Unit 3 Principles of Histology.pdf

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1. Unit 3: Principles of Histology

2. Learning Objectives
At the end of this unit the learners should be able to:
1.To gain understanding of the microanatomy of cells, tissues and organs
and be able to relate this structure to the function of these system
2.Define and describe histological characteristics of different cell types
3.Gain general knowledge of tissue preparation and commonly used
staining techniques

3. Introduction
Histology,alsoknownasmicroscopicanatomy,isthescientificstudyof
themicroscopicstructureoforgansandtissuesinthebody
Thisbranchofscienceinvolvesexaminingtissueswithlightandelectron
microscopestogatherdetailsthatareinvisibletothenakedeye
Histologyalsoincludescellulardetaildowntothemolecularlevelthat
canbeobservedusinganelectronmicroscope
Theimportanceofhistologyisthatitisthestructuralbasisforcell,
tissueandorganbiologyandfunction(physiology)and
disease(pathology)

4. Four Basic Types of Tissues
Histology is organized into four basic types of tissue
Epithelium
Connective tissue including
Cartilage and bone
Blood and blood formation
Muscle tissue
Nervous tissue

5. Introduction
Examination of tissues requires that they be prepared for viewing with a
microscope
This is a multi-step process that includes fixation(preserves the tissue),
embedment(stabilizes the tissue for sectioning), sectioning(cuts the
specimen into thin slices of about 5 um) then placing the sections on a
glass slide so they can be stained for viewing
A note about resolution and detection
Resolution refers to the ability to discriminate between two adjacent
objects
For the light microscope with optimal lenses and sample preparation this
approaches 0.2 um, which is the theoretical limit for light microscopes

6. Introduction
The eye can resolve about 250-500 um and the electron microscope can
resolve about 1 nanometer(nm)
Detection refers to the ability to detect something and this can be much
smaller than the limit of resolution
For fluorescence molecules this can be as little as a few molecules

7. Electron Microscope
Structure Size
Human Ovum 120um
Most Cells 10-30um
Red Blood Cells 7um
Mitochondium 0.4-1.0um
Cillium 0.3 um

8. Introduction
Structure Size
Microvillus 100nm
Microtubule 24nm
Myosin filaments 15nm
Intermediate filaments 10nm
Plasma membrane 9nm
Microfilaments(actin) 5nm

9. Tissue Collection and Preparation

10. Tissue Collection
The primary sources of tissue for research are biopsy, surgery and autopsy
Tissues must be collected under strict ethical and legal guidelines and the
collection samples for histology must never compromise the diagnostic
integrity of a specimen
It is preferable for a pathologist to be involved in the procurement of the
tissue specimen during a surgical or autopsy procedure
Other considerations in collecting collection
Assignment
Readandmakenotesonspecimencollection(urine,blood,semen,nail
clippings,bonemarrow,breastmilk,bronchoalveolarlavage,celllines,
exhaledair,feces,fluidsfromcytology(ascites,pleuralfliud,synovialfliud)
hair)

11. Tissue Collection
Timing
Itisimportanttominimizethetimebetweencollectionandstabilization
andprocessingoftissuespecimens
Thistimewillvaryaccordingtotheintendeduse,sincedifferent
biomoleculesdegradeatdifferentrates
Theeffectsofcollectiontimingontissueandmacromoleculespreservation
havenotbeenwellstudied
Thebestapproachistocollect,stabilize(freezingorfixing)andprocess
tissuespecimensasrapidlyaspossible
Itisrecommendedthatsurgicalorbiopsyspecimensarepreservedwithin
onehourorlessofexcision

12. Tissue Collection
Tissue subject to a delay up to two hours should still be collected
Detailed records of timing of events from excision to fixation or freezing
should be kept
Tissue banking staff must be present in pathology to freeze or fix it
Tissues must be snap frozen(rapid cooling of a substance for the purpose
of preservation) either directly or enclosed in a container immersed in the
freezing medium( precooled iospentane)

