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11

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AP122 Automating the AP122 Automating the Histology LabHistology Lab

Impact on Surgical and Molecular Impact on Surgical and Molecular PathologyPathology

A R Morales, MDA R Morales, MDM Nadji, MDM Nadji, MD

University of Miami, Jackson Memorial HospitalUniversity of Miami, Jackson Memorial Hospital

© College of American Pathologists 2004. Materials are used with the permission of the faculty.

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2

Course ObjectivesCourse Objectives

The most recent automated instruments in histopathology.The most recent automated instruments in histopathology.

Advantages of continuous flow rapid processing method.Advantages of continuous flow rapid processing method.

Impact of the new technologies on patient care.Impact of the new technologies on patient care.

Potential for macromolecular preservation in histologic materialPotential for macromolecular preservation in histologic material..

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33

3

AgendaAgenda

10 minSummary and ClosingMolecular PathologyPost Hematoxylin & Eosin

10 minOpening/ Introductions

Post Embedding30 minBreak

Processing and EmbeddingPre-Processing

TimeTimeTopicTopic

44

4

DisclosureDisclosureDrs Morales and Nadji:Drs Morales and Nadji:Participates in a sponsored research with Sakura Finetek Participates in a sponsored research with Sakura Finetek through the University of Miami. The University has licensed through the University of Miami. The University has licensed their patents to Sakura.their patents to Sakura.

Dr Nadji:Dr Nadji:Is a consultant to DakoCytomation through the University Is a consultant to DakoCytomation through the University of Miami.of Miami.

3

5>3 days

3rd day

2nd day

Same day1st day

Diagnostic Tissue PreparationStandard Practice

Not standardized

Formalin: Toxic, Crosslinks, Slow, Cheap

OvernightToxic: Formalin, Xylene

Processing

Embedding

MicrotomyH&E Slide Microscopy

Surgery

Tissue

Fixation

Grossing

HistochemistryImmunohistochemistryMolecular Assays (limited)

6

Automated Tissue ProcessorsStationary Chamber Rotating Basket

Bavimed HistomasterLeica ASP 300 TP 1020Medite TPCSakura Tissue-Tek VIPTBS ATPThermo Shandon Excelsior CitadelVentana Paraffin Renaissance

4

7

TISSUE PROCESSINGSTATIONARY CHAMBER

50 min EACH

ETHANOL

3.2 L EACH

50 min EACH

PARAFFIN

4 L EACH

50 min 50 min EACHEACH

FORMALIN

3.2 L EACH

HEATVACUUM

AGITATION

50 min EACH

XYLENE

3.2 L EACH

8

5

9

ParaffinFormalin

Alcohol

Formalin

Alcohol

Xylene

Paraffin

Paraffin

Alcohol

Alcohol Alcohol

Xylene

1 houreach

1-2 hourseach

1.5 hourseach

45 minseach

TISSUE PROCESSINGROTATING BASKET

10

6

11

12Arendt 1909

7

13

Same day

14

Procedures Used to AcclerateTissue Processing

• Heat–External–Internal (microwave)

• Agitation• Vacuum

8

15

Convective Heat

16

Microwave

9

17

18

Microwave Processors

Model

Energy Beam H2500,H2800

Milestone Hacker Micromed T/T Mega

Milestone Hacker Micromed URM

Sakura Xpress

10

19

TISSUE PROCESSINGCOMMON MICROWAVE METHODS

Variable Time ETHANOL

and/or ISOPROPANOLor others

Variable TimeHEATED PARAFFIN

AGITATIONAGITATION+/+/-- VACUUMVACUUM

Variable Time ETHANOL

and/or ISOPROPANOLor others

20

11

21

REAGENTS

Excellent morphology

Expedient function

Enhanced macromolecules preservation

Low toxicity

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REAGENTS

Acetone Isopropyl alcohol

Polyethylene glycolMineral oil

Paraffin

12

23

MICROWAVE DESIGN

Automatable

Harness energy uniformly

Low wattage

24

13

25

Common MW= 400Common MW= 400--1000 W1000 WMagnetronMagnetron= ON/OFF, Inhomogeneous MW field= ON/OFF, Inhomogeneous MW field

