1.

0TITLE:PROCEDUREFORNONEGYNESPECIMEN

2.0PRECAUTIONS
PPEshouldbewornatalltimeswhenhandlingpotentiallyinfectiousbodyfluidsinaccordancewiththe
KFHUSafetyPolicy.Allpreparatoryproceduresshouldbeperformedusingafaceshield.Allinfectious
materialsshouldbeproperlydisposedofinbagsidentifiedforbiologicalorinfectiouswastesforproper
disposal.

3.0PROCEDURESFORSPECIMENFIXATION:
Allspecimensprocessedincytologyshouldbefixedbeforestaining.
FixativesUsedinCytopathology
1.80%isopropanol(800mlofabsoluteisopropanol+200mlofwater)
2.Spraycytefixative(Commerciallyprepared)
3.Bouinsfixativeforcellblock(preparedbyHistopathologySection)
4.0PROCEDURECENTRIFUGATIONANDPROCESSINGOFFLUIDSPECIMENS
Centrifugationcanbedoneforanyofthethreepurposes:
1.Toobtainacellconcentrate(pellet),fromwhichtomakecellspreads.
2.Toseparatecellsfromtheirdebrisladensolution,priortofiltration.
3.Toformacellconcentrate,or"button",forCellblockpreparation

4.1Reagents,Instrumentation:Vialwithspecimen.Centrifuge,tubes,PasteurPipettes.
4.2StepbystepDescription
SpecimenlabelsarecheckedagainstdataontheRequestFormforaccuracy.Allrequestformsaregivena
uniquesequentialnumber,andthenloggedinthecomputer.Ifapatienthasapreviouscytologyorhistology
reportthelaboratorynumberandtheresultarenoted.
1.Notedownthevolume,colorandconsistencyofeachspecimenattheupperrighthandcornerof
therequestform.
2.Centrifugethespecimenin1525cccentrifugestubes(dependingonthevolumeofthespecimen)
andcentrifugeatthe18002500revolutionsperminute(RMP)for10minutes.
3.RemovethesupernatantwiththehelpofaPasteurpipette.
4.Placeoneortwodropsfromthesedimentorthebuffycoatontheprelabeledfrostedendglass
slidesandspreadevenlywiththehelpofanothercleanglassslides.
5.Fixthemimmediatelywithspraycyteordipin80%isopropanol/95%ethanol.
6.StainthemwithPapanicolaoutechniqueandmount.
4.3EffectofcentrifugationonCells
Althoughoflittleobservablesignificanceinexfoliativecytology,severaleffectsofcentrifugationonthe
qualityandquantityofcellshavebeendescribed.Reductioninnumbersofcellsalwaysoccurs.Itmaybe
causedbyoneofthefollowingfactors:
4.3.1Ruptureofdelicate,senescent,ordegeneratecellsbyshearstresses.
4.3.2Impactionalongthewallofthecentrifugetube,or"walledeffect".
4.3.3Inadequatecentrifugalforce,therebycausingsomecellstosuspendinthesupernatant.
4.3.4Damagetothecellularmorphologybycentrifugalforceisseenprimarilyinthecytoplasmofthe
cells.Cellsthusdamagedcanexhibitraggedcytoplasmicborders.Inturn,theseborderscancausecellsto
clumpsecondarily.Thepresenceofproteininsomespecimenslargelyprotectsagainsttheseeffects.
4.4ReportofResults:Specimenisscreenedbyseniorcytotechnologistandsubmittedtotheconsultant
cytopathologistforreporting.
4.5MaintainingofCentrifuges/Cytospin
4.5.1DailyCleaningCleaningoutersurfacesandifthereisuglyspillage,immediatelycleanwithhospcide
spray/10%chlorox.Drybucketbeforeuse.Switchoffcentrifugebeforecleaning
4.5.2WeeklyCleaningandmonthlyRemovebucketscleanthefollowing:bowl,carriages,insidecover,
externalsurfaceswithhospicidespray/10%chloroxrinsewateranddrybeforeuse.
4.5.3ChangingCarbonBrushifapplicable.MaintainedbyBioMedDepartment
SpeedCheck Speed Time

Centrifugemachine(Heraeus) 2000rpm 10minutes
Centrifugemachine(Hettich) 2000rpm 10minutes
CytospinShandon 800rpm 3minutes
Precautions:Closethelidbeforestart.Neverusewithopenlid.