13. Tissue Collect Cont.
Surgical Specimens
Remnant samples may be collected from diagnostic procedures or with
proper IRB approval, specimens may be resected specifically for research
Specimens may be transported or frozen immediately
Samples requiring snap freezing can be frozen in liquid nitrogen or on dry
ice at time of collection
Otherwise it is recommended that samples can be transported in saline on
wet ice to the lab for additional processing

14. Tissue Collection Cont.
Autopsy Specimens
Itisimportanttoknowthetimeintervalbetweendeathandcollection
processingofspecimenasspecimensmaydegradequicklyafterdeath
Autopsyprocedure'smayyield“normaltissues”(i.e.normallung)orlarge
quantitiesofaspecimenthatwouldnototherwisebeavailablefomsurgical
procedures
Tissuespecimenscollectedatautopsyshouldbeappropriatelylabelledas
totheorgansite,tissuetypeandtimeofresectionandimmendiately
placedinacontainerofsalineonweticefortransporttotissuerepository
forprocessing

15. Tissue Collection Cont.
TransplantTissueandorgansthatareinappropriatefortransplantmay
sometimesbeavailableforresearch
Transplanttissueisofhigherqualitythaneithersurgicalorautopsy
specimensduetothespecialeffortsmadetopreservetheintegrityofthe
transplantorgans

16. Tissue Processing

17. Introduction
Proper handling of tissue specimens is critical to ensure that an accurate
diagnosis is obtained from patients tissue samples
Regardless of the methodology, tissue samples requiring processing need to
be placed in fixative as soon as possible after excision from the patient
This is essential to prevent autolysis/self-digestion (destruction of a cell
through the action of its own enzymes) which could destroy diagnostic
elements and to prepare the tissue for the rigors of reagents used in
subsequent processing steps

18. Definition
Definition
Tissue processing describes the steps required to take animal or human
tissue from fixation to tissue to the state where it is completely infiltrated
with suitable histological wax and can be embedded ready for section
cutting on the microtome
Aim
The aim of tissue processing is to embed the tissue in a solid medium firm
enough to support the tissue and give it sufficient rigidity to enable thin
sections to be cut and yet soft enough not to damage the knife or tissue

19. Factors Influencing Tissue Processing
Viscosity
Solutionswithlowviscositysmallersizedmoleculesallowforfaster
penetrationrateandviceversa
Meltedparaffinwaxhaslowviscositywhichenhancestheimpregnation
rate
Agitation
Increasestheflowofsolutionsaroundthetissue
Themechanismofagitationisautomatedprocessors:eithervertical/rotary
oscillationorpressurizedremovalandreplacementoffluidsattimed
intervals

20. Factors Influencing Tissue Processing Cont.
Heat
Increase in temperature improves the fluid exchange and penetration rate
Excessive exposure to heat can cause shrinkage and hardening of the tissue
which negatively affects subsequent staining and immunohistochemistry
Vacuum and Pressure
Increase fluid mobility thus decreasing the time necessary to complete
each processing step
Vacuum aids the removal of air pockets in porous tissue i.e. lungs

21. Factors Influencing Tissue Processing Cont.
Processing solvent contamination
Thenumberofblocksoneachrun,tissuetype,size,frequencyoftheruns,
useofspongesandcrosscontaminationofprocessorsolventswill
influencehowoftensolutionsshouldberotatedbetweenstationsor
changedtomainprocessingquality
Size
Tissueslicebelow4mmaresatisfactory

24. Fixation
Fixationisthefirstandmostcriticalstepinspecimenhandling
Fixationdenaturesproteinsrenderingthecellanditscomponentsresistant
tofurtherautolysis
Completefixationallowsthetissuetowithstandthenegativeeffectsof
subsequentprocessingreagents
Althoughsamplesaretypicallyreceivedinfixativestationasnatural
diffusionofwaterfromtissuethetissuewillhavedilutedthefixativeinthe
specimencontainer

