Slide Based Cytometry of

Immunostained Tissue:
"Virtual Flow Cytometry" on a
Slide

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 Background
• The standard of clinical practice is to estimate the percentage of
immunohistochemically (IHC) stained cells

• This practice is subjective and often gives a wide range of

results that depends on the level of the microscopists’ skill.
– limitation of our visual system
– difficulty in counting positive(or negative) cells
• overlapped stained nuclei
• variability of immunostaining presence of other irrelevant objects
• Takes time to manually count these cells
• Wide intra- and interobserver results because of subjectivity

• Developing a cytometry tool that promises similar objectivity as

flow cytometry, will only decrease the incidence of errors in
mensuration and diagnosis.
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Quantitative IHC Report-All iHC are
estimates

Above example is the semiquantitative

report by estimate- subjective, may be
inaccurate,may contribute to error
propagation in diagnosis and prognosis

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Human perception of color is limited.

Although we excell in pattern recognition

our color visual system is influenced by
the saturation and intensity components of color;
we are also poor judge of intensity differences

Although the hue value of the neck

of the hourglass remains the same,
(hue = 0)
we do not see them as equal.
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Computerized algorithms could separate color
into its components for analysis- left panel,magnified view
of immunohistologic stained cells- negative nuclei and positive nuclei, right panel are
the hue, saturation and intensity components of the pixels

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Overlapping and variability of
staining and presence of other
cells of different class(type)
Positive
lymphoid cells

Negative
lymphoidcells
Irrelevant
objects

Non
lymphoid
negative
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Stained and unstained cells of the same class, ie, lymphocytes

unstained

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Flow cytometry, as a model for this virtual flow cytometry, classifies cells of
the same class as positive and negative on a defined set of marker/s

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 Flow cytometry uses fresh cell suspension to give objective results but the observer
loses visual confirmation. In contrast, immunohistochemistry using fixed tissue,
allows observer to see but loses the objectivity in the counting of these cells. The
flow cytometry also loses the location information-location cytomics is not
recovered.
MARKERS
– IMMUNOPHENOTYPE
• flow
cytometry

• Immunohisto-
chemistry

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 Applications of virtual flow cytometry

• Research applications
– Tissue culture biomarkers analysis; location cytomics
• Prognostic markers
– tumor infiltrating lymphocytes (CD8 TILs in B-cell
lymphomas, sarcomas, carcinomas, and others)
• Diagnostic uses
– CD4/CD8 ratio
– Kappa Lambda ratio
– Proliferative Index ( Ki-67, Mib1)
• Therapeautic applications
– Monitoring targeted cell antigens pre- and post-therapy
in PTCLs-AntiCD2,anti CD30, etc
– Monitoring CD20 post-Rituxan therapy

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 Challenges of Slide based virtual Flow Cytometry

1)Thick tissue sections with overlapping of cells,

2) Variability of technics and platforms in immunostaining of tissues,

3) Optimal sampling resolution for obtaining size and staining data per cell
object ( objective magnification 10x vs 20x vs 40x),

4) Issues of optical output properties of image acquisition hardware/s,

5 ) Lack of cell based algorithms able to detect dual populations (positive

immunostained cells and non-immunostained relevant cells of the same
class) and,

6) Arriving at an automated and adaptive thresholding algorithm based on the

characteristics of the objects and the background non objects,

7) Determining image specific parameters that optimize detection of

percentage positive cells

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 Solutions
• 1. Quality Histology ( 2 micron sections and good hematoxylin countestain)

• 2. Using an automated industry standard autostainer

• 3. At 20x magnification, sampling is optimal ; 10x too pixelated, 40x less cells
per frame

• 4. Built in contrast enhancement provided by the three chip CCD blue, green
and red channels

• 5. Enhanced image lends easily to “dual thresholding” of positive and negative

cells of the same class; thresholding divided the image pixels into positive and
negative cell objects; here we threshold for brown and then blue pixels

• 6. Adaptive automated histogram based thresholding algorithm combined with

percentile thresholding; the latter to discard the darkest and brightest pixels
.
• 7. We discovered the parameters obtained from the image itself to automate the
thresholding function using the entropy algorithm. We found a good correlation
between the ratio of positive cell objects with negative cell objects and the value
of this parameter entropy. Hernani Cualing MD
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 Definitions

