FromDonnaWillis,HarrisMethodistHospital,Ft.Worth,Texas

PURPOSE:

Toproviderapidtissueprocessingthatwilldecreaseturn-around-time.

PRINCIPLE:

Microwaveexposureisusedtodehydrate,clearandimpregnatetissuesamples.Fixationoftissuesamplesisachievedpriortoprocessinginthemicrowaveoven.processingschedulesaredevelopedusingtissuethicknessasadeterminingfactor.DehydrationisaccomplishedusingEthylAlcohol.Isopropanolissubstitutedforxyleneasaclearingagent.Theisopropanolis"boiledout"ofthetissueduringtheimpregnationprocessbyheatingparaffinabovetheboilingpointofisopropanol.

EQUIPMENT:

1. Rotator
2. CommercialMicrowaveoperatingatapprox.600watts.
3. Paraffinpot.Temperaturesetat84°C.Thisinstrumentisusedtorecycleparaffin.
4. Paraffinpot.Temperaturesetat60°C.

QUALITYASSURANCE:

FollowdepartmentalMicrowaveTissueprocessingQualityAssuranceprocedure.

REAGENTS:

1. EthylAlcohol:SafetyData:HealthhazardsofFlammableandIrritantshouldbeconsideredwhenusingthischemical.NFPAratingof2,3,0shouldbefollowed.Targetorgansarenervesandliver.PersonalProtectiveEquipmenttobeused:imperviouslabcoat,gloves,eyeprotection,respiratoryprotectionifexposureisabovethresholdlimits.FirstAidprocedures:Swallowing:Ifvictimisconsciousandabletoswallow,havevictimdrinkwaterormilktodilute.Nevergiveanythingbymouthifvictimisunconsciousorhavingconvulsions.CALLAPHYSICIANORCONTROLCENTERIMMEDIATELY.InducevomitingonlyifadvisedbyphysicianorPoisonControlCenter.Inhalation:Immediatelyremovevictimtofreshair.Ifvictimhasstoppedbreathing,giveartificalrespiration,preferablemouth-to-mouth.GETMEDICALATTENTIONIMMEDIATELY.Contactwitheyesorskin:Immediatelyflushaffectedareawithplentyofcoolwater.Eyesshouldbeflushedforatleast15minutes.Removeandwashcontaminatedclothingbeforereuse.GETMEDICALATTENTIONIMMEDIATELY.AllsplashestotheeyesmustbereportedtoEmployeeHealth.Disposal:Donotflushtodrain.Distillandreuseorcollectanddisposeatanapprovedhazardouswastesite.
2. Isopropanol:PurchasedfromAllegienceScientificProducts.SafetyData:HealthhazardsofFlammable,Teratogen,Irritantshouldbeconsideredwhenusingthischemical.NFPAratingof1.3.0.0shouldbefollowed.Targetorgansareeyes,nose,throat,skin,CNS,respiratorytractandGItract.PersonalProtectiveEquipmenttobeused:labcoat,gloves,eyeprotection.FirstAidprocedures-Inhalation:Removefromexposureareatofreshairimmediately.Ifbreathinghasstopped,giveartificialrespiration.Ifbreathingisdifficultgiveoxygen.Getmedicalattentionimmediately.EyeContact:Flusheyesimmediatelywithlargeamountsofwater,occasionallyliftingupperandlowerlidsforatleast15-20minutes.Ifirritationpersists,seekmedicalattention.AllsplashestotheeyesmustbereportedtoEmployeeHealth.SkinContact:Removecontaminatedclothingandshoesimmediately.Washeffectedareawithsoapandmilddetergentandlargeamountsofwater.Ifirritationpersists,seekmedicalattention.Ingestion:Ifswallowed,inducevomitingbygivingtwoglassesofwaterandstickingfingersdownthroat.Keepvictimsheadlowerthanhipstopreventaspirationintolungs.Nevergiveanythingbymouthtoanunconsciousperson.Callaphysicianimmediately.Disposal:Donotflushtodrain.Collectanddisposeatanapprovedhazardouswastesite.
3. Paraffin:SafetyData:Healthhazards:Undernormalworkingconditions,theuseofthisproductisdeemednon-hazardous.AccordingtoOSHA,paraffinfumemaybeconsideredtobehazardousiftheworKPLaceairborneconcentrationexceeds2mg/m3.SkinContact:Exposuretohotproductmaycausethermalburns.Eyecontact:Exposuretohotproductsmaycausethermalburns..Ingestion:Iflargequantitiesareconsumed,nauseaandvomitingmayoccur.NFPAratingof0,1,0shouldbeconsideredwhenusingthisproduct.PersonalProtectiveEquipment:Ventilation;Providegeneralventilationtoinsurefumeconcentrationsdonoexceed3mg/m3.Gloves:Useprotectivegloveswhenhandlinghotmaterial.EyeProtection:Safetyglassesshouldbewornasgoodlaboratorypractices.FirstAid:Inhalation:Removefromexposureareatofreshairimmediately.Ifbreathinghasstopped,giveartificialrespiration.Ifbreathingisdifficult,giveoxygen.Getmedicalattentionimmediately.SkinContact:Incaseofskincontactwithproductundernormalconditions,washthoroughlywithmilddetergentandlargeamountsofwater.Immediatelycontactaphysicianfortreatmenttothermalburns.EyeContact:Ifhotproductissplashedintoeyes,flushimmediatelywithclearwaterandcontactaphysician.Ingestion:Ifverylargeamountsareswallowedseekmedicaladvice.Disposal:Ensurecompliancewithlocal,stateandfederalregulations.