25. Fixation
Thesizeandtypeofspecimeninthetissuecassettedeterminesthetime
neededforcompletefixationandprocessingtooccur
Thetissueshouldbedissectedto2-4mminthicknessandtrimmedtobe
thesizethatallowscompleteflowofreagentsaroundthetissueincassette
Ideally,tissueshouldbeseparatedaccordingtosizeandtypeand
processedusingdifferentschedules

26. Tissue Fixation
Formalinoralcoholbasedfixationandparaffinembeddingmaybeusedto
preservetissuesatrelativelylowcostwhenadequatefreezingprocedures
andstoragefacilitiesarenotavailable
Formalinfixationisalsothestandardpracticeforpreservationoftissues
collectedduringsurgeryorautopsy
Fixedparaffinblocksmaybestoredinlightandhumiditycontrolledfacilites
atroomtemperature(18-22°C)
FormalinfixedtissuesmaybeusedforDNAextraction
TheDNAisusuallyfragmentedbutremainssuitableforPCR-based
analysisofshortDNAfragments

27. Tissue Fixation
Due to degradation issues, formalin-fixed, paraffin-embedded tissues are of
limited use as a source RNA
RNAlateris a commercial aqueous, non-toxic tissue storage reagent rapidly
permeates tissues to stabilize and protect cellular RNA and eliminate the
need to immediately freeze or stabilize tissue samples
Tissue samples can be harvested and submerged in RNAlaterfor storage
for specific periods without jeopardizing the quality and quantity of RNA
extracted at a later time or date
However, specimens processed in RNALatercannot be further used for
histomorphopathologicalanalysis

28. Tissue Fixation
Alternatives to formalin fixation include ethanol, optimal cutting
temperature(OCT) media, methacarn and Carnoys solution among others
Formalin-fixation remains the standard tissue preservation method

37. Commonly Used Clearing Agents
Xylene-ithasarapidaction,biopsyspecimensof3-4mmthicknessare
clearedin2-4hours
Immersiontimemustnotbeprolongedotherwisethetissuebecomebrittle
TolueneandBenzenearesimilarinpropertiestoxylenebutarelessdamaging
tothetissuesonprolongedexposure
Chloroform
Itisslowerinactionbutitcauseslessbrittlenessthereforetissuecanbeleft
initovernight
Itdoesnotaffecttherefractiveindexofthetissueisnotrenderedtranslucent
Itisexpensive
Itisinflammable

38. Commonly used clearing agents
Carbontetrachloride
Itistoxic
Ithassimilarpropertiestochloroformbutischeaper
Non-inflammable
Cedarwoodoil(histological)
Itisgoodfortreatmentofdelicatetissuesasithastheleasthardeningeffect
Itisveryslowinaction
Itisveryexpensive

39. 4. Impregnation
Definition-Itisthecompleteremoval
ofclearingreagentsbysubstitutionof
paraffinoranysuchsimilarmedia
Impregnationwithwax
Impregnationwithparaffinwaxtakes
placeinanovenheatedto56-60°C
dependinguponthemeltingpointof
thewaxinuse
Frequentcheckofthetemperatureof
paraffinbathsisrequiredsince
temperature5°Cabovethemelting
pointoftheparaffinwillcausetissue
shrinkageandhardening

40. Properties of Paraffin Wax
1.Easytopreparelargenumberoftissueblocksincomparativelyshorttime
2.Minimumsupervisionisrequired
3.Itischeaperthanotherimpregnatingmedia
4.Duringstainingthereisverylittledifficultythanothermedia

41. Points to be remembered during use of paraffin wax
1.Itshouldbefreefromdust,gritanotherforeignmatter
2.Itshouldnotcontainwater,whichcausesittocrystallizeandturnit
white
3.Thewaxhastobefilteredbeforeusebyuseofordinaryfilterpaper
4.Highermeltingpointwaxesarehardtoribbon
Forimpregnationthewaxovenhastobekeptathightemperature,making
thetissuehard,toolowmeltingpointwaxmaynotbehardenoughto
supportthetissueduringcutting
Ifthewaxisoverheatedandremainsinthatstateforalongtime,ittends
tocrystallizeandbecomeuseless

