White Blood Cells Identification and Counting From Microscopic Blood Image

White Blood Cells Identification And Counting From Microscopic Blood Image
CHAPTER1
INTRODUCTION
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Eswar College of Engineering
INTRODUCTION
1.1 IMAGE
An image is a two-dimensional picture, which has a similar appearance to some
subject usually a physical object or a person.
Image is a two-dimensional, such as a photograph, screen display, and as well as a
three-dimensional, such as a statue. They may be captured by optical devicessuch as
cameras, mirrors, lenses, telescopes, microscopes, etc. and natural objects and phenomena,
such as the human eye or water surfaces.
The word image is also used in the broader sense of any two-dimensional figure such
as a map, a graph, a pie chart, or an abstract painting. In this wider sense, images can also be
rendered manually, such as by drawing, painting, carving, rendered automatically by printing
or computer graphics technology, or developed by a combination of methods, especially in a
pseudo-photograph.
Figure1: A Digital Image

An image is a rectangular grid of pixels. It has a definite height and a definite width
counted in pixels. Each pixel is square and has a fixed size on a given display. However
different computer monitors may use different sized pixels. The pixels that constitute an
image are ordered as a grid (columns and rows); each pixel consists of numbers representing
magnitudes of brightness and color.
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Figure 1.1: View Of Image As Pixels And Intensities

Each pixel has a color. The color is a 32-bit integer. The first eight bits determine the
redness of the pixel, the next eight bits the greenness, the next eight bits the blueness, and the
remaining eight bits the transparency of the pixel.
Figure 1.2: Color Representation Of Pixels
1.1.1 IMAGE FILE SIZES

Image file size is expressed as the number of bytes that increases with the number of
pixels composing an image, and the color depth of the pixels. The greater the number of rows
and columns, the greater the image resolution, and the larger the file. Also, each pixel of an
image increases in size when its color depth increases, an 8-bit pixel (1 byte) stores 256
colors, a 24-bit pixel (3 bytes) stores 16 million colors, the latter known as true color.
Image compression uses algorithms to decrease the size of a file. High resolution
cameras produce large image files, ranging from hundreds of kilobytes to megabytes, per the
camera's resolution and the image-storage format capacity. High resolution digital cameras
record 12 megapixel (1MP = 1,000,000 pixels / 1 million) images, or more, in true color. For
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example, an image recorded by a 12 MP camera; since each pixel uses 3 bytes to record true
color, the uncompressed image would occupy 36,000,000 bytes of memory, a great amount
of digital storage for one image, given that cameras must record and store many images to be
practical. Faced with large file sizes, both within the camera and a storage disc, image file
formats were developed to store such large images.
1.1.2IMAGE FILE FORMATS

Image file formats are standardized means of organizing and storing images. This
entry is about digital image formats used to store photographic and other images. Image files
are composed of either pixel or vector (geometric) data that are rasterized to pixels when
displayed (with few exceptions) in a vector graphic display. Including proprietary types, there
are hundreds of image file types. The PNG, JPEG, and GIF formats are most often used to
display images on the Internet.
Figure1.3: Raster And Vector Formats Of an Image

