Virtual Microscopy in Hematology:experiences from the Belgian EQA

Bernard CHATELAINU.C.L. Mont-Godinne

External Belgian Quality Control in hematomorphology

• 3 times a year• Unstained slide fixed in methanol

– Laboratory staining procedure• Software on CD-ROM

– Wide-field to evaluate red blood cells and plateletsmorphologies

– 210 pictures of nucleated cells to establish a WBC differential count

– Clinical information about the case– Calibration procedure and virtual normal smear (105

pictures)

Smears: Blood 28 / Bone Marrow 2

Hairy Cell Leukemia, t(8;21) AML (didactic)2009 MarchT Prolymphocytic leukemia, MDS Ider20Q (didactic)2008 NovemberSecondary ALL, BiN Polyclonal Hyperlymphocytosis (didactic) 2008 JuneNormal, TTP2008 MarchNormal, Hairy Cell Leukemia2007 November

Normal, Reactive Lymphocytosis2007 JuneNormal, CML2007 MarchB12 deficiency, Secondary leukemia(didactic) (bone marrow aspirate)Myeloma, Chediak-Higashi (didactic)2006 NovemberNormal, Drepanocytosis, May Hegglin (didactic) 2006 JuneNormal, B12 deficiency2006 March Normal, Secondary leukemia2005 NovemberNormal, Hereditary Spherocytosis2005 JuneNormal, B Prolymphocytic Leukemia2005 MarchALL2004 November

November 2004

March 2005

June 2006

November 2006

May 2008

May 2008

How to reach the”high quality”microscope image

How to reach the”high quality”microscope image

• Colors

R + B = M (magenta)R + B = M (magenta)

R + V = Y (R + V = Y (yellowyellow))

B + V = C (cyan)B + V = C (cyan)

WhiteWhite

RR MMBB

CCYY

GG

RGB colors

Retina photoreceptors >>> CCD

• Retina: – L cones (Large) => Red (600 à 700 nm)

– M (Medium) => Green (500 à 600nm)

2X more frequent– S cones (Short) => Blue (400 à 500nm)

• CCD: 1R / 2G / 1B

ConeCone

RodRod

How many levels for a smoothfeeling?

2277 = 128= 128

2288 = 256= 256

200

2277 = 128= 128

2288 = 256= 256

RGB

G

How to reach the”high quality”microscope image

Gamma = brightness and contrast

Gamma dynamic range:eye 24 IL >< CCD 8 IL

Gamma curve correction

• Increase dynamicrange for darkobject

• But request more than 3 X 8 bit colordefinition (Olympus DP 71: 11)

How to reach the”high quality”microscope image

• Image resolution

Based on optical resolution you can calculate how many pixels you need!

X N.A. L x 2.3 B x 2.30.5 0.025 3265 2458

1 0.05 3265 24582 0.1 3265 24584 0.2 3265 2458

10 0.45 2913 219320 0.75 2444 184040 0.95 1525 114840 1 1623 122260 0.95 1017 76560 1.4 1486 1119

100 1.4 1486 1119Magnification

Numerical Aperture

Image pixels

density

Obj. 100 XNA 1.4

Obj. 40 XNA 1.3

Obj. 100 XNA 1.4

Obj. 40 XNA 1.3

+ resampling and accentuation (g=126; r=3.6; s=3.0

How to reach the”high quality”microscope image

• Size of the picture

Wide field Image

Stitching of 80 images (100 x)

Obj. 40 XNA 1.3

Material and methods

• Olympus PROVIS system– Olympus AX-70 microscope with autofocus and XYZ

• Camera– Olympus DP71– DP manager for image acquisition

• Home made software for wide field capture• Stitching unlimited (Realviz) to merge pictures• Adobe Photoshop for image editing• Stitching software for wide-field elaboration

www.zoomify.com

PID=Pixel On Demand

Today maximum size of the digitized field by manual technique

Full resolution image (N.A.=1,4 100X equiv)3 mm / 2 mm

Low resolution image (N.A.=0,4 15X equiv)15 mm / 10 mm

Limit is related to the size of the TIF picture (1Gb). And the size of the computer RAM for post stitching correction.

