Cytopreparatory TechniquesCytopreparatory Techniques

Teresa Alasio, MD

August 18, 2010

Teresa Alasio, MD

August 18, 2010

Why do we need to understand cytopreparatory techniques?Why do we need to understand cytopreparatory techniques?

The diagnosis of cytology specimens begins with specimen preparation

Know difference between types of specimens

Know difference between types of stains Advantages and

disadvantages to each

Know about cell block and its purpose

The diagnosis of cytology specimens begins with specimen preparation

Know difference between types of specimens

Know difference between types of stains Advantages and

disadvantages to each

Know about cell block and its purpose

Apply these techniques to specimens and know how to modify them to obtain the highest specimen yield

Communicate with laboratory technicians and cytotechnology supervisors/cytotechnologists regarding specimen processing

Clinical history and relevant patient information utilized to determine how specimen will be processed

Technical component – hospital bills

Apply these techniques to specimens and know how to modify them to obtain the highest specimen yield

Communicate with laboratory technicians and cytotechnology supervisors/cytotechnologists regarding specimen processing

Clinical history and relevant patient information utilized to determine how specimen will be processed

Technical component – hospital bills

Specimen TypesSpecimen TypesExfoliative specimens

Shed spontaneously from internal or external body surfaces

EffusionsAscitesWashings (pelvic, bronchial)SputumBrushings (bronchial, endoscopic)Pap smear

Exfoliative specimensShed spontaneously from internal or

external body surfacesEffusionsAscitesWashings (pelvic, bronchial)SputumBrushings (bronchial, endoscopic)Pap smear

Specimen TypesSpecimen TypesSpecimens obtained by aspiration

SuperficialSmears obtainedFluids (cyst)

DeepSmearsCell blocks

Specimens obtained by aspirationSuperficial

Smears obtainedFluids (cyst)

DeepSmearsCell blocks

Fixation MethodsFixation MethodsNo fixative

Optimal if specimen is immediately delivered or can be refrigerated

Fixative (EtOH) coagulates protein, causes clogging of filter, prevents adherence of cells to slide

RPMI/Hank’s solutionNeutral solution that allows cells to remain

viable

No fixativeOptimal if specimen is immediately

delivered or can be refrigeratedFixative (EtOH) coagulates protein, causes

clogging of filter, prevents adherence of cells to slide

RPMI/Hank’s solutionNeutral solution that allows cells to remain

viable

Fixation MethodsFixation MethodsLess desirable methods

Limited options for specimen manipulationAlcohol

Can be used if delay in specimen transport or specimen cannot be refrigerated

FormalinLeast desirableFixes not only cells but proteins, debris,

blood

Less desirable methodsLimited options for specimen manipulation

AlcoholCan be used if delay in specimen transport

or specimen cannot be refrigeratedFormalin

Least desirableFixes not only cells but proteins, debris,

blood

Smear FixationSmear Fixation Smears are fixed either by spraying with fixative or

immersing slide in liquid fixative Slide should remain in fixative for minimum of 15-30

minutes, not more than 1 week Liquid fixatives

EthanolMost common is 95%

Methanol – less cell shrinkage, cytogenetics, 100% methanol comparable to 95% ethanol

Coating fixativesMust soak slides in 95% alcohol for up to 10 minutes to

remove coating fixative and then stain slides Methanol/diethyl ether (not used) Acetone

Smears are fixed either by spraying with fixative or immersing slide in liquid fixative

Slide should remain in fixative for minimum of 15-30 minutes, not more than 1 week

Liquid fixatives Ethanol

Most common is 95% Methanol – less cell shrinkage, cytogenetics,

100% methanol comparable to 95% ethanol Coating fixatives

Must soak slides in 95% alcohol for up to 10 minutes to remove coating fixative and then stain slides

Methanol/diethyl ether (not used) Acetone

Smear FixativesSmear FixativesSpray fixatives are commercially availableUsually contain ethanol or isopropyl alcohol

with carbowax (polyethylene glycol)After alcohol evaporates, cells are left coated

with carbowax, which prevents cell shrinkageOther spray fixatives include commercially

available aerosol fixativesHair spray – not recommended

Smears must be fixed immediately – regardless of the method of fixation

Air drying artifact renders slides unsuitable for interpretation

Spray fixatives are commercially availableUsually contain ethanol or isopropyl alcohol

with carbowax (polyethylene glycol)After alcohol evaporates, cells are left coated

with carbowax, which prevents cell shrinkageOther spray fixatives include commercially

available aerosol fixativesHair spray – not recommended

Smears must be fixed immediately – regardless of the method of fixation

Air drying artifact renders slides unsuitable for interpretation

Liquid Based Preparations (LBP)Liquid Based Preparations (LBP)ThinPrep and SurePath

