TISSUEPROCESSINGINSURGICALPATHOLOGY

SerenaBoninDSM-Dip.ScienzeMediche

ClinicalSamples

BiologicalFluids

Blood

CSF

Urine

Synovialfluid

Lymphaticdrainage

Ascites

TissuesFFPE(99%)

Freshorfrozen(1%)

SOURCESOFCLINICALMATERIAL

Clinicalsamples

Cytologicalspecimens

Cellularsuspension

Smears

Cytocentrifugationand

Cytoinclusion

Formalin

AlternativeFixatives

BiologicalFluids

CTCs orfcDNA

Biopsy orsurgicalresection

Formalin

SAMPLE’STYPE

BIOPSIESExcisional

Incisional

Curettage

FNA

PUNCHBIOPSIES

AUTOPSIES

ALLPREANALYTICALFACTORSAFFECTRNAINTEGRITYMORESEVERELYTHANDNA

BoninS,StantaG.Nucleicacidsextractionmethodsinfixedandparaffin-embeddedtissuesincancerdiagnostics.ExpRevMol. Diagn.2013,13.

TissueSAFEVacuumUnit

Dedicatedvacuumunitinstalledin“dirtyroom“adjacenttosurgerysuite.

Eliminationofformalininsurgerytheatre.

Transportbiospecimens in“asfreshconditions”tothePathologylab.

SAMPLING

• WASHINGOFTISSUESAMPLES(inneutral bufferinphysiologicalconditions toremove blood - withsucrose ordextran forgoodmorphological preservation forfreezing)• TISSUETRANSPORT(infixative orundervacuum)• SHORTTIMES:autolysis (type oftissue)andputrefaction• INSTRUMENTS:(knives,scalpels,scissors,cork board,...)• TECHNIQUEOFCOLLECTIONANDBIOLOGICALRISKSTYPEOFMATERIALSENT:(surgical piece,biopsy,needle biopsy,autopsy,cytological material)• MACROSCOPICDESCRIPTIONDIMENSIONS(suitable forhistologicalpreparations withthickness suitable forfixation)• LOCALIZATIONANDORIENTATION(anatomy,lesion)

FIXATION• AIM:provide ahistological imageas faithful as possible totherealityorconstantly reproducible (EQUIVALENTIMAGE)

• FIXATIONRAPIDITY:toavoid autolysis (intracellular releaseoflysisenzymes)andputrefaction (saprophytic andenvironmental bacteria).

• PENETRATIONOFFIXATIVE:rateofpenetration into thetissues(depending onthetemperature,which also increases degradation)

• FIXATIONTIME:depends onthetype offixative,thetype offabric andthesize ofthesample

• FIXATIVEVOLUME:1:20forformalin• FIXATIONCONDITION:tissue immersion,pH 7.3-7.4,osmosis pressure0.5osm.

• FIXATIONFAILURE:sampleloss

TYPEOFFIXA

TIVE

S Chemical

Coagulative

Addictive

Physical

Heating (flame-fiamma)

Dessication at r.t.

Freezing

Sublimation (ofwaterundervacuum)

Microwave Oven

Chemical Fixatives(Acting onproteins)

PROTEINHYDRATION:• COAGULATIVEFIXATIVES:thefixative replaces thehydrationwateroftheproteins that denature andprecipitate(COAGULATION)

REACTIONWITHTISSUECOMPONENTS::• ADDICTIVEFIXATIVES:thefixative molecules react chemicallywiththeTISSUEcomponents,withconsumption ofthefixative

ADDICTIVEFIXATIVESFORMALDEHYDE:HCOH–colorless gas,watersoluble.Formalinistheacqueous solutionofformaldehyde(37%).Inteh fixationprocessformalinsolutionisbufferedwithphosphatebuffere atphysiologicpH.

MECHANISM:-proteincrosslinks(methylen bridges-CH-)

R-H+HCHO> R-CH2-OH+H-R’> R-CH2-R’+H2OPENETRATION:0.8mm/hUSE:-SOLUTIONDILUTED10X(knownasformalin10%,realamountofformaldehyde4%)- Lightcanconvertformaldehydeintoformicacid(darkbottleandCalciumcarbonateaddiction)-Fixationlastsfrom12hto4-5days.-Byhaemoglobindegradationformaadarkbrownpigmentisformed.Itcanbeeliminatedaddingethanol70%(95pp)orammonia5%(5pp)

-Conservation(inmuseumswithmarblefragments(Casalts)andafterbubblingwithcitygas,CO>methaemoglobin,tomaintaincolors)

-Mummification-RESULTS:- FATFIXATION-poorcoercion-partiallydissolvesglycogenanduricacidPARAFORMALDEHYDE: polymerofFormaldehyde

ADDICTIVEFIXATIVES

FIXATIVE MIXTURES with picric acid:

•Bouin’s Fixative: a mixture of 15 parts of picric acid in a saturated aqueous solution, 5 parts of 40% formaldehyde and 1 part of glacial acetic acid

•Duboscq – Brasil Fixative: 150 ml EtOH 80%, 60 ml 40% formaldehyde, 15 ml of glacial acetic acid and 1 g picric acid

Both are very penetrating fixatives; however, the presence of picric and acetic acid isan obstacle to any retrospective investigations and DNA extraction and, moreover, iteasily dissolves most of the calcifications.

COAGULATIVEFIXATIVES

ALCHOLIC FIXATIVES• Ethanol 95°• Ethanol 95° and ethyl ether in equal parts• Methanol• Methanol + acetone in equal parts

Alcohol itself, due to its low oxidation potential and moderate penetration capacity, is not a goodhistological fixative (but it is better than nothing); determines excessive coarctation andhardening of the tissues, denature the proteins and coagulates coarsely the cytoplasm;therefore it is preferred to use it in association with other components in order to obtain a moreeffective and homogeneous fixative.