42. Various Waxes Used
ParaffinWax
Paraplast
Paraplastplus
Gelatin
Celloidin

43. Types of Impregnation Ovens
Electricheatedoven
Vacuumembeddingoven
Gasheatedoven

44. Factors Affecting Impregnation
Dependsonthefollowing3factors
•Thesizeandtypeoftissue
•Theclearingagentemployed
•Theuseofvacuumembeddingoven

45. Technique of Impregnation
Thetissueistransferredfromclearingagenttomoltenparaffinwax
Theamountofwaxshouldbe25-50timesthevolumeoftissue
Thetissuemustbesubmittedto3changesinwax
Thetemperatureofthewaxbathshouldbe2-3°Cabovethemeltingpointof
wax

47. 5. Embedding
Embedding
Itistheorientationoftissueinmeltedparaffinwhichwhensolidified
providesafirmmediumforkeepingintactallpartsofthetissuewhen
sectionsarecut

50. Types of Moulds

51. 6. Section Cutting

67. Principles of Microscopy

68. Principles of Microscopy
Biochemicalanalysisisgenerallygoalongwithmicroscopicinvestigation
ofcell,tissueororganellepreparations
Suchtypeofinvestigationareusedinvariousapplications,fore.g.forthe
biochemicalcharacterizationortoexaminetheintegrityofsamples
duringtheexperiment
Microscopyisatechniqueuseformakingverytinythingstovisibletothe
nakedeyesandtheinstrumentusedtomakethingsvisibletotheunaided
ornakedeyeisknownasmicroscope
Themicroscopeistheinstrumentmostfrequentlycharacteristicsof
microbiologylaboratory
Themagnificationprovidedbythemicroscopeenablesviewingof
microorganismsandtheirstructuresotherwiseinvisibletothenakedeye

69. Types of Microscopes
Therearetwofundamentaltypesofmicroscope:
Lightmicroscope
Electronmicroscope
Lightmicroscopeinvolvestheuseofseriesofglasslensestofocuslightin
ordertocreateanimagewhereas,electronmicroscopeuses
electromagneticlensestofocusbeamofelectrons
Lightmicroscopeshaveabilitytomagnifyobjectsuptoamaximumof
approximately1500timeswhereaselectronmicroscopesareabletomagnify
theobjectsmaximumofapproximately200,000times
Magnificationisnotthebestmeasureofmicroscope,rather,resolutionthat
istheabilitytodistinguishbetweentwocloselyspacedobjectsinaspecimen
whichisthemorereliableestimateofamicroscope’sutility

70. Types of Microscopes Cont.
Resolutionlimitofstandardlightmicroscopeisabout0.5micrometersbutin
contrastlateralresolutionlimitofelectronmicroscopeisupto1nanometer
(nm)
Lightmicroscopesareusedtoviewbothlivinganddeadspecimensandoften
inrealcolor,butinelectronmicroscopesonlydeadspecimensareviewedand
neverinrealcolor

72. Light Microscopy
Itisatypeofmicroscopyinwhich
magnificationsisobtainedbyasystemof
opticallensesusinglightwaves
Lightmicroscopeabideofasinglelens
mountedinametalframeisthesimple
formofmicroscope-amagnifyinglens
Lightmicroscopeusuallyusessunor
ambientindoorlightasasourceof
illuminations
Becauseofthetravellingoflightthrough
thespecimens,thisinstrumentisalso
calledastransmissionlightmicroscope

73. Light Microscope
Thelightmicroscopeformsamagnifiedimageofaspecimenwhichis
basedontheprinciplesofabsorption,transmission,diffractionand
refractionoflightwaves
Allmodernlightmicroscopesareusuallymadeupofmorethanoneglass
incombinationsinwhichthemajorcomponentsaretheeyepiecelensor
ocularlens,theobjectivelens,andthecondenserlens,andinstrumentsof
suchcombinationsarethereforecalledcompoundmicroscopes..