Both gyn and non-gyn specimens

Pap stain only

Reflex HPV testing

ThinPrep and SurePath

Both gyn and non-gyn specimens

Pap stain only

Reflex HPV testing

Advantage to LBP SpecimensAdvantage to LBP SpecimensSignificantly more satisfactory samples

than conventional smears

More lesions are detected because cells are evenly distributed with less overlap

Smaller area to screen

Blood is lysed, cleaner background

Significantly more satisfactory samples than conventional smears

More lesions are detected because cells are evenly distributed with less overlap

Smaller area to screen

Blood is lysed, cleaner background

Disadvantages to LBPDisadvantages to LBPEquipment is expensive

Consumables (ThinPrep) for both gyn and non-gyn specimens

Specialized training/certification for LBPs

Equipment is expensive

Consumables (ThinPrep) for both gyn and non-gyn specimens

Specialized training/certification for LBPs

Comparison of LBP MethodsComparison of LBP MethodsThinPrep

Methanol-based fixative

20mm diameter deposit

Filter used, vacuum cylander

Throw away collection brush

ThinPrepMethanol-based

fixative20mm diameter

depositFilter used, vacuum

cylanderThrow away

collection brush

SurePathEthanol-based

fixative13mm diameter

depositNo filter, sample is

vortexedCollection brush

retained in specimen

SurePathEthanol-based

fixative13mm diameter

depositNo filter, sample is

vortexedCollection brush

retained in specimen

StainsStains

Papanicolaou

Romanowsky

H&E

Papanicolaou

Romanowsky

H&E

Papanicolaou StainPapanicolaou Stain Used for fixed smears Uses hematoxylin for

nuclear staining Followed by Orange-G,

which stains keratin If keratin is not present,

then Orange-G will not stain

Useful in squamous differentiation

Used for fixed smears Uses hematoxylin for

nuclear staining Followed by Orange-G,

which stains keratin If keratin is not present,

then Orange-G will not stain

Useful in squamous differentiation

Papanicolaou Stain - AdvantagesPapanicolaou Stain - AdvantagesSuperb nuclear detail

NucleoliInclusionsChromatin

Identification of keratinNormal squamous cells vs.

dysplastic/carcinoma

Cytoplasmic transparency

Superb nuclear detailNucleoliInclusionsChromatin

Identification of keratinNormal squamous cells vs.

dysplastic/carcinoma

Cytoplasmic transparency

Papanicolaou StainingPapanicolaou Staining4 main steps

FixationNuclear staining with hematoxylinCytoplasmic staining with counterstains Orange G

(keratin) and EA (cytoplasm, nucleoli, cilia)EA: eosin, light green, Bismarck brownEA-36 (original formula), EA-50 (commercial

prep), EA-65 (reduced light green) Clearing

Progressive vs. regressive staining techniquesProgressive stains nuclei to desired intensity while

regressive overstains nuclei initially and then removes excess stain with HCl

4 main stepsFixationNuclear staining with hematoxylinCytoplasmic staining with counterstains Orange G

(keratin) and EA (cytoplasm, nucleoli, cilia)EA: eosin, light green, Bismarck brownEA-36 (original formula), EA-50 (commercial

prep), EA-65 (reduced light green) Clearing

Progressive vs. regressive staining techniquesProgressive stains nuclei to desired intensity while

regressive overstains nuclei initially and then removes excess stain with HCl

Keratin – benign vs. malignantKeratin – benign vs. malignant

Papanicolaou Staining – Nuclear detailPapanicolaou Staining – Nuclear detail

Air-Dried SmearsAir-Dried SmearsUseful for immediate assessmentEliminates the fixation step – more

rapid assessmentDried smears are preferable to smears

that have been badly fixed and then Pap-stained

Useful for immediate assessmentEliminates the fixation step – more

rapid assessmentDried smears are preferable to smears

that have been badly fixed and then Pap-stained

Stains for Air-dried SmearsStains for Air-dried SmearsMost common:

Romanowsky stains traditionally used for air dried smearsDiff-QuikOriginally a hematology stain

Less common:Toluidine BlueUltrafast PapProvides nuclear detail that Romanowsky

stains do not have

Most common:Romanowsky stains traditionally used for

air dried smearsDiff-QuikOriginally a hematology stain

Less common:Toluidine BlueUltrafast PapProvides nuclear detail that Romanowsky

stains do not have

Romanowsky Stain (Diff-Quik)Romanowsky Stain (Diff-Quik)Methanol fixation (Step 1)Eosin (Step 2)Methylene blue or Azure A, B or C