Virtual scan www.aperio.com

Olympus Dotslide1 slide= 50 Gb

Z-Axis

CombineZ picture

CombineZ picture

Global appreciation of the CD-ROM for the first and second QCs

General appreciation of the software and the virtual slide

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120

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Appreciation against actual smear

%ag

e of

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ries

2004-3

2005-1

Evaluation of both methods on 5 consecutive assays

-7,50-8,7116124,7Other cells

-5,41-10,4192,1202,5Blast cells

5,0012019Monocytes

3,855130125Lymphocytes

0,00088Polynuclear eosinophils

7,3510136126Polynuclear neutrophils +

Band cells

13,041,6512,6511Band cells

4,155,2125,2120Polynuclear neutrophils

Virtualsmear

Actualsmear

Relative error (%)

Diff (Virtual -Actual)

Median (total of 5 surveys)

Cell types

Actual and virtual smears : diagnosis accuracy for 5 surveys

Diagnosis Accuracy : Comparison between actual and virtual smears

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2004-3 2005-1 2005-2 2005-3 2006-1

Survey

Dia

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ccur

acy

(%)

Actual smear

Virtual smear

H/6916H/6916 MayMay--HegglinHegglin

H/7100H/7100 ChediakChediak--HigashiHigashi

H/7100H/7100 MDS ider20qMDS ider20q

H/9353H/9353

AML t(8;21)AML t(8;21)

H/7597H/7597 CMLCML

Mai 2008

Bone marrow virtual smeal

We will try to optimize our materialregarding your precious remarks

and evaluations

Authoring Tool For The External Quality

Assessment

1. State of the art

Questions – answers mapping(ex. hematology : cell by cell analysis)

No questions - answers mapping

Answers inside the system(answers analysis possible)

Answers outside the system(paper, web forms)

Composition autonomyDependence on a developper

Any domainOne domain

Offline - OnlineOffline

Content managed by the expertsContent management delegated to a developper

JavaFlash

AFTERBEFORE

2. Content Logical Representation

• Content Transmission Scheme

The experts ofa domain

AuthoringTool

LogicalContentLaboratories

??

3. Content Manifestation

• Content Transmission Scheme

The experts ofa domain

AuthoringTool

LogicalContent

Laboratories

OK

ContentManifestation

4. Transformations (Scenario Application)Structured Datas Datas Manifestation (full or partial) Datas Presentation

GeneralLogicalContent

AdaptedLogicalContent

Target Independant Target Specific

Adaptation Transformations Presentation Transformations

XML file XML file

XSL

XML

HTML

FO

SMIL

. . .

PDF

TEXT

PS

. . .

Smi

Program

Browser

Applet

XSL

XSL

XSL

XSL

XSL

PDF reader

Text Editor

Printer

Presentation

. . .

. . .FOP

Pro

cess

ing

SM

ILE

ngin

e

Spat

ial

Dim

ensi

on

Spat

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lD

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In

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imen

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Don’t forget the limitation of classical microscopic slides!

ReagentsSorensen Phosphate Buffer pH 6.8 : Na2HPO4.2H2O M/15 (2.56 g/l) and KH2PO4M/15 (6.63 g/l)

Buffered water : dilution 1/20e of Sorensen Phosphate Buffer in distilled water

May-Grünwald solution

Giemsa solution

StainingMethanol 10 minutes

Pure May-Grünwald solution 5 minutes

May-Grünwald (50/50 in buffered water buffer pH 6.8) 2-3 minutes

Giemsa (1/10 in buffered water pH 6.8) 15 minutes

Rinse with running water

Buffered water pH 6.8 30 seconds

Let dry smears upright

Guidelines for blood smear preparation and staining procedure for setting up an external quality assessment scheme for blood smear interpretation. Part I: control material (EQALM), J-L Vives Corrons, S. Albarède, G. Flandrin, S. Heller, K. Hovarth, B. Houwen, G. Nordin, E. Sarkani, M. Skitek, M. Van Blerk, J-C. Libeer, Clin Chem Lab Med, 2004, 42(8):922-926

Adapted from EQLAM MGG staining recommandations

EQALM : External Quality Assurance Programmes in Laboratory Medecine

Don’t adjustthe pH of the

bufferedwater!

Check the pH of the

Sorensen buffer!

1 : Darkness + Turbovac 800s (vacuum + N2) + Room temperature2, 3, 4, 5 : Darkness + Room temperature

Stainability alteration during the smear storage

Turbovac (800s)

1. Packaging of the opaque box containing smears in a plastic bag2. 98-99 % vacuum3. Nitrogen gas injection4. Heat closure of the plastic bag5. Storage at room temperature

Oxygen depletion + N2 injection

Immediate staining

H/6517 (+ Turbovac)H/2654 (- Turbovac)

MGG staining

Staining after 1 ½ month

e-Hematimage.eu50 belgian participants/more than

2500 around the world

many thanks for theircontribution to

Marc ChatelainYvan Cornet

Agathe DebliquisDamien DenizzaHubert Meurisse

Jan PhilippéJérôme Pruvot

Marjan Van Blerk and the hematology expert comiteeJean-Claude Libeer