74. Types of Light Microscope
Brightfieldmicroscope:inthistypeofmicroscopy,theareaobservedor
themicroscopicfieldisappearsbrightlylightedwhereasthe
microorganismsappearsdarkbecausetheyabsorbsomeofthelight
Darkfieldmicroscope:inthis,effectsproducedbythedarkfield
techniquesisthatofadarkbackgroundagainstwhichobjectsarebrightly
illuminated
Phasecontrastmicroscope:inprinciple,thistechniqueisbasedonthefact
thatlightpassingthroughonematerialandintoanothermaterialofa
slightlydifferentrefractiveindexand/orthicknesswillundergochange
Fluorescencemicroscope:inthisthespecimensitselfactsasalightsource,
thespecimensusedtostudyareeitherfluorescentmaterialsorstained
withfluorescentdye

75. Electron Microscope
Electronmicroscopeisusedwhenthegreatestresolutionisrequired,and
whenthelivingstatecanbeignored.
Theimageproducedinanelectronmicroscoperevealstheultrastructure
ofcells.
Thesourceoflightinanelectronmicroscopeistheelectrongunor
electronbeam.
Atungstenfilamentemitselectrons,whenahighvoltageofbetween40
000and100000volts(theacceleratingvoltage)ispassedbetweenthe
cathodeandtheanode..

77. Types of Electron Microscopes

78. Types of Electron Microscopes
Transmissionelectronmicroscope(TEM):intheTEM,electronsthat
passesthroughthespecimenareimaged
Scanningelectronmicroscope(SEM):intheSEMelectronsthatare
reflectedbackfromthespecimen(secondaryelectrons)arecollected,and
thesurfacesofspecimensareimaged

79. Principles of an Electron Microscope
Electronmicroscopesusesignalsarisingfromtheinteractionofan
electronbeamwiththesampletoobtaininformationaboutstructure,
morphologyandcomposition
Theelectrongungenerateselectrons
Twosetsofcondenserlensesfocustheelectronbeamonthespecimen
andthenintoathintightbeam
Tomoveelectronsdownthecolumn,anacceleratingvoltage(mostly
between100kiloVoltage-1000kiloVoltage)isappliedbetweenthe
tungstenfilamentandanode
Thespecimentobeexaminedismadeextremelythin,atleast200times
thinnerthanthoseusedintheopticalmicroscope.Ultra-thinsectionsof
20-100nmarecutwhichisalreadyplacedonthespecimenholder

80. Principles of an Electron Microscope Cont.
Theelectronicbeampassesthroughthespecimenandelectronsare
scattereddependinguponthethicknessorrefractiveindexofdifferent
partsofthespecimen
Thedenserregionsinthespecimenscattermoreelectronsandtherefore
appeardarkerintheimagesincefewerelectronsstrikethatareaofthe
screen
Incontrast,transparentregionsarebrighter
Theelectronbeamcomingoutofthespecimenpassestotheobjective
lens,whichhashighpowerandformstheintermediatemagnifiedimage
Theocularlensesthenproducethefinalfurthermagnifiedimage

81. Medical Imaging

82. Definition of Imaging
Whatisanimage?
Isarepresentationoftheexternalformofapersonorthinginart
Itistherecordingthefeatureofanobjectformanyengineering,medical,
scientific,securityand/orsocialpurposes
Howcananimagebeobtained?
Thefeatureofanobjectcouldberecordedmanuallyinartsandmost
widelybyimagingsystemseithercamerasoranymodality

83. Definition of Imaging
Whatisanimagingsystem?
Itisasystemthatsendssomesortofenergytotheobjectofinterestand
thenreceivestheenergyreflectedortransmittedfromtheobjecttoafilm
sensitivetothistypeofenergy
Thereceivedenergycarriesdatafromtheobjectwhichwillappearonthe
filmasdifferenceinintensities
Itmaybeassimpleasthatormayrequiresometransformation
mechanismsgetsuchintensitydifferences
Recently,thereareawidevarietyofimagingsystemsthatuse
sophisticatedtechnologiestogetmoreinformationabouttheobjects
beingimagedaccordingtothetypeofenergy,thetypeoffilmwillbeused