In addition to straight image formats, Metafile formats are portable formats which can
include both raster and vector information. The metafile format is an intermediate format.
Most Windows applications open metafiles and then save them in their own native format.
1.1.3RASTER FORMATS
These formats store images as bitmaps (also known as pixmaps).
JPEG/JFIF
JPEG (Joint Photographic Experts Group) is a compression method. JPEG
compressed images are usually stored in the JFIF (JPEG File Interchange Format) file format.
JPEG compression is lossy compression. Nearly every digital camera can save images in the
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JPEG/JFIF format, which supports 8 bits per color (red, green, blue) for a 24-bit total,
producing relatively small files. Photographic images may be better stored in a lossless nonJPEG format if they will be re-edited, or if small "artifacts" are unacceptable. The JPEG/JFIF
format also is used as the image compression algorithm in many Adobe PDF files.
EXIF
The EXIF (Exchangeable image file format) format is a file standard similar to the JFIF
format with TIFF extensions. It is incorporated in the JPEG writing software used in most
cameras. Its purpose is to record and to standardize the exchange of images with image
metadata between digital cameras and editing and viewing software. The metadata are
recorded for individual images and include such things as camera settings, time and date,
shutter speed, exposure, image size, compression, name of camera, color information, etc.
When images are viewed or edited by image editing software, all of this image information
can be display.
TIFF
The TIFF (Tagged Image File Format) format is a flexible format that normally saves
8 bits or 16 bits per color (red, green, blue) for 24-bit and 48-bit totals, respectively, usually
using either the TIFF or TIF filename extension. TIFFs are lossy and lossless. Some offer
relatively good lossless compression for bi-level (black & white) images. Some digital
cameras can save in TIFF format, using the LZW compression algorithm for lossless storage.
TIFF image format is not widely supported by web browsers. TIFF remains widely accepted
as a photograph file standard in the printing business. TIFF can handle device-specific color
spaces, such as the CMYK defined by a particular set of printing press inks.
PNG
The PNG (Portable Network Graphics) file format was created as the free, opensource successor to the GIF. The PNG file format supports true color (16 million colors)
while the GIF supports only 256 colors. The PNG file excels when the image has large,
uniformly colored areas. The lossless PNG format is best suited for editing pictures, and the
lossy formats, like JPG, are best for the final distribution of photographic images, because
JPG files are smaller than PNG files. PNG, an extensible file format for the lossless, portable,
well-compressed storage of raster images. PNG provides a patent-free replacement for GIF
and can also replace many common uses of TIFF. Indexed-color, grayscale, and true color
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images are supported, plus an optional alpha channel. PNG is designed to work well in online
viewing applications, such as the World Wide Web. PNG is robust, providing both full file
integrity checking and simple detection of common transmission errors.
GIF
GIF (Graphics Interchange Format) is limited to an 8-bit palette, or 256 colors. This
makes the GIF format suitable for storing graphics with relatively few colors such as simple
diagrams, shapes, logos and cartoon style images. The GIF format supports animation and is
still widely used to provide image animation effects. It also uses a lossless compression that
is more effective when large areas have a single color, and ineffective for detailed images or
dithered images.
BMP
The BMP file format (Windows bitmap) handles graphics files within the Microsoft
Windows OS. Typically, BMP files are uncompressed, hence they are large. The advantage is
their simplicity and wide acceptance in Windows programs.
1.1.4 VECTOR FORMATS

As opposed to the raster image formats above (where the data describes the
characteristics of each individual pixel), vector image formats contain a geometric description
which can be rendered smoothly at any desired display size.
At some point, all vector graphics must be rasterized in order to be displayed on
digital monitors. However, vector images can be displayed with analog CRT technology such
as that used in some electronic test equipment, medical monitors, radar displays, laser shows
and early video games. Plotters are printers that use vector data rather than pixel data to draw
graphics.
CGM
CGM (Computer Graphics Metafile) is a file format for 2D vector graphics, raster
graphics, and text. All graphical elements can be specified in a textual source file
that can be compiled into a binary file or one of two text representations. CGM
provides a means of graphics data interchange for computer representation of 2D
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graphical information independent from any particular application, system,
platform, or device.
SVG
SVG (Scalable Vector Graphics) is an open standard created and developed by the
World Wide Web Consortium to address the need for a versatile, scriptable and all purpose
vector format for the web and otherwise. The SVG format does not have a compression
scheme of its own, but due to the textual nature of XML, an SVG graphic can be compressed
using a program such as gzip.
1.2 IMAGE PROCESSING

Digital image processing, the manipulation of images by computer, is relatively recent
development in terms of mans ancient fascination with visual stimuli. In its short history, it
has been applied to practically every type of images with varying degree of success. The
inherent subjective appeal of pictorial displays attracts perhaps a disproportionate amount of
attention from the scientists and also from the layman.
Digital image processing like other glamour fields, suffers from myths, mis-connect
ions, mis-understandings and mis-information. It is vast umbrella under which fall diverse
aspect of optics, electronics, mathematics, photography graphics and computer technology. It
is truly multidisciplinary endeavor ploughed with imprecise jargon.
Several factor combine to indicate a lively future for digital image processing. A
major factor is the declining cost of computer equipment. Several new technological trends
promise to further promote digital image processing. These include parallel processing mode
practical by low cost microprocessors, and the use of charge coupled devices (CCDs) for
digitizing, storage during processing and display and large low cost of image storage arrays.
There are ten basic fundamental steps in digital image processing as shown in the
bellow diagram. The process through this ten steps is entirely done in two parts. One is that
both the inputs and outputs are images. And in the second process, input is image and output
is attribute. This is the whole process to be done in image processing to be done to extract the
features of an image or to do enhancement on that image.
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FUNDAMENTAL STEPS IN DIGITAL IMAGE PROCESSING
Figure 1.4: Fundamental Steps In Digital Image Processing
1.2.1 IMAGE ACQUISATION