PROCEDURE:

MicrowaveBiopsy(1mmthick)procedure(processtime45minutesincludingfixation.)

1. Tissuesamplesareplacedinplasticprelabeledcassettesandallowedafixationtimeof30minutesforfreshtissue.(Mostsamplesarefixedadequatelyuponreceipt.)Freshsamplesshouldbeplacedonarotomixeragitatortoenhancefixation.
2. RinsecassetteswithwatertoeliminatethepossibilityofasaltprecipitationwhenthecassettesareplacedintheEthylAlcohol.
3. PlacecassettesintotheTeflonprocessingrack.20cassetteswillfitin1rackleavingthetoplevelemptytoallowforevaporation.80cassettescanbeprocessedatatimeusing4racksand4plasticprocessingcontainers.(Ifyouareprocessinglessthan8cassettesyoushouldplaceatleast8emptycassettesinthebottomoftherack.Thiswillregulatethetemperatureandensureproperprocessing.
4. Placeprocessingrackinplasticcontainerandfillwith100%EthylAlcoholtorinseoffwater.Discardalcoholandfillwith400mL.offresh100%EthylAlcohol.
5. Placetheplasticcontainerinthemicrowaveovenandplacethetemperatureprobecentrallyintothecontainer.Makesuretheprobedoesnottouchthecassettes.
6. Whenstartingthemicrowavefollowthesesteps:
   1. Depressthepowerswitchandobservethatafterabriefperiod,duringwhichtheventsystemcomesuptospeed,thestopswitchindicatorextinguishes.
   2. DepressthelampswitchandobservethatthechamberlightisIlluminated.
   3. Theturntableisnotusedthereforetheswitchshouldnotbedepressed.
   4. Theairflowbubblierisnotusedthereforetheswitchshouldnotbedepressed
   5. Setthetimerselectpushbuttontotheminutesselection.
   6. Setthetime-attemperaturemodeusingthetimermodeselectswitch.
   7. Setthetimepauseposition(out)oftimer/doorbutton.
7. Microwaveatatemperatureof67°C.for5minutes.
8. Taketheplasticcontainerandprocessingrackfromthemicrowaveandremovetherackfromthecontainerdrainingtherackonapapertowel.DumptheEthylAlcohol.
9. PlacetheprocessingrackbackintotheplasticcontainerandfillwithIsopropylAlcohol.
10. Placetheplasticcontainerinthemicrowave.Addthetemperatureprobecentrallyintothebathandmicrowaveatatemperatureof74°C.for3minutes.
11. Removetheplasticcontainerandrackfromthemicrowave.Placetheprocessingrackintoanemptycontainerandfillwithparaffinthatisatatemperaturesettingof60°C.AgitatetheracksothattheexcessIsopropanolwillmixwiththeParaffin.Pourthisparaffinintoaparaffinpotthatisatatemperatureof84°C.RefillthecontainerwithfreshParaffinthatis60°C.
12. Placethecontainerinthemicrowave.Addthetemperatureprobecentrallyintothebathandmicrowaveatatemperatureof65°C.for2minutes.
13. Openthemicrowavedoorandagitatetherackseveraltimestoensuretemperatureconsistency.Adjustthetemperaturesettingto80°C.for5minutes.
14. Removethecontainerfromthemicrowaveanddumpparaffinintotheparaffinpotthatissetatatemperatureof84°C.
15. Removetheprocessingrackfromtheplasticcontainerandplacecassettesatembeddingcenter.
16. Placeprocessingrackintoxylenetoremoveexcessparaffin.Bothplasticcontainerscanbereusedforthenextrun.

PROCEDURENOTES:

1. Themicrowavemustbeventedjustlikeachemicalfumehood.Donotventtoregularreturnairvents.
2. Cassettesshouldbeequallydistributedincassetterack.
3. Whenprocessinglessthan8cassettesuseaminimumof8emptycassettesonthebottomoftheracktoregulatetemperature.

REFERENCE:

H2800MicrowaveprocessorUser"sManual.

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