5.0PROCEDURECYTOSPINPREPARATIONS:
5.1MaterialandEquipment
BiohazardhoodCytocentrifuge
Gloves(Nitrite)Pencil
CytospinchambersCytospinfunnels
CytospinfiltercardsSpraycyte/80%isopropanol
Glassfrostedend(superfrostplusslides)

5.2Stepbystepprocedureforsmearpreparation
5.2.1Thevolumeanddescriptionofthespecimenisnotedinthecorrespondingnumberedrequestform.
ThisnumbershouldbeindicatedinthespeciallymadeslidesfortheCytospini.e.,slideswithwhiterings.It
shouldbenotedthattheslidesarecleanedwithalcoholpriortouse.
5.2.2SwitchtheCytospinon,openthelid,andlifttheheadassemblyoutoftheinstrumentbeforeloading
thesamples.Theheadassemblycanthenbeopenedbypullingupthecentralbutton.Theslideclipsarethen
removedfromtheheadassemblyandloadedwiththeglassslidefilterpapersamplechambercombination
andseeingtoitthattheoutletportcoincideswiththefiltercardholesandthepredrawncircleontheslide.
Oncechecked,thisslideassemblyisthensecurelyheldbytheretaininghooksoftheslideclips.This
assemblyshouldbemadeinduplicateandplaceinoppositeslotsoftheheadassemblytobalance.
5.2.3Threetofivedropsofthesampleareplacedinsideineachofthesamplechambersandthehead
assemblybeingclosedsecurely.Theheadcontainingthesamplesisthenloadedinthecenterconeofthe
machine.Withthesafetycoverinposition,andlockedthecorrectspeedprogramisentered,and
centrifugationisstarted.Abuiltintimerandactualspeedindicatorenablestheoperatortomonitorthe
centrifugationprocess,andalarmstoindicatespeeddroporcyclecompletion.
5.2.4Theslideassemblyisseparatedfromeachotherwiththefiltercarddetachedfromtheslideinaslow
peelingmotiontopreventsmudgeinotherareasoftheslideotherthaninsidethedrawncircle.
5.2.5ThepreparedslidesareimmediatelysprayedwithSpraycytetofix.Thesearethenallowedtodryfor
5minutesandreadytostain.TheCytospin4isaspecialinstrumentwhichconcentratessmallnumberofcells
inminutevolume.Itusescentrifugalforcetospincellsuspensionsandsedimentsuntoaglassslidewiththe
mediumbeingabsorbedbyafiltercard.Theresultisamonolayerofcellsspreadwithina6.0mm.areaon
theslide.

6.0PROCEDURESPECIMENWITHBRUSHES
6.1SpecimenRequired
SmearsaremadefromEsophageal,gastricandbronchialbrushspecimensintheclinics.Alternativelythese
canbesentfresh,insmallamountsofsalineorRPMIsolution,justenoughtocoverthebrush.
Allspecimensaresentfreshtothecytologylaboratory

6.2Reagents,Instrumentation:Disposablepipette,Centrifuge,Centrifugetube.

6.3StepbystepDescription
6.3.1Transferthespecimentoanappropriatecentrifugetube,preferably(1525ml).
6.3.2Agitatethesample(vortex)withthebrushonit.
6.3.3Centrifugeat2,000rpmfor10minutes.
6.3.4Removethebrushfromthecentrifugetubewithforceps.Discardthebrushinthebiohazardcontainer.
6.3.5Pouroffsupernatant.
6.3.6Agitatethecellpellet(vortex).
6.3.7Proceedwiththeappropriatesmearpreparation.

7.0PROCEDURETOELIMINATEREDBLOODCELLSFROMCYTOLOGICSPECIMENSBY
HEMOLYZINGFIXATIVE(CYTORICHRED):
Mix/shakethesamplewell.
Pourarepresentativeamountintoaproperlylabeled50mlcentrifugetube.
Centrifugethesamplefor10minutesat2000rpm.
Decantthesupernatantfluidandadd25mlofcytorichredfluid.
Capthetubeandvortexfor1015seconds
Allowthesampletofixandlyseforatleast15minutes.
Processspecimenaccordingtolabpreparationtechnique.
Precautions
UsePPE.
Followbiohazardprecautionswhenhandlingsamples.

