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1.2 Histology > Histopathological Techniques > Flashcards

Histopathological Techniques Flashcards

1
Q

What is neuropatholgy?

A

CNS & PNS disease. Skeletal muscle disease, ophthalmic pathology, & temporal arteritis investigation

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2
Q

What is cytopathology?

A

investigation of diseased cells, serious effusions, CSF, urine, FNAs, bronchial lavages, biliary brushings.

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3
Q

What is immunohistochemistry?

A

exploiting the principle of antibodies specific to antigens.

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4
Q

What is electron microscopy?

A

renal tissue disease, neuromuscular biopsies

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5
Q

What is the core diagnostic laboratory?

A

biopsies from endoscopy, breast clinic, skin – larger samples from surgery e.g. breast, lung, colon – perinatal pathology e.g. placenta, POCs.

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6
Q

What is molecular pathology?

A

melanoma, lung, colorectal muscle – determine the suitability of targeted therapies.

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7
Q

What type of pathology is carried out on a patient posthumously?

A

Postmortem pathology

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8
Q

What are samples received in?

A

10% buffered formalin

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9
Q

What is every sample given before testing?

A

Accession no. - allowed to be tracked

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10
Q

What is minimum acceptance criteria for a sample?

A

patient name, MRN, DOB, nature of specimen & site, relevant clinical information (suspected problem), if an urgent report is needed

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11
Q

What are types of large specimens?

A

colon, breast mastectomy/lumpectomy, kidney, ovary & uterus, & prostate

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12
Q

What are small specimen types?

A

core needle biopsies (lung, breast, liver, lymph node, prostate), stereotactic biopsies (breast), skin biopsies (punch, shaves, excisional, incisional, curetting, wide local excisions)

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13
Q

Describe the histology workflow?

A

fixation of tissue → processing of tissue → embedding of tissue → microtomy → staining → diagnosis/result

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14
Q

Describe cell break down.

A

lysosomes release enzymes in cytoplasm – break down proteins, nucleic acid, & lipids – self-digestion & autolysis.

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15
Q

What is putrefaction?

A

bacteria & fungal spores grow on cell – microbial spoilage.

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16
Q

What is fixation and its purpose?

A

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.

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17
Q

What are features of fixatives?

A

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.

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18
Q

what is the difference between a simple & compound fixative?

A

simple = 1 chemical, compound = >1 chemicals

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19
Q

Write a note on aldehydes as fixatives?

A

create covalent chemical bonds between protein & tissue – protect secondary & tertiary structure – strength & rigidity.

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20
Q

What are2 type of aldehyde fixatives?

A

Formaldehyde & glutaraldehyde

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21
Q

Write a note on formaldehyde.

A

commonly used – mixed with other reagents

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22
Q

Write a note on glutaraldehyde.

A

Electron microscopy (small renal biopsies) – similar chemical properties to formaldehyde – inactivates all enzymes – unsuitable for downstream staining techniques.

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23
Q

Give an example of an oxidising agent as a fixative.

A

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.

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24
Q

What are 2 examples of denaturing agent?

A

acetic acid & ethanol

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25
Q

Write a note on acetic acid.

A

counter the shrinkage effect of fixatives – preserve DNA – many compound fixatives.

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26
Q

Write a note on ethanol as a fixative.

A

cytology – preserves proteins without denaturation – increase penetration by other fixatives.

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27
Q

Write anote on Mercuric chloride as a fixative

A

in many compound fixatives – precipitates proteins – penetrates & hardens tissue rapidly – leave a black precipitate needed to be removed.

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28
Q

Write a note on picric acid as a fixative.

A

binds with histones & basic proteins – form crystalline picrates with AA & precipitate protein – enhance cytoplasmic staining – explosive properties.

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29
Q

What are examples of routine compound fixatives?

A

10% buffered formalin, Bouins Fluid, Carnoys Fixative

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30
Q

Write a note on 10% buffered formalin as a fixative.

A

10% formaldehyde, 90% saline – most common – formaldehyde can cause dermatitis – irritation in eyes, nose & throat.

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31
Q

Write a note on Bouins Fluid as a fixative.

A

Picric acid, formalin, glacial acetic acid – demonstrate glycogen – liver.

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32
Q

Write a note Carnoys fixative.

A

absolute alcohol, chloroform, glacial acetic acid – preserve neurons.

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33
Q

What is needed for grossing?

A

Cutting board, scalpel, sharp knife, scissors, forceps, ruler, weighing scales, downdraft bench, inks, sharps bin, formalin container.

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34
Q

What does the scientist grossing the sample make?

A

Gross report – info. on specimen, description, size, shape, colour, weight, measurement of lesions/tumours, measurement from lesions to margins

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35
Q

What does the scientist do to the sample during grossing?

A

margins inked – serial sections from lateral to medial.

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36
Q

What is an example of an automated tissue processor?

A

Tissue Tek VIP

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37
Q

What are the steps of tissue processing?

A

Placed in cassette – dehydration, clearing, impregnation & infiltration with paraffin wax & embedded further with wax.

38
Q

Tissue Processing: What is dehydration?

A

series of alcohols – dispel water – acetone (urgent small biopsies) – dioxane (diethylene dioxide) (rarely used – toxic & expensive)

39
Q

Tissue Processing: What is clearing?