CRTP = 50W (<160 W)CRTP = 50W (<160 W)Magnetron= ON , Uniform MW fieldMagnetron= ON , Uniform MW field

MW ≠ HeatMW = Energy

26

15 min

MW agitation

AcetoneIsopropanol

PEG

15 min

MW agitation

AcetoneIsopropanol

Mineral Oil

15 min

Heat Heat VacuumVacuum

Mineral OilParaffin

15 min

HeatVacuum

Paraffin

CRTP

14

27

28

15

29

30Time (min)

Temperature(centigrade)

Power(watt)

61

320

160

NewBasket

NewBasket

20 min 15 min

~60

CRTP

16

31

Processing Agents

RTPYesYesYesYes

NoNoYesYesYesYesYesYesYes

PhysicalConvection HeatMicrowaveVacuumAgitation

ChemicalFormaldehydeXylene AlcoholAcetonePEGDMSOMineral OilParaffin

ConventionalYesNoYesYes

YesYesYesNoNoNoNoYes

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GROSSING

Standardize slice thickness at <1.5mm

500x3 microns thick sections300x5 microns thick sections

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33

34

18

35

GROSSING

Develop appropriate grossing tools

Board

Fork

36

19

37

38

TAT Surgical SpecimensJMH

0

10

20

30

40

50

60

SAME DAY 1 DAYS 2 DAYS 3 DAYS > 3DAYS

1996 (23349 Cases)

2003 (30124 Cases)

20

39

TAT Of Surgical Cases

• Australia• China

– Biopsies– Complex

• Italy• Jamaica• Spain• US

– Biopsies– Complex

• UM/JMH• UMHC

Same Day

5944

1- Day73

79562531

2- Days

1012

3- Days

93

3766

45

>3- Days

1006334

100

5928

40

Diagnostic Tissue Preparation

CRTP

Standardized

Molecular Friendly

Surgery

Tissue

Fixation

Grossing

Microtomy

Microscopy

Same day1st day

2nd day

>2 days

Processing

EmbeddingH&E Slide

HistochemistryImmunohistochemistryMolecular Assays

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41

Paraffin

Automated Embedding

22

43

Diagnostic Tissue Preparation

CRTP

Standardized

Molecular Friendly

Surgery

Tissue

Fixation

Grossing

Microtomy

VirtualMicroscopy

Same day1st day

2nd day

>2 days

Processing

EmbeddingH&E Slide

HistochemistryImmunohistochemistryMolecular Assays

4444

44

Post Post -- H&EH&E

HistochemistryHistochemistryImmunohistochemistryImmunohistochemistryIn Situ HybridizationIn Situ HybridizationFlow CytometryFlow Cytometry

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4545

45

HistochemistryHistochemistry

Pipette SystemPipette SystemCartridge SystemCartridge System

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4747

47

Immunohistochemistry InstrumentsImmunohistochemistry Instruments

Rotary or LinearRotary or LinearWith heat or Without heatWith heat or Without heatContinuous or BatchContinuous or BatchRandom Access or NonRandom Access or Non--randomrandomAll Steps or Select StepsAll Steps or Select Steps

TYPES-MECHANISMS-FUNCTIONS

25

4949

49

IHC instruments; look for:IHC instruments; look for:

Slide capacitySlide capacityClosed/Open SystemClosed/Open SystemUser friendlinessUser friendlinessBench spaceBench spaceExpandabilityExpandabilityEconomicsEconomics

5050

50

IHC InstrumentsIHC Instruments

Available nowAvailable nowComing soonComing soon

26

5151

51

In Situ HybridizationIn Situ Hybridization

Stand Alone instrumentsStand Alone instrumentsCombined with HC / IHCCombined with HC / IHC

27

5353

53

Imaging SystemsImaging Systems

Quantitative Image AnalyzersQuantitative Image AnalyzersDigital Scanning MicroscopyDigital Scanning Microscopy

5454

54

Quantitative ImagingQuantitative Imaging

Events, intensity, or both?Events, intensity, or both?Internal or external control?Internal or external control?Clinically relevant information?Clinically relevant information?