Image Acquisition is to acquire a digital image. To do so requires an image sensor and
the capability to digitize the signal produced by the sensor. The sensor could be monochrome
or color TV camera that produces an entire image of the problem domain every 1/30 sec. the
image sensor could also be line scan camera that produces a single image line at a time. In
this case, the objects motion past the line.
Figure 1.5: Devices Used For Acquisition Of Images

Scanner produces a two-dimensional image. If the output of the camera or other
imaging sensor is not in digital form, an analog to digital converter digitizes it. The nature of
the sensor and the image it produces are determined by the application.
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Figure 1.6: Image Of A Scanner
1.2.2 IMAGE ENHANCEMENT

Image enhancement is among the simplest and most appealing areas of digital image
processing. Basically, the idea behind enhancement techniques is to bring out detail that is
obscured, or simply to highlight certain features of interesting an image. A familiar example
of enhancement is when we increase the contrast of an image because it looks better. It is
important to keep in mind that enhancement is a very subjective area of image processing.
Figure 1.7; Original And Enhanced Image
1.2.3 IMAGE RESTORATION

Image restoration is an area that also deals with improving the appearance of an
image. However, unlike enhancement, which is subjective, image restoration is objective, in
the sense that restoration techniques tend to be based on mathematical or probabilistic models
of image degradation.
Figure 1.8: View Of Restorated Image

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Enhancement, on the other hand, is based on human subjective preferences regarding
what constitutes a good enhancement result. For example, contrast stretching is considered
an enhancement technique because it is based primarily on the pleasing aspects it might
present to the viewer, where as removal of image blur by applying a deblurring function is
considered a restoration technique.
1.2.4 COLOUR IMAGE PROCESSING

The use of color in image processing is motivated by two principal factors. First, color
is a powerful descriptor that often simplifies object identification and extraction from a scene.
Second, humans can discern thousands of color shades and intensities, compared to about
only two dozen shades of gray. This second factor is particularly important in manual image
analysis.
Figure1.9: View Of Colour Image Processing
1.2.5 WAVELETS AND MULTI RESOLUTION FACTORING

Wavelets are the formation for representing images in various degrees of resolution.
Although the Fourier transform has been the mainstay of transform based image processing
since the late1950s, a more recent transformation, called the wavelet transform, and is now
making it even easier to compress, transmit, and analyze many images.
Unlike the Fourier transform, whose basis functions are sinusoids, wavelet transforms
are based on small values, called Wavelets, of varying frequency and limited duration.
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Figure 1.10: Multi Resolution Of An Image

Wavelets were first shown to be the foundation of a powerful new approach to signal
processing and analysis called Multiresolutiontheory. Multiresolution theory incorporates and
unifies techniques from a variety of disciplines, including sub band coding from signal
processing, quadrature mirror filtering from digital speech recognition, and pyramidal image
processing.
1.2.6 COMPRESSION
Compression, as the name implies, deals with techniques for reducing the storage
required saving an image, or the bandwidth required for transmitting it. Although storage
technology has improved significantly over the past decade, the same cannot be said for
transmission capacity. This is true particularly in uses of the Internet, which are characterized
by significant pictorial content. Image compression is familiar to most users of computers in
the form of image file extensions, such as the jpg file extension used in the JPEG (Joint
Photographic Experts Group) image compression standard.
1.2.7 MORPHOLOGICAL PROCESSING

Morphological processing deals with tools for extracting image components that are
useful in the representation and description of shape. The language of mathematical
morphology is set theory. As such, morphology offers a unified and powerful approach to
numerous image processing problems. Sets in mathematical morphology represent objects in
an image. For example, the set of all black pixels in a binary image is a complete
morphological description of the image.
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Figure 1.11: Morphological Processed Image