8.0PROCEDUREFORCELLBLOCK
Thisprocedureutilizeshistologicproceduresforprocessingcytologicmaterial,thusallowingmultiple
sectionsofthesamematerialforusewithroutineaswellasspecialstainapplication.Thistechniquecanbe
appliedtoallcytologicspecimensbutisespeciallyusefulforfluidsandaspiratesorwhenevertissue
fragmentsareincidentallyobtainedduringcytologicprocedure.Cellblockingprocedureshouldbeprepared
wheneveritisneededespeciallyinaspecimenwithclotandsubstantialamountofcellpelletafterthesmear
preparation.
Precautions
8.1Solution:Bouinssolution
8.2Materials
Cassette
Spatula/applicatorstick
Pipette/forceps
2550mLcentrifugetube/universalcontainer
Lenspaper/filterpaper

8.3Stepbystepprocedure
1.SedimentsandtissuefragmentsareplacedinBouinsfixative.Itshouldbenotedthatfibrinclots
onthesideofthefibrincontaineraregentlytwistedandremovedbyapplicatorstickwithfluidwrung
outwiththetrappedcellsinthementionedfixative.Allowtostandforatleasttwo(2)hourstofix
(optional).
2.Centrifugeat1,8002,500RPMfor10minutes.
3.Pouroffsupernatantorremovesupernatantbydrainingthetubeontheabsorbentpaperleaving
thecellbuttonorsediment.
4.Carefullyremovethesedimentfromthecentrifugetubeusingaspatula/applicatorstickandwrap
inlenspaper/filterpaper.Thewrappedsedimentsarethenplacedintissuecassettewiththeir
correspondingcytologyaccessionnumber.(Usepencilonly).
5.Takespecimentohistopathologyforprocessing.

SPECIALPROCEDUREFORCERTAINSAMPLES

9.0PROCEDUREFORRESPIRATORYTRACTSPECIMEN(sputum,brush,lavage):
9.1Materials
1.Biohazardhood
2.PPE
3.Frostedendmicroscopicslides
4.Pencil

9.2Reagents:
1.Spraycyte
2.80%isopropanol
3.Papanicolaoustain

9.3Samples(Sputum,Bronchialbrush,Bronchiallavage)

9.4Procedures:(Processing)
ProcedureforPreparation&fixationofslideswillbedifferentforeachtypeofspecimensDetailsaregiven
below:
9.4.1BronchialBrush:
Smearsarepreparedandfixedinbronchoscopyroomandsenttothecytologylabforprocessingand
reporting.
9.4.2BronchialLavage:
Specimeniscentrifugedat2,000RPMfor10minutesandsmearspreparedfromthesedimentasfromfluids
&fixedimmediatelywithspraycyteor80%isopropanol.
9.4.3Sputum:
Sputumisamixtureofsalivaandsecretionsformthelowerrespiratorytract.Inaddition,nasopharyngeal
mucusmaybeadmixedwiththespecimen.Therefore,somespecimensdesignatedassputummayinfact
beonlynasopharyngealmaterial.
Thetracheobronchialtreepresentsaverylargeareaforsampling,andmucusfromdifferentlocationsinthe
patientslowerrespiratorytractmaypossessdifferentgrossphysicalcharacteristicswithinagivenspecimen.
Forthisreason,materialfromsputumsmearsshouldbeselectedbypickingoutsmallportionsofthematerial
withdifferentcolorandtexture.Thesesmallsamplesarepooledononeslideandsmearedtogether.Sucha
selectivesamplingprocedureisnot,ofcourse,necessaryforspecimensthatappeargrosslyhomogeneous.

Thefollowingstepsaretobeusedinthepreparationofsputumspecimensformicroscopic
examination:
1.Specimenmustbefreshorprefixedsputum.
 2.Labeltwoslideswiththepatientsname,accessionnumberandsourceonthefrostedendofthe
slide.
3.Selectsuspiciousparticlesfromthesample.
4.Transferparticletotheslide.
5.Gentlycrushsamplebetween2slides,distributingmaterialthinlyandevenlyoversurfaceofthe
slide.(Seediagram).