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5555

55

Virtual MicroscopyVirtual Microscopy

Continuous FeedContinuous FeedScanning TimeScanning TimeImage ResolutionImage ResolutionUser FriendlinessUser Friendliness

5656

56

Molecular PathologyMolecular PathologySlide Based InstrumentsSlide Based Instruments

In Situ PCRIn Situ PCRMicrodissectionMicrodissection

29

5757

57

MICRODISSECTIONMICRODISSECTION

P.A.L.M. SYSTEM

5858

58

Molecular Path Molecular Path InstrumentsInstruments

DNA/RNADNA/RNAProteinProtein

AmplificationAmplificationQuantitationQuantitationIdentificationIdentification

30

5959

59

Molecular Path InstrumentsMolecular Path Instruments

DNA/RNADNA/RNAProteinProtein

AmplificationAmplificationQuantitationQuantitationIdentificationIdentification

6060

60

Molecular Pathology TechniquesMolecular Pathology TechniquesDNA/RNADNA/RNA

HybridizationHybridizationSequencingSequencingElectrophoresisElectrophoresisExpression profileExpression profile

31

6262

62

Conventional (CPM)

Rapid (RTP)

G F P M 18 HRS

G P M 1.5 HRS

32

6363

63

Formalin fixation and conventional tissue processing destroy most macromolecules.

FACT

6464

64

Surgery Fresh Tissue

Surgery Fresh Tissue

FormalinFixationFormalinFixation

CPMCPM

HistologyHistology

AncillaryTests

AncillaryTests

Molecular Studies

Molecular Studies

33

6565

65

Surgery Fresh Tissue

Surgery Fresh Tissue

UMFIXUMFIX

Molecular Studies

Molecular Studies

6666

66

1- Fresh2- UMFIX 1 hr3- UMFIX 24 hr4- Formalin 1 hr5- Formalin 24 hr

34

6767

67

Surgery Fresh Tissue

Surgery Fresh Tissue

UMFIXUMFIX

CPMCPM

HistologyHistology

Molecular Studies

Molecular Studies

AncillaryTests

AncillaryTests

XX

6868

68

Surgery Fresh Tissue

Surgery Fresh Tissue

UMFIXUMFIX

RTPRTP

HistologyHistology

Molecular Studies

Molecular Studies

AncillaryTests

AncillaryTests

35

6969

69

UMFIX/RTP Control

18S

28S

Fluore

scen

ce

Time (seconds)

0.0

2.5

5.0

7.5

10.0

12.5

19 24 29 34 39 44 49 54 59 64 69

7070

70

Real Time RTReal Time RT--PCRPCR

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7171

71

Molecular PathologyMolecular PathologyProteinProtein

Western BlotWestern Blot2D 2D -- GelGelMas SpectroscopyMas SpectroscopyXX--ray Crystallographyray CrystallographyProtein ProfileProtein Profile

7272

72

Western BlotWestern BlotFresh UMFIX 1hr UMFIX 24 hr Form. 1hr Form. 24 hr

37

Fresh

UMFIX

Formalin

2D-GEL

7474

74

The Laboratory of the 21st The Laboratory of the 21st Century Should:Century Should:

Become familiar with the new technologies.Become familiar with the new technologies.

Provide “molecular friendly” archival tissue.Provide “molecular friendly” archival tissue.

Play an active role in patient management.Play an active role in patient management.

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7575

75

The Practice of Pathology TodayThe Practice of Pathology Today

Ability to consistently extract intact RNA and Ability to consistently extract intact RNA and protein from paraffin blocks used for diagnosisprotein from paraffin blocks used for diagnosis

Ability to complete surgical pathology reports within Ability to complete surgical pathology reports within twotwo--three hours after surgerythree hours after surgery

Elimination or reduction of toxic reagentsElimination or reduction of toxic reagents

7676

76

The Practice of Pathology TodayThe Practice of Pathology TodayEliminate or reduce patient’s anxiety related Eliminate or reduce patient’s anxiety related to days of waiting for diagnosisto days of waiting for diagnosisProvide option to receive diagnosis during Provide option to receive diagnosis during outpatient surgical biopsy appointmentoutpatient surgical biopsy appointmentAllow preparation of diagnostic material at Allow preparation of diagnostic material at regular working hours regular working hours Eliminate batching of tissue samples and Eliminate batching of tissue samples and stressful loads of slidesstressful loads of slides