In binary images, the sets in question are members of the 2-D integer space Z2, where
each element of a set is a 2-D vector whose coordinates are the (x,y) coordinates of a
black(or) white pixel in the image. Gray-scale digital images can be represented as sets
whose components are in Z3. In this case, two components of each element of the set refer to
the coordinates of a pixel, and the third corresponds to its discrete gray-level value.
1.2.8 SEGMENTATION
Segmentation procedures partition an image into its constituent parts or objects. In general,
autonomous segmentation is one of the most difficult tasks in digital image processing. A
rugged segmentation procedure brings the process a long way toward successful solution of
imaging problems that require objects to be identified individually.
Figure1.12: Segmentation Of An Image

On the other hand, weak or erratic segmentation algorithms almost always guarantee
eventual failure. In general, the more accurate the segmentation, the more likely recognition
is to succeed.
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1.2.9 REPRESENTATION AND DESCRIPTION

Representation and description almost always follow the output of a segmentation
stage, which usually is raw pixel data, constituting either the boundary of a region (i.e., the
set of pixels separating one image region from another) or all the points in the region itself. In
either case, converting the data to a form suitable for computer processing is necessary. The
first decision that must be made is whether the data should be represented as a boundary or as
a complete region. Boundary representation is appropriate when the focus is on external
shape characteristics, such as corners and inflections.
Regional representation is appropriate when the focus is on internal properties, such
as texture or skeletal shape. In some applications, these representations complement each
other. Choosing a representation is only part of the solution for transforming raw data into a
form suitable for subsequent computer processing. A method must also be specified for
describing the data so that features of interest are highlighted. Description, also called feature
selection, deals with extracting attributes that result in some quantitative information of
interest or are basic for differentiating one class of objects from another.
1.2.10 OBJECT RECOGNISATION
The last stage involves recognition and interpretation. Recognition is the process that
assigns a label to an object based on the information provided by its descriptors.
Interpretation involves assigning meaning to an ensemble of recognized objects.
1.2.11 KNOWLEDGEBASE
Knowledge about a problem domain is coded into image processing system in the
form of a knowledge database. This knowledge may be as simple as detailing regions of an
image when the information of interests is known to be located, thus limiting the search that
has to be conducted in seeking that information. The knowledge base also can be quite
complex, such as an inter related to list of all major possible defects in a materials inspection
problem or an image data base containing high resolution satellite images of a region in
connection with change deletion application. In addition to guiding the operation of each
processing module, the knowledge base also controls the interaction between modules. The
system must be endowed with the knowledge to recognize the significance of the location of
the string with respect to other components of an address field. This knowledge glides not
only the operation of each module, but it also aids in feedback operations between modules
through the knowledge base. We implemented preprocessing techniques using MATLAB.
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1.3 COMPONENTS OF AN IMAGE PROCESSING SYSTEM

As recently as the mid-1980s, numerous models of image processing systems being
sold throughout the world were rather substantial peripheral devices that attached to equally
substantial host computers. Late in the 1980s and early in the 1990s, the market shifted to
image processing hardware in the form of single boards designed to be compatible with
industry standard buses and to fit into engineering workstation cabinets and personal
computers. In addition to lowering costs, this market shift also served as a catalyst for a
significant number of new companies whose specialty is the development of software written
specifically for image processing.
Network
Image displays
Hard copy
computer
Mass storage
Specialized image
processing
hardware
Image processing
software
Image sensor
Problem domain
Figure 1.13: Components Of An Image Processing System
Although large-scale image processing systems still are being sold for massive
imaging applications, such as processing of satellite images, the trend continues toward
miniaturizing and blending of general-purpose small computers with specialized image
processing hardware. Figure 1.13 shows the basic components comprising a typical generalpurpose system used for digital image processing. The function of each component is
discussed in the following paragraphs, starting with image sensing.
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IMAGE SENSORS
With reference to sensing, two elements are required to acquire digital images. The
first is a physical device that is sensitive to the energy radiated by the object we wish to
image. The second, called a digitizer, is a device for converting the output of the physical
sensing device into digital form. For instance, in a digital video camera, the sensors produce
an electrical output proportional to light intensity. The digitizer converts these outputs to
digital data.
SPECIALISED IMAGE PROCESSING HARDWARE