Note:
Thick,slipperyoftenaciousspecimensaredifficulttosmearintotheproperfinishedproductandmustbe
manipulatedforsomelengthoftimebeforethematerialbeginstospreadproperlyandadheretotheslide
evenly.Specimensthatarethinorwatery,ontheotherhandrequireonlyaminimumoftimetosmear.
6.Sprayfixorfixslide(s)immediatelyin80%isopropanolforaminimumof10minutes.
Note:
Somespecimensdonotadheretotheslidewhenplacedinfixativeandconsequentlythespecimen
willfloataway.Whenthishappens,additionalsmearsshouldbepreparedifpossibleandsprayfix
andleavetodrybeforestaining.

10.0PROCEDUREVOIDEDURINE,CATHETERIZEDURINEORBLADDERWASH.
1.Notethegeneralappearanceofthesampleandindicatethedescriptionintherequestform.
2.Pourthespecimeninto1550ml.centrifugetubesandspinforten(10)minutesatrecommendedspeedof
1,8002,500rateperminute.Whiledoingthecentrifugation,twosuperfrostplusslidesarepreparedand
labeledaccordingly.
3.Pourofthesupernatantfluidbackintotheoriginalcontainer,keepingtheremainingfluid(withthe
sediment)atthebottomofthecentrifugetubeforthenextstep.
4.Mixthesedimentbyslightlytappingthetubeorforthickerfluidbytheuseofthevortexmixer.
5.Adroportwoisplacedononeoftheslidesand,usingthepullapartmethod,thesedimentisspread
evenlyonthesurfaceofbothslides.Remainsofthesedimentinthecentrifugetubesarestoredinthe
refrigeratoruntilthediagnosishasbeenreleased.
6.ThesmearsarefixedimmediatelybyCytosprayorimmersedin80%/isopropanol/95%ethylalcohol.The
slidesareallowedtostandforatleast1015minutesbeforestaining.

11.0PROCEDURECEREBROSPINALFLUID(CSF)
11.1Sample:
Cerebrospinalfluidisobtainedthroughlumbartaporventriculartapasaclinicalprocedureintheclinicsand
immediatelysenttothelabwithoutanydelay.Becauseanydelayinprocessingmorethananhourwillaffect
thecellsnegatively.

11.2Regents:Spraycyte/80%isopropanol,MGGstain

11.3ProcedureforSmearPreparation
1.Thevolumeanddescriptionofthespecimenisnotedinthecorrespondingnumberedrequestform.This
numbershouldbeindicatedinthespeciallymadeslidesfortheCytospini.e.,slideswithwhiterings.It
shouldbenotedthattheslidesarecleanedwithalcoholpriortouse.
2.SwitchtheCytospinon,openthelid,andlifttheheadassemblyoutoftheinstrumentbeforeloadingthe
samples.Theheadassemblycanthenbeopenedbypullingupthecentralbutton.Theslideclipsarethen
removedfromtheheadassemblyandloadedwiththeglassslidefilterpapersamplechambercombination
andseeingtoitthattheoutletportcoincideswiththefiltercardholesandthepredrawncircleontheslide.
Oncechecked,thisslideassemblyisthensecurelyheldbytheretaininghooksoftheslideclips.This
assemblyshouldbemadeinduplicateandplaceinoppositeslotsoftheheadassemblytobalance.
3.Threetofivedropsofthesample(CSF)areplacedinsideineachofthesamplechambersandthehead
assemblybeingclosedsecurely.Theheadcontainingthesamplesisthenloadedinthecenterconeofthe
machine.Withthesafetycoverinposition,andlockedthecorrectspeedprogramisentered,and
centrifugationisstarted.Abuiltintimerandactualspeedindicatorenablestheoperatortomonitorthe
centrifugationprocess,andalarmstoindicatespeeddroporcyclecompletion.
4.Theslideassemblyisseparatedfromeachotherwiththefiltercarddetachedfromtheslideinaslow
peelingmotiontopreventsmudgeinotherareasoftheslideotherthaninsidethedrawncircle.
5.ThepreparedslidesareimmediatelysprayedwithSpraycytetofix.Thesearethenallowedtodryfor5
minutesandreadytostain.TheCytospin4isaspecialinstrumentwhichconcentratessmallnumberofcellsin
minutevolumee.g.,CSF.Itusescentrifugalforcetospincellsuspensionsandsedimentsuntoaglassslide
withthemediumbeingabsorbedbyafiltercard.Theresultisamonolayerofcellsspreadwithina6.0mm.
areaontheslide.
6.SmearsthenaregiventoHistosectionforGiemsastain.