• Image segmentation- is the process of dividing an image into regions;

in digital images based on pixels (picture elements) properties into
objects and into background. Most use either thresholding of pixel
histogram or localizing object boundaries using edge detection.
• The simplest and most widely used segmentation method is
thresholding- where all the pixels below a certain darker threshold is
assigned the object and above, the background –grayscale and color
• Cellular logic operations- to further define objects

Image analysis components for slide based cytometry

1. Segmentation- need dual mode for population of positive and
negative
2. Feature extraction-size, perimeter, staining density, etc.
3. Classification- positive and negative, percent positive, stain
density of positive
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 Edge vs Thresholding
Two major approaches in segmentation use the property of color and gray
level values of objects: discontinuity and similarity.

Objects with well-defined discontinuities usually benefit from edge-based

detection and
those with poorly defined discontinuities may benefit from similarity
approach such as thresholding. Accurate segmentation remains one
of the problems in pixel based image ratio with up to 15% error on both
falsely positive and falsely negative results (USPTO patent #
6,553,135).

Because of the complex, discontinuous, multicolor, large scale

images in immunohistochemically stained biologic cells, We
implemented cell-based thresholding algorithm.

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The generic method in extracting information from an image by
segmentation is by thresholding the brightness of pixels-
divides the image into component objects or regions based on
their distribution in the brightness scale; Objects==below
threshold and background==above threshold, that is for gray
scale images. For colored images, the issue is more complex.

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Approaches:
1) simple gray scale thresholding?

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 Raw color frame for manual interpretation

CD8 positive
T-lymphocytes

CD8 negative
lymphocytes

Tumor nuclei

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 corresponding computer generated
interpretation

CD8 positive
T-lymphocytes

CD8 negative
lymphocytes

Tumor nuclei

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Strategy for reducing the theory of a slide
cytometer or virtual flow cytometer to practice

1. Count positive and negative cells of the

same cell type-phase I
2. Extract the size and color intensity
information and generate a two parameter
histogram distribution-phase II
3. Validate the results in relation to
manual count and flow cytometry results on a
defined cancer type-phase III.
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 Results of Phase I Study : CD8 Tumor
infiltrating Lymphocytes with Positive and
Negative Cells
Case x
Immunostai Lymphocyte Lymphocyte Lymphocyte
nDensity Number s (+) stain stain (+) (%)
1 4 58 23 39.655174
2 4 52 22 42.307693
3 4 57 43 75.438599
4 4 56 22 39.285713
5 4 61 44 72.131149
6 4 106 48 45.28302
7 4 54 25 46.296295
8 4 74 13 17.567568
9 4 43 33 76.744186
10 4 45 22 48.888889
11 4 36 18 50
12 4 73 8 10.958904
13 4 131 13 9.923664
14 4 86 24 27.906977
15 4 42 18 42.857143

Min 4 36 8 9.923664
Max 4 131 48 76.744186
Median 4 57 22 42.857143
Total 974 376
Average 4 64.933334 25.066668 38.603695
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 Phase II extraction of cell
objects data
• The formula below was used to convert pixel area to cell diameter in
microns ( Y data), for a 20x magnified image:

Cell diameter = 2 * (sqrt of (Area in pixels /  )) / 1.5

= sqrt of (Area in pixels * P) ;where diameter is in microns,  is conversion factor (
0.56588424212) for 20x

• The optical density data per cell object was given by the sum of optical density
of the colored pixels comprising the previously labeled Cell objects ( X data).
• Each cell object then was labeled and contained size and density numbers
which were used for the dot plot display. The final resulting data then consisted
of an array of labeled objects with size and corresponding stain density.
• These two parameters defined the frequency distribution of the dual cell objects
in the two parameterl dot plot: a display commonly used in flow cytometry.

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Flow cytometry immunophenotypic analysis is applied to cell suspension to
give the percentage values of stained and unstained cells based on their
fluorescence intensities.

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 iHCFlow ™ Tissue Cytometry
• A method and approach is developed-
preservation of location cytomics.