Specialized image processing hardware usually consists of the digitizer just
mentioned, plus hardware that performs other primitive operations, such as an arithmetic
logic unit (ALU), which performs arithmetic and logical operations in parallel on entire
images. One example of how an ALU is used is in averaging images as quickly as they are
digitized, for the purpose of noise reduction. This type of hardware sometimes is called a
front-end subsystem, and its most distinguishing characteristic is speed. In other words, this
unit performs functions that require fast data throughputs (e.g., digitizing and averaging video
images at 30 frames) that the typical main computer cannot handle.
COMPUTER
The computer in an image processing system is a general-purpose computer and can
range from a PC to a supercomputer. In dedicated applications, sometimes specially designed
computers are used to achieve a required level of performance, but our interest here is on
general-purpose image processing systems. In these systems, almost any well-equipped PCtype machine is suitable for offline image processing tasks.
IMAGE PROCESSING SOFTWARE

Software for image processing consists of specialized modules that perform specific
tasks. A well-designed package also includes the capability for the user to write code that, as
a minimum, utilizes the specialized modules. More sophisticated software packages allow the
integration of those modules and general-purpose software commands from at least one
computer language.
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MASS STORAGE
Mass storage capability is a must in image processing applications. An image of size
1024*1024 pixels, in which the intensity of each pixel is an 8-bit quantity, requires one
megabyte of storage space if the image is not compressed. When dealing with thousands, or
even millions, of images, providing adequate storage in an image processing system can be a
challenge.
Digital storage for image processing applications fall into three principal categories:
(1) short-term storage for use during processing, (2) on-line storage for relatively fast recall,
and (3) archival storage, characterized by infrequent access. Storage is measured in bytes
(eight bits), Kbytes (one thousand bytes), Mbytes (one million bytes), Gbytes (meaning giga,
or one billion, bytes), and Tbytes (meaning tera, or one trillion, bytes).
One method of providing short-term storage is computer memory. Another is by
specialized boards, called frame buffers that store one or more images and can be accessed
rapidly, usually at video rates. The latter method allows virtually instantaneous image zoom,
as well as scroll (vertical shifts) and pan (horizontal shifts). Frame buffers usually are housed
in the specialized image processing hardware unit.
Online storage generally takes the form of magnetic disks or optical-media storage.
The key factor characterizing on-line storage is frequent access to the stored data. Finally,
archival storage is characterized by massive storage requirements but infrequent need for
access. Magnetic tapes and optical disks housed in jukeboxes are the usual media for
archival applications.
IMAGE DISPLAYS
Image displays in use today are mainly color (preferably flat screen) TV monitors.
Monitors are driven by the outputs of image and graphics display cards that are an integral
part of the computer system. Seldom are there requirements for image display applications
that cannot be met by display cards available commercially as part of the computer system. In
some cases, it is necessary to have stereo displays, and these are implemented in the form of
headgear containing two small displays embedded in goggles worn by the user.
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HARD COPY
Hardcopy devices for recording images include laser printers, film cameras, heatsensitive devices, inkjet units, and digital units, such as optical and CD-ROM disks. Film
provides the highest possible resolution, but paper is the obvious medium of choice for
written material. For presentations, images are displayed on film transparencies or in a digital
medium if image projection equipment is used. The latter approach is gaining acceptance as
the standard for image presentations.
NETWORK
Networking is almost a default function in any computer system in use today. Because
of the large amount of data inherent in image processing applications, the key consideration
in image transmission is bandwidth. In dedicated networks, this typically is not a problem,
but communications with remote sites via the Internet are not always as efficient. Fortunately,
this situation is improving quickly as a result of optical fiber and other broadband
technologies.
1.4 AREA OF RESEARCH

The observation of blood samples by expert operators is one of the diagnostic
procedures available for the recognition of different diseases. The human visual inspection is
tedious, lengthy and repetitive, and suffers from the presence of a non-standard precision:
that is because it depends on the operator skills. These reasons have limited its statistical
reliability. On the other side, the automated analysis by computer requires only one image
and not a blood sample; for this, it turns out to be less expensive but at the same timemore
scrupulous in providing precise standards. The main goal of this work is the analysis and
processing of a microscopic image, in order to provide an automated procedure to support the
medical activity. On the market there are various systems for the automatic quantification of
blood cells that allow counting the number of different types of cells within the blood smear.
These counters make use of techniques of flowcytometry to measure some physical
characteristics and/or chemical properties of the blood cells, passing through a light detector
which, through the fluorescence or electrical impedance, allows to identify the type of cell.
Although the results of quantification are very precise, morphological abnormalities of the
cells are not detected by the machine and, therefore, it is seen necessary a subsequent analysis
of blood under the microscope.
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The use of image processing techniques has grown rapidly in the recent years. These
techniques help to count the cells in the human blood and, at the same time, provide
information on the cells morphology. Unfortunately, there are not standard techniques for the
analysis and processing of the images valid for each type of them, but the processing must be
adapted to the context. Regarding the microscopic images, processing techniques vary
depending on the type of blood cells to be analyzed. Our work focuses on the analysis of
white blood cells or WBCs. A typical blood image usually shows four components: plasma,
red blood cellsor erythrocytes, white blood cells or leukocytes, and platelets. The most
represented cells in the blood are red blood cells and white blood cells. Leukocytes are easily
identifiable, as their nucleus appears darker than the background.
Figure. 1.14: Original Blood Sample Image

