipitate protein – enhance cytoplasmic staining – explosive properties.
What are examples of routine compound fixatives?
10% buffered formalin, Bouins Fluid, Carnoys Fixative
Write a note on 10% buffered formalin as a fixative.
10% formaldehyde, 90% saline – most common – formaldehyde can cause dermatitis – irritation in eyes, nose & throat.
Write a note on Bouins Fluid as a fixative.
Picric acid, formalin, glacial acetic acid – demonstrate glycogen – liver.
Write a note Carnoys fixative.
absolute alcohol, chloroform, glacial acetic acid – preserve neurons.
What is needed for grossing?
Cutting board, scalpel, sharp knife, scissors, forceps, ruler, weighing scales, downdraft bench, inks, sharps bin, formalin container.
What does the scientist grossing the sample make?
Gross report – info. on specimen, description, size, shape, colour, weight, measurement of lesions/tumours, measurement from lesions to margins
What does the scientist do to the sample during grossing?
margins inked – serial sections from lateral to medial.
What is an example of an automated tissue processor?
Tissue Tek VIP
What are the steps of tissue processing?
Placed in cassette – dehydration, clearing, impregnation & infiltration with paraffin wax & embedded further with wax.
Tissue Processing: What is dehydration?
series of alcohols – dispel water – acetone (urgent small biopsies) – dioxane (diethylene dioxide) (rarely used – toxic & expensive)
Tissue Processing: What is clearing?
alcohol & wax soluble – xylene (toxic) most common – benzene, chloroform, & cedar wood oil – consider safety, cost, speed, min. tissue distortion.
Tissue Processing: What is infiltration?
paraffin wax (mixture of n-alkanes – 20-40 C chains) – wax solid at RT, melts at 65˚
Tissue Processing: What is the processing procedure?
1)70% ethanol (1 hour)
2) 95% ethanol (1 hour)
3) 100% ethanol (1 hour)
4) 100% ethanol (1.5 hour)
5) 100% ethanol (1.5 hour)
6) 100% ethanol (2 hour)
7) 1st clearing agent (1 hour)
8) 2nd clearing agent (1 hour)
9) 1st wax infiltration @ 58˚ (1 hour)
10) 2nd wax infiltration @ 58˚ (1 hour)
How does embedding work?
Cassette removed from processor – placed in metal mold that corresponds to the size of tissue – hot wax dispelled into mold – placed in cold plate and topped up with wax - cool for 30 mins – wax solidified, mold removed
What is important to carry out when embedding tissue?
Ensure clearing agent & dust cleared – rapid cooling (smallest crystals)
What is microtomy?
cutting of a thin section of tissue - one cell thick
What is microtomy carried out in?
a microtome - a mechanical instrument used to cut biological specimens into thin section of a predetermined thickness on the character of tissue
Who carries out microtomy?
a microtimist
What is needed for microtomy?
Microtomes
Blades
Floating out water baths
Slides – twin frost and adhesive
Slide rack and attachment arm
Fine pointed forceps/paintbrush/ Pencil, probe
Medical wipes
Calbonex / Rapid Decalifier
10% Formic Acid
30% Alcohol
KOH for softening nail specimens
What thickness is them specimen cut to?
2 - 25 micrometers
What are 2 types of microtomes?
Rocking microtome and automatic microtome
How does a rocking microtome work?
Block of embedded tissue moves in arc against fixed knife - section small paraffin blocks
What is an automatic microtome?
Advanced rocking microtome - rotation of hand wheel sets in motion of a cycle of up & down strokes - device allows automatic advancement of block after a cycle - block moved vertically, knife @ 90° - high speed precision electrically driven as small as 1μm
What is an ultra microtome
Section resin-embedded blocks for EM - diamond/glass knife - 5-100nm
Very brief description of microtomy?
Blocks trimmed to 20μm to remove excess wax & expose full face of block - Tissue placed on cold plate & sectioned to 2-4μm - floated in 40° water and picked up by charged slide
Explain the water bath in microtomy
Deionised - 45°-50° - cleaned between cutting (paper dragged through bath to remove debris)
Supportive reagents in microtomy?
Calbonex/rapid decalcifier - soften calcified areas
10% Formic acid - decalcify bone marrow
10% KOH - soften nails/nail beds
Troubleshooting in microtomy
Crunchy/brittle tissue - indicate over processed tissue - ensure new section of blade is used - ensure block is cold when cutting - may benefit from water/softening agent
Calcium/Hard deposits - soak in decalcifying agent
Outer parts of tissue will cut but inside wont - under processed or incomplete wax infiltration
What is case assembly?
Matched with corresponding request form and sent to pathologist
History of common stains
Carmine - botanists
Haematoxylin - Wilhelm von Waldeyer in 1863 - nuclear stain - short staining time & resists acidic solution
Gram stain - bacteria - 1875
Giemsa stain - blood smears - 1891
Zielh-Nielsen - TB - 1883
What is a H&E stain?