• The conversion of immunohistochemistry

(IHC) data to a flow cytometry-like two
parameter dot-plot display, is called
IHCFLOW technic and because the dot plot
is obtained from a slide using single cell
analysis of the same population; is also called
a virtual flow cytometry.

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 How we differ from previous studies

– Other systems use multispectral instrumentation and adaptive

wavelength filters
– confocal immunofluorescent microscopy to obtain separation of
positive stain from nonstained pixels.
– few hybrid microscope-flow cytometry were developed to obtain
single cell information to mimic flow cytometry.
– Immunofluorescent technics were applied on slide substrate to
obtain cell populations using laser scanning instrumentation

• The IHCFLOW technic does not need complex, fluorescent and

expensive instrumentation. It needs a 3 chip RGB CCD equipped
microscope

• Some studies looked at counting immunohistochemistry stained slides,

but none converted the results to a flow cytometry paradigm.

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Virtual Flow cytometry

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Segmentation and Result

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Results of Flow Cytometry
and Virtual Flow Cytometry

6 % CD8 +
5.08 % CD8
+

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CD3
CD3 = 24%

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BCL-1

85%

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Ki-67

28%

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CD5

55%

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 Slide Based image cytometry
Slide based cytometry promises to be a tool to complement flow cytometry
and enhance objectivity in histologic analysis

Niche: For tissue and cell-based quantitative analysis of

immunohistochemistry stained cells

Other applications:
Quantitative tissue microarrays
Stromal marker studies
Quantitative biomarker profile of epithelia
Tumor infiltrating lymphocytes in mantle cell lymphomas
Tumor infiltrating lymphocytes in colonic carcinomas

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Tissue MoAb Microarray ( bcl-1 in mantle cell
lymphoma)

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Tissue Microarray Slide Based Flow Cytometry

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 Area X Y Perim Angle Circ. Feret IntDe Skew %Are XStart YStart
. n a
1 51 253 6 25 0 -1 9 0 0 100 250 2

2 118 503 7 48 174 1 18 0 0 100 505 2

3 173 468 10 47 172 1 17 10455 1 76 464 3

4 73 56 12 32 56 1 13 0 0 100 55 7

5 167 220 22 49 66 1 19 1530 5 96 221 13

6 299 128 29 68 69 1 24 0 0 100 123 19

7 54 145 26 66 20Hernani Cualing

0 MD 16 0 0 100 145 20
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8 830 191 38 129 33 1 50 85425 0 60 193 21
Ki67 in tonsilar epithelium- 31 %

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Ki67 in tonsilar epithelium- 18 %

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VALIDATION OR PHASE III: PROJECT on
MANTLE CELL LYMPHOMAS AND Tumor
Infiltrating lymphocytes ( TIL’S)

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 DESIGN AND RESULTS
• Segmentation of a 512 x 474 RGB image and tabular display of statistical results
table took 2-3 seconds using proprietary developed algorithms ( by IHCFLOW,
Inc).
• We used a panel of 7 antibodies for validation on 14 cases of mantle cell
lymphoma giving percentage positive, total lymphocytes, and staining density. A
total of 2,027 image frames with 810,800 cell objects (COBs) were evaluated.
Antibodies to CD3, CD4, CD8, Bcl-1, Ki-67, CD20, CD5 were subjected to virtual
flow cytometry on tissue. The results of Tissue Cytometry were compared with
manual counts of expert observers and with the results of flow cytometric
immunophenotyping of the same specimen.
• The correlation coefficient and 95 % confidence interval by linear regression
analysis yielded a high concordance between manual human results (M), flow
cytometry results (FC), and Tissue Cytometry (TC) results per antibody, (r
=0.9365 manual vs TC, r =0.9537 FC vs TC). The technical issues were
resolved and the solutions and results were evaluated and presented

• Reference: Cualing HD,Moscinski LC, Zhong E. “Virtual Flow Cytometry” of Immunostained Lymphocytes
on Microscopic Tissue Slides, Clinical Cytometry Jan 15, 2007.

