Virtual Slides in Haematology:

Past, Present and Future

Szu-Hee Lee

St George Hospital

SEALS Central, NSW

I have no financial or consultative relationships with any commercial entities

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“Digital Haematology”

• Single frame images

• Automated cell image analysers

• Telehaematology

• Virtual Slides

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Digital Haematology

• Automated cell image analysers (ca 1960’s)

– CellaVision, EasyCell, Vision Hema etc.

– Capture images of cells in blood films

– Pre-classify RBC and WBC (5-part diff)

– Manual review and adjustment

– Acceptable accuracy, some limitations, high costs

• Telehaematology

– Transmission of static images

– Networked automated image analysers

– Networked slide scanners

– Real-time robotic microscopy over a network

– Remote site expertise required, high costs

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Virtual Microscopy: Definitions

• Virtual microscopy :

– The simulation of microscopy using a computer to display,

pan, zoom and focus images

– Synonym: “Whole Slide Imaging” (WSI)

• Virtual slide :

– A digital image file of a real (glass) slide

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1930 Aerial reconnaissance maps

1969 Charge-coupled device (CCD; Boyle & Smith, Nobel Prize 2009)

1985 Digital image tiles of histology sections (Silage & Gil)

1994 Robotic workstation for scanning X40 virtual slides (Bacus Labs)

1996 Multi-resolution pyramidal image file format (Kodak)

2000 Virtual slides introduced to medical schools (Uni. Iowa)

2003 ABP: X40 virtual and glass slides for AP examinations

2004 Introduction of Z-stacks for focusing virtual slides

2005 Robotic workstation for scanning X100 virtual slides (Aperio)

History

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2006 RCPAQAP: X40 virtual slides for Anatomical pathology

2007 RCPAQAP: X100 virtual slides for Haematology

2008 ABP: Virtual and glass slides for cytopathology examinations

2009 ABP: Virtual and glass slides for haematology examinations

2009 NSW Haematology Network: X100 virtual slides for teaching

2013 RCPA Haematology Online Virtual Slide Library

2013 CAP: Guidelines for Whole Slide Imaging in diagnostic pathology

2015 RCPA: Guidelines for digital microscopy in AP and cytology

History

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Virtual Slides

• Technology

• Applications

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What is the filesize required to capture a whole slide at X40?

• Dimensions of glass slide = 75mm x 25mm

• Resolving power (X40) = 0.3 microns2 / pixel

• In 24-bit RGB colour, 1 pixel = 3 bytes

• Filesize to capture entire slide

– 60GB (uncompressed)

– 4GB (15x compression)

• Large storage capacities required

• Rapid processing times required

• Not necessary to capture entire slide

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Capture of X100 PB or BMA slides

75mm

25mm

10mm x 10mm

~1.0GB (compressed)

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Automated Slide Scanners: Robotic Tiling

• BLISS system (Bacus Labs,1994)

– Motorized stage

– 1cmx1cm captured at 40X = 1600 images

– Automated image stitching

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Automated Slide Scanners: Line Scanning

• Scanscope (Aperio Technologies, 2000)

– Line scan camera coupled to objective lens

– Continuous stage motion

– Image seams limited to 1 axis

– Focus is adjusted every scan line

– X100 (oil) images (2005)

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Automated Slide Scanners: Multiple Microlenses (X40)

3mm/sec

3000 frames/sec

Weinstein 2009

8x10

lens array

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Pyramidal File Format

– A series of images at different resolutions are stored in a single file

– Image tiles at the selected resolution are streamed to the viewer

Fred Dee, University of IowaSzu-Hee Lee 2016

Image viewer: BM Aspirate (X100)

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Scanscope OS:

• Oil immersion objective X60 / 1.35 N.A.

• 0.14 micron2/pixel

• 10x10 mm scan area

• Total processing time 10-15 min

• .TIFF format (.svs)

• Pyramidal file structure

• Filesize ~ 1.0GB (compressed 15x)

PB or BMA slides at X100: Specifications

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Some Technical Issues

• Speed of online display

– Depends on size of window

– Independent of filesize

– Dependent on processor, graphics card, RAM

– Network bandwidth, traffic

• Depth of field and focus

– Lenses of wide numerical aperture result in reduced depth of field

– Z-stacks

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Z-Stacks

• “Z-axis”

• Acquire series of images at different focal planes to form Z-stack

• Use a pointing device to simulate focusing

• Increases the scanning time and filesize of virtual slide

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Z-stacks demo

www.microbrightfield.com

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Extended Focus Module

www.zeiss.com

1) Acquire Z-stacks.

2) Combine the sharp areas of each plane to create an

image that is sharp throughout.

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Virtual Slides

• Technology

• Applications

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Comparison of Virtual with Glass Slides

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Virtual Slides Glass Slides

ADVANTAGES

Anytime, anywhere One user at one location

All users view the same image Glass slides are never identical

Unlimited copies Cannot duplicate rare slides

Easy to store, retrieve, distribute Cumbersome

Do not deteriorate Fading, breakages, losses

Annotate areas of interest Difficult

Less physical storage space More storage space

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Virtual Slides Glass Slides

DISADVANTAGES

Need Z-stacks (increases file size) Able to focus 3-D specimen

Limited field Able to view whole specimen

Students do not learn to use microscope Students learn to use microscope

Controls may not be optimal Microscopes are ergonomically

designed

Validation necessary Gold standard for diagnostic work

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NSW Pilot Survey of Online

Virtual Haematology Slides (2010)

• Four virtual slides:

– 2x PB (X100)

– 1x BMA (X100)

– 1x BMTB (X40)