However, the analysis and the processing of the data related to the white blood cells
can present some complications due to wide variations in cell shape, dimensions and edges.
The generic term leukocytes refers to a set of cells quite different each other.
In human blood is very common the presence of lymphocytes, with a percentage of

20-45% and a size of 7-15microns, characterized by a rounded nucleus and a cytoplasm poor.
Monocytes are the most voluminous white blood cells, with a diameter of 12-18 microns and
representing 3-9% of circulating leukocytes. Therefore, in this paper, we present a method to
identify all types of white blood cells present in the microscopic images, which need to
various steps to reach the goal.
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Figure.1.15: Comparison Between Different Types Of White Blood Cells: Neutrophil,

Basophil, Eosinophil, Lymphocyte, Monocyte
The leukocyte cells containing granules are called granulocytes and include
Neutrophils, basophils and eosinophils. The cells withoutgranules are called agranulocytes
and include the lymphocytes and monocytes. Thus we can distinguish between them, notonly
according to the shape or size, but also thanks to the presence of granules in the cytoplasm
and also by the number of lobes in the nucleus. The lobes are the most substantial part of the
nucleus and are connected to each other by thin filaments.
Neutrophils are mainly present in human blood with a percentage ranging between 50
and 70%, have sizes around10-12 microns and are distinguishable due to the number of lobes
present in the nucleus, which can be up to a maximum of5. Basophils instead represent only
0-1% of lymphocytes inhuman blood, have a diameter of about 10 microns and, for the 1-5%
in human blood, have predominantly rounded shape with dimensions around 10-12 microns,
and have anucleus with more lobes, but not greater than 2. They differ from other white blood
cells for the presence of granules, which include paracrystalline structures in the form of
coffeebean.
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CHAPTER2
LITERATURE SURVEY
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LITERATURE SURVEY
2.1 A NEW WHITE BLOOD CELL SEGMENTATION USING MEAN SHIFT
FILTER AND REGION GROWING ALGORITHM
The differential counting of white blood cells (WBCs) provides invaluable
information to hematologist for diagnosis and treatment of many diseases. However,
manually counting the WBCs is a time consuming and susceptible to error procedure. Then
an automatic and efficient system seems necessary and helpful. In the automatic process, the
segmentation of WBC image is one of the most important stages. In this paper, we propose a
new algorithm to segment the image.
The algorithm consists of two tasks. Firstly, the Mean Shift (MS) filter is used to remove
noise. Secondly, we propose a simple but effective region growing algorithm to segment the
image. The proposed algorithm was tested in RGB color space and CIE L*a*b* color space,
and the experimental results show the excellent performance in both color spaces.
2.2. A FAST SEGMENTATION SCHEME FOR WHITE BLOOD CELL IMAGES

Presents a fast segmentation scheme for automatic differential counting of white
blood cells. The segmentation procedure consists of three phases. First a novel simple
algorithm is proposed for localization of white blood cells. The algorithm is based on a priori
information about blood smear images.
In the second phase the different cell components are separated with automatic thresholding.
The thresholds are selected with a simple recursive method derived from maximizing the
interclass variance between dark, gray and bright regions based on the method proposed by
Otsu (1979). Finally the segmented regions are smoothed by morphological operations. The
segmentation scheme works successfully for classification of white blood cells. Some
experimental results are also presented.
2.3. THE ACUTE LYMPHOBLASTIC LEUKEMIA IMAGE DATABASE FOR