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 Correlation

r = 0.95 r = 0.93

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Mantle cell lymphoma and TIL’s

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 S04-A Ya S04-c y c
TC manual flow TC manual flow TC
bcl1 94 95 81 85 79
cd3 36 30 34 24 25 29 31
cd4 19 20 16 19 20 21 25
cd5 86 50 89 80 85 75 99
cd8 14 20 15 15 9 8 7
cd20 96 90 86 84 90 76 94
ki67 8 10 10 12 56

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CD8 and CD20 tumor infiltrating lymphocytes (TILs)
and prognosis of adenocarcinoma of the colon

carcinoma

CD8 CD20

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 Quantitative microscopy redefined
 Quantitative microscopy is increasingly used in both
pathology and biomedical research applications
 Applications needing morphometry values from digital
images of microscopic sections of tissue-”Tissue Cytomics
or Tissomics”
 Where morphometry is defined beyond the commonly
used pixel area of the target objects per image frame(“pixel
based analysis”) ;
 But that of quantitative information obtained per cell-
“Cell-based analysis or Cytomics”
 Delineation of % of cells carrying the targeted biomarker
in a population of the same class of cells “ Class targeted
cytomics”
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 Tissomics
• Combining diverse data streams across
different levels of observations-such as
molecular, cellular, clinical data-is a system
wide approach
• Tissue cytomics –high throughput high
content phenotyping methodology provides
data rich profile of cellular heterogeneity in
tissue enabling correlative statistics over
biologics targets
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 Class-targeted Cytomics
• Need for slide based:
– Non consumptive analysis= re-analysis
– Allows visual validation
– Cell parameters could be correlated
– Fixed tissue
– Amenable to routine and experimental
setup

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 Virtual flow cytometry applied to an
immunostained tissue analysed on a slide
matrix
• Reduced to practice, patent pending
• Not the same as antigen quantitation
• Has real world applications in daily
pathology practice
• CPT code ready- ie.88361- computer
assisted software analysis of IHC
• Similar to flow cytometry but performed
on paraffin fixed histologic sections
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 Cytomics tool
• The iHCFlow ™ Tissue Cytometry fulfills the major Cytomics
criteria of
• 1) relating multiple parameters to each other,
• 2) within large population of cells,
• 3) on a single-cell basis,
• 4) on a quantitative and observer-independent manner.
• But unlike the other cytomics systems which use
immunofluorescent tissue stains, the system differs in using
routine immunohistochemistry on tissue generating flow
cytometry-like results.
• Ecker R C,Steiner GE. Microscopy-based multicolor tissue cytometry at the single-cell level. Cytometry A
2004;59:182-190.

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 summary
population statistics and dot plot two-dimensional histogram results show a
reliable, robust, accurate reporting similar to that of a flow cytometer.
We differ from the current methods of image analysis systems by focusing on cell-
based population statistics instead of pixel-area data.
The correlation between each case run in flow cytometry and estimated by experts
and by the advanced image Tissue Cytometry is high and suggest a valid
approach to objectively quantifying immunostaining

REFERENCES
Cualing HD,Moscinski LC, Zhong E. “Virtual Flow Cytometry” of Immunostained Lymphocytes on Microscopic Tissue Slides, Clinical Cytometry Jan 15,
2007.
Khalil F*, Heekin J, Lili Miles, Cualing H.The rate and morphometric pattern of bone marrow recovery post-myelosuppressive therapy for acute leukemia: a
quantitative study-submited to Archives of Pathology and Lab Medicine Aug 2007
Cualing H. Automated Analysis in Flow Cytometry. Cytometry, 2000 , 42:p.110-113.
Young IT, Quantitative Microscopy. IEEE Engineering in Medicine and Biology, 1996, 15(1): p.59-66.
Ridler TW ,Calvard S. Picture Thresholding Using Iterative Selection Method. IEEE Trans. On Systems, Man, and Cybernetics, 1978. SMC-8(8):p
630-632.
Cualing H. Kothari R, Balachander T. Immunophenotypic Diagnosis of Acute Leukemia Using Decision Tree Induction. Lab Investigation, 1999,
79:p.205-212.

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 Collaborators
• IHCFLOW, Inc
• GreenGreat, Inc.
• E Zhong
• Marshall Kadin
• L Moscinski

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