Haematoxylin & Eosin stain - tissue architecture & morphology - regressive stain - Haematoxylin = (acidic molecules) well delineated, blue/purple crisp nuclear staining - Eosin= stains RBC red/pink & (basic molecules) collagen, muscle & cytoplasm orange/pink
Charge-based, general purpose stain
What type of nuclei do plasma cells have?
Cart-wheel nuclei
Stain used for iron (Haemochromatosis diagnosis)
Perl’s Prussian blue - stains haemosiderin blue
Why is staining carried out?
Good contrast between different cells & tissue - diagnosis
What is the process of staining?
Tissue (solid phase) interact with applied stain (liquid phase)
What chemical bonds are involved in staining?
Hydrophobic bonding, Van der Waals forces, hydrogen bonding, electrostatic attraction, covalent bonding, ionic bonds
What is a chromophore?
Any chemical group - alters the absorption range & make an organic compound such as benzene coloured
What is chromogen?
Substance that will dissolve to give coloured molecules
What are most synthetic dyes produced from?
Benzene
Benzene + chromophore = chromogen
What is an auxochrome?
Ionisation of chromogen - ionising groups e.g. NO2 (nitro), SO2 (sulphono), & COOH (carboxyl) - turns coloured compounds into dyes & enable to bind to tissue
Chromagen + auxochrome = true dye
What can alter final colour of a dye?
Modifiers - ethyl / methyl groups deepen final colour
What are the different staining methods?
Dye method
vital staining
lysochrome methods
histochemistry
immunohistochemistry
metallic impregnation
What are vital stains?
Stain living cells
- Supra -vital: active uptake of dye particles by living entity e.g. methylene blue uptake by reticulocytes
- Intra-vital: colouring of enclosed spaces - injection vital strain - mitochondia with Janus green
What is the lysochrome method?
Colouring agent dissolves into tissue - colouring of lipids with oil soluble dyes - more soluble in lipids than solvent - pass along a partition gradient via elective solubility - Sudan Black & Oil Red O
Dyes used in the lysochrome method?
Sudan Black & Oil red O
Histochemisty staining
Use chemical or enzymatic reaction to demonstrate tissue
1 chemical reagents - coloured end product in tissue
2 colourless dye - colour restores by tissue components
Examples of chemical reactions used in histochemistry?
Perl’s reaction - ferric ions react with ferrocyanide give blue reaction - presence of iron in bone marrow tissue
Fouchet’s Test - bilirubin - ferric chloride react with bilirubin to form cholecyanin (blue)
Example of enzymatic reaction in histochemistry?
Presence/absence of alkaline phosphatase - demonstrated by neutrophils - enzymes cause a colour change on the addition of substrate - decreased in CML
What is immunohistochemistry/ immunofluoroescence?
Demonstarte specfic antigen/antibodies found in diseased state - e.g. tumour specific antigens - autoimmune diseases antibodies in plasma or attached to antigens in human cells e.g. coeliac Ig to tissue transglutaminase
What is metallic impregnation?
Reduction of metal salts to the metallic state by tissue constituents - silver nitrate reduced to black silver deposits - used in Reticulin stain (reticular fibres in liver - reduced in hepatocellular carcinoma & acute hepatic necrosis)
How are results presented?
Pathology report - results e.g. benign/mailgnant & bacterial infection
Histology Lab Workflow
Day 1 Sample Reception - sample received & accessioned
Day 2 Cut up - macroscopic grossing, inking, overnight processing
Day 3 Main Lab - embedding. microtomy, staining, case assembly
Day 4 Pathologist - Slide review, further tests ordered
Day 5 IHC/ Special Stains - microtomy, staining, slide review
Day 6 Pathologist - reporting
Dye method
Direct - direct affinity for tissue of dissimilar charge - eosin, acid fuchsin, methlyene blue, neutral red
Indirect - poor affinity between dye & tissue - intermediate layer + mordant & combines with dye to form a lake - lake has good affinity & attaches to tissue
dye + mordant > lake + tissue > stained tissue
What are mordants usually?
Salts of metals
Explain mordants MOA
Binding sites attach to tissue & dye - metals affinity for tissue high in glycols, carboxylic acids & certain phenol compounds - used in 3 ways 1 combines in staining solutions 2 used prior to staining solutions 3 used after staining solutions
What are examples of mordant dyes?
Ehrlich’s haemotoxylin (potassium)
Best’s Carmine (aluminum
What are accelerators in staining?
Improve staining reactions - enhance affinity between metals & tissue - metallic impregnation (KOH)
What are trapping agents?
Large aggregates with dye - dye percipiataing in the tissue - difficult to differentiate - trapping reagent increase the hold the tissue has for the stain
What is differentiation?
Removing excess stain from tissue (Gram stain)
What forms a ribbon?
successive sections stuck edge to edge
What can be done to help establish a ribbon?
Blowing on section, return to cold plate
Achieving a slide
1st section held by foreceps, teased out from knife edge with small brush - floating out to avoid folds by placing tail of ribbon in water first (folds removed with paint brush), 30% solution alcohol assist floating out sections - drawn up on charges slide - labelled block number on frosted end with pencil - filter paper remove excess water