84. Definition of Imaging
Therearemoreadvancedimagingsystemthatutilizedcomplicated
technologytoconveyinternalaswellasexternalfeature

85. Definition of Medical Imaging
Medicalimagingisaprocessoftakinganimageforinteriorpartsofthe
humanbodywithoutsurgery.Thisimageprovideinformationaboutthe
differentproperties(eitheranatomicaland/orphysiological)oftheorgans
Thephysiciancanusetheseinformationmakeproperdiagnosisandplan
fortreatment
Manymedicalimagingmodalitiesarecurrentlyavailablewhichallowto
obtainimagesandvaluableinformationforalmostallpartsofthehuman
body

86. Medical Imaging
MajorImagingModalitiesinclude:
X-rays(radiographs)
Mammography
Fluoroscopy
Angiography
ComputedTomography(CT)
Ultrasound(withDoppler)
MagneticResonanceImaging(MRI)
NuclearMedicine(PET/CT)
Theimagemodalityischosenaccordingtotheorganandpropertiestobeinvestigated.
Thedegreeofhazardisalsoanimportantfactorwhenchoosingcertainimaging
modality

87. Assignment: Read and make notes on the major imaging modalities: mention the
indications of each of them
MajorImagingModalitiesinclude:
X-rays(radiographs)
Mammography
Fluoroscopy
Angiography
ComputedTomography(CT)
Ultrasound(withDoppler)
MagneticResonanceImaging(MRI)
NuclearMedicine(PET/CT)
Theimagemodalityischosenaccordingtotheorganandpropertiestobeinvestigated.
Thedegreeofhazardisalsoanimportantfactorwhenchoosingcertainimaging
modality

89. Main Methods of Obtaining Medical Images
Measuringthereflectedradiations(e.g.opticalandultrasoundimaging)
Measuringthetransmittedradiations(e.g.x-rayimaging)
Measuringtheemittedradiation(e.g.magneticresonanceimaging)

90. History of Medical Imaging

124. Imaging Modalities

125. Mammogram
Mammographyimagesarecreatedbytheprocessofx-rayspassing
throughbreasttissueandinteractingwithadigitaldetector
Theanodeinthex-raytubeusedformammographyismadeof
molybdenum,orrhodium,ratherthantungstenwhichiscommonlyusedin
generalx-raytubes
Thisphysicalchangetothex-raytubeanoderesultsinadifferent
spectrumofx-raysthatarebettersuitedtoassessingthefat,connective
tissue,andmammarytissuefoundinthebreast
Thisallowsforverydetailedimagesofsofttissuesusingx-raysasthe
sourceofradiation
Thisimagingmodalityispredominantlyusedtoimagethefemalebreast
tissuebutitisalsocapableofimagingmalebreasttissueforassessmentof
apalpablenoduleormass

127. •The standard positions for image
acquisition in mammography is
the cranial-caudal view and an
oblique view
•Additional mammographic
positions, such as the lateral view,
are used to problem solve
complex abnormalities
•Newer mammography machines
routinely obtain images as the x-
ray tube and the detector plate
rotate resulting in a process called
tomosynthesis

130. •These screening images depict normal breast tissue, fat, and connective tissue
•The more opaque tissue at the back edge of the images is the pectoralismajor muscle
•The breast tissue is predominantly fat replaced and there is no evidence of atypical
calcifications, a nodule, mass, or architectural distortion
•Normal lymph nodes are seen in the axillary area

131. Indications For Mammogram
Mammographyischieflyrecommendedforeverywomanabovetheageof
40,inevery1or2years
Itisrecommendedearlierinlifeincaseofpersonalorfamilyhistoryof
breastcancer