Virtual Microscopy in Hematology:
experiences from the Belgian EQA
Bernard CHATELAIN
U.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 platelets
morphologies
– 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
2004 November
ALL
2005 March
Normal, B Prolymphocytic Leukemia
2005 June
Normal, Hereditary Spherocytosis
2005 November
Normal, Secondary leukemia
2006 March
Normal, B12 deficiency
2006 June
Normal, Drepanocytosis, May Hegglin (didactic)
2006 November
Myeloma, Chediak-Higashi (didactic)
B12 deficiency, Secondary leukemia(didactic) (bone marrow aspirate)
2007 March
Normal, CML
2007 June
Normal, Reactive Lymphocytosis
2007 November
Normal, Hairy Cell Leukemia
2008 March
Normal, TTP
2008 June
Secondary ALL, BiN Polyclonal Hyperlymphocytosis (didactic)
2008 November
T Prolymphocytic leukemia, MDS Ider20Q (didactic)
2009 March
Hairy Cell Leukemia, t(8;21) AML (didactic)
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
RGB colors
M
R
B
R + B = M (magenta)
White
Y
C
G
R + V = Y (yellow)
B + V = C (cyan)
Retina photoreceptors >>>
CCD
Rod
•
Retina:
– L cones (Large) => Red (600 à 700 nm)
Cone
– M (Medium) => Green (500 à 600nm)
2X more frequent
– S cones (Short) => Blue (400 à 500nm)
•
CCD: 1R
/ 2G / 1B
How many levels for a smooth
feeling?
200
27 = 128
28 = 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 dynamic
range for dark
object
• But request more
than 3 X 8 bit color
definition (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!
Magnification
Numerical Aperture
X
N.A. L x 2.3 B x 2.3
0.5 0.025 3265
2458
1 0.05 3265
2458
2
0.1 3265
2458
4
0.2 3265
2458
10 0.45 2913
2193
20 0.75 2444
1840
40 0.95 1525
1148
40
1 1623
1222
60 0.95 1017
765
60
1.4 1486
1119
100
1.4 1486
1119
Image pixels
density
Obj. 100 X
NA 1.4
Obj. 40 X
NA 1.3
Obj. 100 X
NA 1.4
Obj. 40 X
NA 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 X
NA 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 Dotslide
1 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
120
2004-3
2005-1
100
%age of laboratories
80
60
40
20
0
Bad
Less good
Appreciation against actual smear
Good to very good
Evaluation of both methods on 5
consecutive assays
Cell types
Median (total of 5
surveys)
Actual
smear
Virtual
smear
Diff (Virtual Actual)
Polynuclear neutrophils
120
125,2
5,2
Band cells
11
12,65
1,65
Polynuclear neutrophils +
Band cells
126
136
10
8
8
0
Lymphocytes
125
130
5
Monocytes
19
20
1
Blast cells
202,5
192,1
-10,4
Other cells
124,7
116
-8,7
Polynuclear eosinophils
Relative
error (%)
4,15
13,04
7,35
0,00
3,85
5,00
-5,41
-7,50
Actual and virtual smears :
diagnosis accuracy for 5 surveys
Diagnosis Accuracy : Comparison between actual and virtual smears
100
Actual smear
Virtual smear
90
80
Diagnosis accuracy (%)
70
60
50
40
30
20
10
0
2004-3
2005-1
2005-2
Survey
2005-3
2006-1
H/6916
May-Hegglin
H/7100
Chediak-Higashi
H/7100
MDS ider20q
H/9353
AML t(8;21)
H/7597
CML
Mai 2008
Bone marrow virtual smeal
We will try to optimize our material
regarding your precious remarks
and evaluations
Authoring Tool For The
External Quality
Assessment
1. State of the art
BEFORE
AFTER
Flash
Java
Content management delegated to
a developper
Content managed by the experts
Dependence on a developper
Composition autonomy
One domain
Any domain
Offline
Offline - Online
No questions - answers mapping
Questions – answers mapping
(ex. hematology : cell by cell analysis)
Answers outside the system
Answers inside the system
(paper, web forms)
(answers analysis possible)
2. Content Logical Representation
• Content Transmission Scheme
The experts of
a domain
Authoring
Tool
??
Laboratories
Logical
Content
3. Content Manifestation
• Content Transmission Scheme
The experts of
a domain
Authoring
Tool
OK
Laboratories
Content
Manifestation
Logical
Content
4. Transformations (Scenario Application)
Structured Datas
Datas Manifestation (full or partial)
Adaptation Transformations
Datas Presentation
Presentation Transformations
XML
Applet
XSL
XSL
. . .
XML file
Browser
...
PDF
Target Specific
FO
PDF reader
TEXT
Text Editor
PS
Printer
. . .
...
Smi
Presentation
Spatial
Dimension
XSL
XSL
SMIL
Spatio –
temporal
Dimension
Target Independant
HTML
FOP Processing
XML file
XSL
XSL
Adapted
Logical
Content
SMIL
Engine
General
Logical
Content
Interactive
Dimension
Program
Don’t forget the limitation of
classical microscopic slides!
Adapted from EQLAM MGG staining recommandations
Reagents
Sorensen Phosphate Buffer pH 6.8 : Na2HPO4.2H2O M/15 (2.56 g/l) and KH2PO4
M/15 (6.63 g/l)
Check the pH
of the
Sorensen
buffer!
Buffered water : dilution 1/20e of Sorensen Phosphate Buffer in distilled water
May-Grünwald solution
Don’t adjust
the pH of the
buffered
water!
Giemsa solution
Staining
Methanol
Pure May-Grünwald solution
10 minutes
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):922926
EQALM : External Quality Assurance Programmes in Laboratory Medecine
1 : Darkness + Turbovac 800s (vacuum + N2) + Room temperature
2, 3, 4, 5 : Darkness + Room temperature
Stainability alteration during the smear storage
Oxygen depletion + N2 injection
Turbovac (800s)
1. Packaging of the opaque box containing smears in a plastic bag
2. 98-99 % vacuum
3. Nitrogen gas injection
4. Heat closure of the plastic bag
5. Storage at room temperature
MGG staining
Immediate staining
Staining after 1 ½ month
H/2654 (- Turbovac)
H/6517 (+ Turbovac)
e-Hematimage.eu
50 belgian participants/more than
2500 around the world
many thanks for their
contribution to
Marc Chatelain
Yvan Cornet
Agathe Debliquis
Damien Denizza
Hubert Meurisse
Jan Philippé
Jérôme Pruvot
Marjan Van Blerk and the hematology expert comitee
Jean-Claude Libeer