12.0PROCEDURETZANCKSMEARPROCEDURE:
TheTzancksmearprocedureisusedtoidentifycertaincharacteristiccells(multinucleatedSyncytialgiant
cells)orcellinclusions,usuallyviral.
Material:Frostedendmicroscopeglassslide.80%isopropanolinCoplinjarsorsprayfixative.

13.0PROCEDUREPNEUMOCYSTISCARINIIPROCEDURE
Pneumocystiscariniipneumonia(PCP),astheconditioniscommonlytermed(althoughthecausativeorganism
hasbeenrenamedPneumocystisjiroveci)isthemostcommonopportunisticinfectioninpersonswithHIV
infection.Pneumocystisisagenusofunicellularfungifoundinrespiratorytractofmammalsandhumans.It
issometimesfoundconcurrentwiththeotheropportunisticinfectionssuchasaspergillosisorcryptococcosis.
Althoughlatentinfectionexistsinanimalsandman,thediseaseisalmostrestrictedtoprematureand
debilitatedinfants,olderindividualoncaloriedeficientdiets,andparticularlychildrenandadultsunder
treatmentwithcytotoxicdrugs(forleukemiaandlymphoma)orotherimmunologicallycompromisedhosts.
13.1Equipment&Material
Equipment:(Microscope,Centrifuge)
Materials:
GlassSlides,coverslips
Centrifugetube
Stainingdish,Coplinjarsandbasin
Slideholderandtray
Mountant
Distilledwater
Spraycyte
Tissuepaper(Kimwipes)

Reagents:Grocottsmethenaminesilverstain,Papstain

13.2StepbyStepProcedureforSmearPreparation:
Someofthespecimensreceivedinslides,howeverifamucoidspecimenreceivedthepreparationisthesame
asinsputum.Forfluidisspecimen,thesameasintheprocessingofeffusionfluid.
Microscopicscreening
Thescreeningofslidesisusuallyperformedstartingfromthelowpowerobjective(x10),tothehighpower
objective(x40).ThehighpowerobjectiveisutilizedforcloseinspectionofthePneumocystiscarinii
organisms.Thecystsresemblescrushedpingpongballsandarepresentinaggregatesof2to8(notto
confusedwithHistoplasmaorCryptococcuswhichtypicallydonotformaggregatesofsporesorcells).
Organismsareoftenseeninfoamyalveolarcasts(roundedmassesoforganisms).Castsstaineosinophilicto
basophilicinPapanicolaoustain.InGrocottsmethenaminestain,thecellwallofthecyststainsblack,often
withacentraldarkdot.

14.0Responsibility:
Appliestocliniciansorlaboratorystaffresponsibleforhandlingthespecimens.

15.0Attachment:
PapStainingMonitoringForm01

16.0Distributions:
LMDAdministration
CytologyLaboratory
AllClinicalDepartments

17.0References:
Koss,LeopoldG.KosssdiagnosticCytologyanditshistopathologicalbases,1992.
ComprehensiveCytopathology,editedbyMarluceBibbo,1991.
ManualofCytotechnologyeditedbyCatherineM.Keebler,AmericanSocietyofClinical
Pathologist,1983.
CytologyDiagnosticPrinciplesandClinicalCorrelatesThirdEdition.ByEdmundS.Cibasand
BarbaraS.Ducatman
ManualforCytology,NationalCancerControlProgramme,DirectorateGeneralofHealth
Services.MinistryofHealthandFamilyWelfare.GovernmentofIndia2005