Ethanol 95° or Ethanol 50° are used as pre-fixatives in cytology (equal volume of the samples).

FIXATIVESMIXTURESWITHALCOHOL

• Serra’s Fixative: 2 parts of Ethanol 95% and 1 part of 40% formaldehyde + sme drops of glacial acetic acid.

• Carnoy and MethaCarnoy Fixatives : 6 parts of abs Ethanol(Methanol), 3 parts of CHCl3 and 1 part of glacial acetic acid

• Clarke’s Fixatives: 3 parts of abs Ethanol and 1 part of glacial aceticacid

• Alcholic formalin of Lillie: 9 parts of abs Ethanol and 1 part of 40% formaldehyde

FIXATIVEWASHING

AFTERFIXATIONTISSUEFRAGMENTSMUSTBEWASHEDTOREMOVETHERESIDUESOFTHEFIXATIVETHATOTHERWISEMAYIMPACTONTHEFOLLOWINGPROCESSES

INCLUSION- FIXEDTISSUES,INORDERTOBEOBSERVEDATTHEOPTICALMICROSCOPE,MUSTBESECTIONEDINTHINSECTIONS2-8µm

-TOOBTAINTHINSECTIONSTISSUESMUSTBESUFFICIENTLYHARDANDCOMPACT>INCLUSIONINSEMI-SOLIDSUBSTANCES-TISSUESMUSTBEEMBEDDEDINTHEINCLUSIONSUBSTANCE>PARAFFIN

PARAFFINCH3-CH2- - - -CH2-CH3 (CnH2n+2)

-ITISOBTAINEDFROMTHEOILDISTILLATIONRESIDUES

-THEMELTINGPOINTRISESWITHTHELENGTHOFTHECHAIN

-INHISTOLOGYC22-C28PARAFFINSAREUSED,DIVIDINGTHEPARAFFINSINLOWMWLTING(45- 54° C)ANDHIGHMELTING(58- 60° C)- NOWADAYSNONATURALPARAFFINAREUSED(MIXTUREOFDIFFERENTLENGTH),BUTSYNTHETICPARAFFINS,PUREANDHOMOGENEOUS(ES.PARAPLAST)

-PARAFFINSOLVENTS::XYLENE,CHCL3,BENZENEANDTOLUENE

TISSUES’INCLUSIONPROCESS1-FIXATION2-WASHING-ETHANOL70% - 4h I- ETHANOL95% - 4h I 3-DEHYDRATION- ETHANOL100% - 4-6h I- ETHANOL100% - 4-6h I

-XYLENE - 1-2h I 4-CLEARING-XYLENE - 1-2h I

-PARAFFIN2-3X - 3-4hX1 5-INCLUSION6-WAXCASTING (PARAFFINGBLOCKINCLUDINGORIENTEREDTISSUE)

7-COOLING(HOMOGENEOUSPARAFFIN)

HYSTOLOGICSECTIONSMICROTOMES: -SLEDGEMICROTOMES(MOBILEORFIXBLADE)

-ROTATORYMICROTOME(SERIALSECTIONS)

-CRYOMICROTOME

-CRYOSTAT(ROTATOTARYMICROTOMEINREFRIGERATEDCHAMBER)

MICROTOMEBLADES:- THEYHAVESECONDARYCUTTINGFACES-

- INCLINATIONCOMPAREDTOTHECUTTINGSURFACE10- 15°

- LOWERTHAN10° THEBLADEBRUSHESWITHOUTCUTTING,OVER15° THEBLADEBREAKSTHEPARAFFIN

MicrotomoaslittaSLEDGEMICROTOME Microtomorotativo

ROTATORYMICROTOME

DEPARAFFINIZATIONOFTHESECTION

-XYLENE - 5min-XYLENE - 5min

-ETHANOL100% - 5min- ETHANOL100% - 5min- ETHANOL95% - 5min HYDRATION- ETHANOL70% - 5min-DISTILLEDWATER

STAINING

TISSUEDECALCIFICATIONFIRSTOFALLTISSUESSHOULDBEFIXED,OTHERWISETHEYUNDERGOMACERATION.Decalcification is carried outbymeans ofstrongacids,toobtain soluble Casalts.Decalcification methods that employ acids are most widely used in pathology laboratories. Since calcium is soluble at a pH of 4.5, acids quickly and easilydissolve the calcium salts. There are two types of acids used in decalcification procedures: Strong mineral acids OR Weak organic acids. The most commonacids used for decalcification are 5-10% solutions of HCl, nitric acid, and formic acid. These acids can be used alone or in combinations. The followingshould be considered before implementing an acid decalcifying protocol in the laboratory. Tissue must be trimmed small and fixed first. Decalcifiers withhigher concentration of acids act rapidly and affect tissue staining the most. Tissue left in acid too long will lose nuclear staining. Decal. solution must bechanged frequently because calcium that has leached out will become a barrier to further decalcification. Agitate tissue during decalcification to expose allsurfaces to fresh decal. agent. Heat should be avoided with strong acid decalcification as swelling of tissue and possible digestion of bone collagen willoccur. Tissue must be rinsed in water prior to processing, otherwise acids will continue to decalcify tissue; will also prevent possible chemical reactions withsubsequent reagents and contamination of processor reagents.

CaCO3+2HNO3 >Ca(NO3)2+H2O+CO2

DECALCIFIERS:- NITRICACID 5-7.5%-TRICLORIDEACETICACID 5%-FORMICACID CONC.

Decalcification solution should bechanged 2Xin24h.Theprocess lasts somedays.Aneedl is used totestthedecalcification reaction.