(Step 3)Rinse (Step 4)

Methanol fixation (Step 1)Eosin (Step 2)Methylene blue or Azure A, B or C

(Step 3)Rinse (Step 4)

Romanowsky Stains - AdvantageRomanowsky Stains - AdvantageReact metachromatically with variety of

tissue componentsShift of dye’s absorption spectrum in

presence of negatively charged entities to give reddish-purple color. Nucleic acidsEpithelial mucinsExtracellular matrix components

React metachromatically with variety of tissue componentsShift of dye’s absorption spectrum in

presence of negatively charged entities to give reddish-purple color. Nucleic acidsEpithelial mucinsExtracellular matrix components

Metachromatic staining - ECMMetachromatic staining - ECM

Metachromatic Staining - MucinMetachromatic Staining - Mucin

Romanowsky Staining – AdvantageRomanowsky Staining – AdvantageThyroid aspiration biopsies

Identification of colloidThick colloid vs. thin colloid

CarcinomaPapillaryMedullary

Thyroid aspiration biopsiesIdentification of colloid

Thick colloid vs. thin colloid

CarcinomaPapillaryMedullary

ColloidColloid

Papillary CarcinomaPapillary Carcinoma

Medullary CarcinomaMedullary Carcinoma

LymphomaLymphoma

B-Cell lymphomaB-Cell lymphoma Mantle Cell Lymphoma

Comparison of Papanicolaou and Romanowsky Stains*Comparison of Papanicolaou and Romanowsky Stains*

Cytologic Finding Papanicolaou Stain

Romanowsky Stain

Nuclear detail +

Cytoplasmic keratin

+

Cytoplasmic mucin

+

Cytoplasmic granules

+

Thyroid colloid +

ECM +

Diagnosis of hematopoietic malignancies

+

* Adapted from Geisinger, et al. Modern Cytopathology, 2004

Types of processingTypes of processingDirect smearsCytospinCell block

Direct smearsCytospinCell block

Direct SmearsDirect SmearsEffusionsWashingsThick specimens

EffusionsWashingsThick specimens

Effusions and washingsEffusions and washingsCan be bloody and/or thick Need to lyse blood and get the cells out!

Smears are usually done for these specimens

Can also make a cell block

Can be bloody and/or thick Need to lyse blood and get the cells out!

Smears are usually done for these specimens

Can also make a cell block

Direct SmearsDirect SmearsSpin down specimen without fixativePour off supernatantAdd agent to lyse blood, spin againDirect smears from button

PapPut in 95% alcohol

Diff-QuikAir dried smear

Ultrafast PapAir dried smear

Spin down specimen without fixativePour off supernatantAdd agent to lyse blood, spin againDirect smears from button

PapPut in 95% alcohol

Diff-QuikAir dried smear

Ultrafast PapAir dried smear

CytospinCytospinUrineCSFSputum/LavageCell block not usually made, but can be

made from these specimens

UrineCSFSputum/LavageCell block not usually made, but can be

made from these specimens

CytospinCytospin

Cell BlockCell Block

May be useful in certain circumstancesSpecial stains for microorganismsIHC

Cheating?

May be useful in certain circumstancesSpecial stains for microorganismsIHC

Cheating?

Advantages of Cell BlockAdvantages of Cell BlockLooks like histology

Stained with H&ESome architecture may be preservedCan do special stains

Blanks for immunohistochemistry

Looks like histologyStained with H&ESome architecture may be preservedCan do special stains

Blanks for immunohistochemistry

When to order Cell BlockWhen to order Cell BlockCell block is made automatically with

deep FNAsMake cell block from fluids where you

need to look at histology or perform special stains or immunohistochemical stains

Cell blocks usually not needed for urine, CSF or bronchial specimens

Cell block is made automatically with deep FNAs

Make cell block from fluids where you need to look at histology or perform special stains or immunohistochemical stains

Cell blocks usually not needed for urine, CSF or bronchial specimens

Preparation of Cell BlockPreparation of Cell BlockAdd fixative to button, spin down

CytoRich RedTM (CRR)

Lyses blood and leaves cellsFixes specimen

Add plasma (FFP)Add thrombinForm small ball comprised of cells suspended

on the plasma/fibrin matrixProcess like surgical biopsyAble to do IHC if desired

Add fixative to button, spin downCytoRich RedTM

(CRR)

Lyses blood and leaves cellsFixes specimen

Add plasma (FFP)Add thrombinForm small ball comprised of cells suspended

on the plasma/fibrin matrixProcess like surgical biopsyAble to do IHC if desired

Laboratory:How to Make Perfect Smears

Laboratory:How to Make Perfect Smears