• Mounted on RCPAQAP server, viewed with WebScope®

• Specialist haematologists with no prior experience were invited to

review the slides

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Virtual Slide Survey: Image Quality

Mean Score (n=26)

1 = very poor, 2 = poor,

3 = average, 4 = good,

5 = excellent

Speed of Display 3.15

Image Resolution 3.77

Image colours 3.92

Ease of Panning 3.73

Ease of Zooming 3.69

Overall Ease of Use 3.77

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Virtual Slide Survey: Potential Applications

Mean Score (n=26)

1 = very poor, 2 = poor,

3 = average, 4 =good,

5 = excellent

Teaching 3.85

Quality Assurance 3.52

Morphology Examinations 3.31

Second Opinion 3.50

Routine Diagnosis 3.31

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• Issues:

– Speed of display: Hardware and network bandwidth are

important

– Less enthusiasm for use in morphology examinations and

primary diagnosis

• An acceptable alternative to glass slides for haematology education

and QA

Virtual Slide Survey (2010): Conclusions

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Education

• Beginning to replace real microscopy:

– Histology

– Anatomical pathology

– Cytopathology

– Haematology

– Microbiology

• Many pedagogic studies:

– Students like it

– Easier than real microscopy

– Improves students’ performance

– Enables tutor to highlight and annotate objects

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Quality Assurance

• Glass slides

– Difficult to prepare

– Never identical

• Virtual slides

– The same image is viewed by all participants

– Easier to distribute than glass slides

– Images do not deteriorate with time

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RCPA Haematology Online Virtual Slide Library

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• Started in 2013

• All RCPA Haematology Fellows invited to submit cases

• PB, BMA, BMTB

• Web based platform-independent slide viewer

RCPA Haematology Virtual Slide Library

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• Data:

– Blood count

– Clinical details

– Morphology description

– Investigations

– Additional images

– Diagnosis

– Comments

– References

• Search by:

– Text

– Topic (WHO 2008 Classification)

– Specimen type

– Contributor

RCPA Haematology Online Virtual Slide Library

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• 2014:

– 1772 case views

– 1463 returning users

– 309 new users

• 2015: >150 slides of PB, BMA, BMTB

• Surveys of trainees (2013 to 2015):

– The Library is a useful tool for morphology education

– Usage would increase if more cases were added

RCPA Haematology Virtual Slide Library

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Haematology Virtual Slides: Implementation

Education

Quality Assurance

Proficiency Testing

Secondary Consults?

Primary Diagnosis?

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Virtual Slides: Guidelines (AP)

• Guidelines for Digital Microscopy in AP and Cytology (RCPA 2015):

– “Validation must be performed on the digital microscopy system

to … ensure it meets the intended use and process”

– “If … intended for diagnostic use, validation must include

demonstrating the equivalent diagnosis is made between

conventional and digital microscopy”

• Diagnostic equivalence (“concordance”) in Anatomical Pathology:

– Many studies

– Validating WSI for Pathology (meta analysis, CAP 2015):

• Good levels of diagnostic accuracy and concordance

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Validating WSI for Pathology (CAP 2015)

Conclusions:

• No significant difference in accuracy between WSI or glass slides

• Good concordance between diagnoses made using WSI and glass slides

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Virtual Slides: Standardization

• Many variables:

– File formats, processing, compression, storage

– Colours: specimen processing, staining, optics, image sensors

– Display hardware: screen size, resolution, colours

– Network bandwidth

– Privacy, medico-legal issues

• DICOM (Digital Imaging and Communications in Medicine)

– Working Group-26 for Pathology (WG-26)

– Technical standards for single frame images and virtual slides

• International Color Consortium (ICC; ISO 15076-1:2005)

– Device profile format translates colour data from one device to

another

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• Pathologists are skilled at handling glass slides and microscopes

• Pathologists feel uncomfortable with virtual slides for diagnosis

• Unfamiliarity with viewing digital images

• Unfamiliarity with software controls

• Inadequate computer hardware – screen size, RAM, bandwidth

• Some virtual slides may not be perfect e.g. focus

• Need for training – a digital competency test?

Virtual Slides: Implementation Issues

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Competence Assessment of Residents

in Virtual Microscopy (Bruch 2009)

• Performance correlated with length of training

• Learning curve is steeper early in training Szu-Hee Lee 2016

Validating WSI for Pathology (CAP 2015)

• The validation process should include:

• a sample set of at least 60 cases for one application (e.g. hematology)

• another 20 cases for each additional application (e.g. IHC)

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Novice or Expert? Eye Movement Studies

Krupinski et al 2006

Eye Movement

Detector:

Infra-red beam

reflected off

pupil and

cornea

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Krupinski et al 2006Szu-Hee Lee 2016

• Adjunct to real microscopy

• Enhance teaching, learning and QA

• Facilitate peer-to-peer consultative process

• Enable long-distance consultation and diagnosis

• Enable flexible working hours and locations

• Virtual microscopy must do the job better, cheaper or faster

Virtual Microscopy: The Future

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Virtual Microscopy : The Future

Technological Advances

Software advances

Hardware advances

Network bandwidth

Refinement and Testing

Validation

Standardization

User Competency Tests

Diagnostic Equivalence Tests

Implementation

Education

Quality Assurance

Proficiency Testing

Secondary Consults

Primary Diagnosis

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Acknowledgements

Fifin Intan, RCPAQAP

Sarah Halawani, RCPAQAP

Jorge Verastegui, RCPAQAP

Marion Higginson, RCPA

Poomahal Kumar, NSW Haematology Training Network

All contributors to the Haematology Virtual Slide Library

Geoff Greene, Microbrightfield, USA

Fred Dee, University of Iowa, USA

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