A

alcohol & wax soluble – xylene (toxic) most common – benzene, chloroform, & cedar wood oil – consider safety, cost, speed, min. tissue distortion.

40
Q

Tissue Processing: What is infiltration?

A

paraffin wax (mixture of n-alkanes – 20-40 C chains) – wax solid at RT, melts at 65˚

41
Q

Tissue Processing: What is the processing procedure?

A

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)

42
Q

How does embedding work?

A

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

43
Q

What is important to carry out when embedding tissue?

A

Ensure clearing agent & dust cleared – rapid cooling (smallest crystals)

44
Q

What is microtomy?

A

cutting of a thin section of tissue - one cell thick

45
Q

What is microtomy carried out in?

A

a microtome - a mechanical instrument used to cut biological specimens into thin section of a predetermined thickness on the character of tissue

46
Q

Who carries out microtomy?

A

a microtimist

47
Q

What is needed for microtomy?

A

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

48
Q

What thickness is them specimen cut to?

A

2 - 25 micrometers

49
Q
A
50
Q

What are 2 types of microtomes?

A

Rocking microtome and automatic microtome

51
Q

How does a rocking microtome work?

A

Block of embedded tissue moves in arc against fixed knife - section small paraffin blocks

52
Q

What is an automatic microtome?

A

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

53
Q

What is an ultra microtome

A

Section resin-embedded blocks for EM - diamond/glass knife - 5-100nm

54
Q

Very brief description of microtomy?

A

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

55
Q

Explain the water bath in microtomy

A

Deionised - 45°-50° - cleaned between cutting (paper dragged through bath to remove debris)

56
Q

Supportive reagents in microtomy?

A

Calbonex/rapid decalcifier - soften calcified areas
10% Formic acid - decalcify bone marrow
10% KOH - soften nails/nail beds

57
Q

Troubleshooting in microtomy

A

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

58
Q

What is case assembly?

A

Matched with corresponding request form and sent to pathologist

59
Q

History of common stains

A

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

60
Q

What is a H&E stain?

A

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

61
Q

What type of nuclei do plasma cells have?

A

Cart-wheel nuclei

62
Q

Stain used for iron (Haemochromatosis diagnosis)

A

Perl’s Prussian blue - stains haemosiderin blue

63
Q

Why is staining carried out?

A

Good contrast between different cells & tissue - diagnosis

64
Q

What is the process of staining?

A

Tissue (solid phase) interact with applied stain (liquid phase)

65
Q

What chemical bonds are involved in staining?

A

Hydrophobic bonding, Van der Waals forces, hydrogen bonding, electrostatic attraction, covalent bonding, ionic bonds

66
Q

What is a chromophore?

A

Any chemical group - alters the absorption range & make an organic compound such as benzene coloured

67
Q

What is chromogen?

A

Substance that will dissolve to give coloured molecules

68
Q

What are most synthetic dyes produced from?

A

Benzene

Benzene + chromophore = chromogen

69
Q

What is an auxochrome?

A

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

70
Q

What can alter final colour of a dye?

A

Modifiers - ethyl / methyl groups deepen final colour

71
Q

What are the different staining methods?

A

Dye method
vital staining
lysochrome methods
histochemistry
immunohistochemistry
metallic impregnation

72
Q

What are vital stains?

A

Stain living cells

  1. Supra -vital: active uptake of dye particles by living entity e.g. methylene blue uptake by reticulocytes
  2. Intra-vital: colouring of enclosed spaces - injection vital strain - mitochondia with Janus green
73
Q

What is the lysochrome method?

A

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

74
Q

Dyes used in the lysochrome method?

A

Sudan Black & Oil red O

75
Q

Histochemisty staining

A

Use chemical or enzymatic reaction to demonstrate tissue

1 chemical reagents - coloured end product in tissue
2 colourless dye - colour restores by tissue components

76
Q

Examples of chemical reactions used in histochemistry?

A

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)

77
Q

Example of enzymatic reaction in histochemistry?

A

Presence/absence of alkaline phosphatase - demonstrated by neutrophils - enzymes cause a colour change on the addition of substrate - decreased in CML

78
Q

What is immunohistochemistry/ immunofluoroescence?

A

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

79
Q

What is metallic impregnation?

A

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)

80
Q

How are results presented?

A

Pathology report - results e.g. benign/mailgnant & bacterial infection

81
Q

Histology Lab Workflow

A

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

82
Q

Dye method

A

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

83
Q

What are mordants usually?

A

Salts of metals

84
Q

Explain mordants MOA

A

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

85
Q

What are examples of mordant dyes?

A

Ehrlich’s haemotoxylin (potassium)
Best’s Carmine (aluminum

86
Q

What are accelerators in staining?

A

Improve staining reactions - enhance affinity between metals & tissue - metallic impregnation (KOH)

87
Q

What are trapping agents?

A

Large aggregates with dye - dye percipiataing in the tissue - difficult to differentiate - trapping reagent increase the hold the tissue has for the stain

88
Q

What is differentiation?

A

Removing excess stain from tissue (Gram stain)

89
Q

What forms a ribbon?

A

successive sections stuck edge to edge

90
Q

What can be done to help establish a ribbon?

A

Blowing on section, return to cold plate

91
Q

Achieving a slide

A

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

1.2 Histology (3 decks)

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