IMAGE PROCESSING
The visual analysis of peripheral blood samples is an important test in the procedures
for the diagnosis of leukemia. Automated systems based on artificial vision methods can
speed up this operation and increase the accuracy and homogeneity of the response also in
telemedicine applications. Unfortunately, there are not available public image datasets to test
and compare such algorithms. In this paper, we propose a new public dataset of blood
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samples, specifically designed for the evaluation and the comparison of algorithms for
segmentation and classification. For each image in the dataset, the classification of the cells is
given, as well as a specific set of figures of merits to fairly compare the performances of
different algorithms. This initiative aims to offer a new test tool to the image processing and
pattern matching communities, direct to stimulating new studies in this important field of
research.
2.4. ROBUST RECOGNITION OF WHITE BLOOD CELL IMAGES
The objective of this work is to investigate the white blood cell (WBC) image
recognition problem at all stages. A robust and effective method for automatic WBC
differentiation, based on both statistical pattern recognition and neural net approaches, is
presented.
We demonstrate well-evaluated results ranging from image scene segmentation techniques to
recognition details. Recognition accuracy on the test set of 662 images of five WBC types
obtained by different imaging systems from 22 bloodstains is not less than 98%.
2.5. A THRESHOLD SELECTION METHOD FROM GRAY-LEVEL HISTOGRAMS

A nonparametric and unsupervised method of automatic threshold selection for
picture segmentation is presented. An optimal threshold is selected by the discriminate
criterion, namely, so as to maximize the separability of the resultant classes in gray levels.
The procedure is very simple, utilizing only the zeroth- and the first-order cumulative
moments of the gray-level histogram. It is straightforward to extend the method to
multithreshold problems. Several experimental results are also presented to support the
validity of the method.
2.6. DRAWBACKS
Slowness of the analysis and
A nonstandard accuracy,
Dependent on the operator skills.
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CHAPTER 3
PROPOSED METHOD
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PROPOSED METHOD
3.1 OBJECTIVE OF PRESENT STUDY
This technique presents a complete and fully automatic method for white blood cells
identification from microscopic images. The proposed method firstly individuates white
blood cells from which, subsequently, nucleus and cytoplasm are extracted.
3.2 INTRODUCTION
The proposed method, differently from other methods in the literature, does not
present separate steps of pre-processing and segmentation, but uses methods of preprocessing inserted between the various stages of the segmentation, in order to make the
latter more simple and more robust.
The identification of the leukocytes is carried out in the first stage, this requires also
identifying and removing the image background, in order to make the identification of
leukocytes more efficient. The result consists of a binary image showing only leukocytes.
The second step is the identification of leukocytes groups and produces a binary image
showing the individual leukocytes and a binary image showing the adjacent leukocytes.
The third phase takes care of separating the adjacent leukocytes and removing the elements
localized on the border of the image.
The fourth phase proceeds with a shape control, through which all the abnormal components
are removed from the image.
The fifth and last phase deal with the selection of the nucleus and the cytoplasm of each
leukocyte.
The whole process can be schematized as showed in Figure 3.1.
Proposed method entirely will be done in above five steps as shown above , the entire process
can be done using the basic image processing techniques like converting to gray images,
enhancement techniques, thresholding ,morphological operations like region growing using
opening and dilation techniques.
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Figure 3.1: Proposed Method Scheme
In the proposed scheme ,the simple five steps are enough. In the first step, leukocytes
identification
and background removal is done. Next step is group of leukocytes are
identified . In the third step , the adjacent leukocytes are get separated. Fourth stage includes
the shape control and abnormal components are removed. Finally, the selection of nucleus
and cytoplasm of each leukocytes is done.
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3.3 METHODOLOGY
A. Background Identification
Since that the images captured at the microscope suffer from uneven lighting, it becomes
necessary to remove the background because the segmentation methods based on threshold
may suffer heavily for this problem. Some methods for background extraction are present in
literature, but they use a collection of images captured with the same camera and the same
microscope for estimation of the pixels belonging to the background, while others have a
very high computational cost, not necessary for this application. The proposed approach
involves the use of an automatic threshold to the original image in gray level (or along the
green component, in this case the image chosen is the one presenting the greatest difference
between minimum and maximum intensity level). There are many threshold techniques
available in literature. Here, we use the threshold value based on triangle method or Zack
algorithm. The triangle method is applied to the image histogram, constructing a straight line
that connects the highest histogram value h[ ] and the lowest histogram value h[ ],
where and indicatethe values of the gray levels where the histogram h[x] reaches
its maximum and minimum, respectively. The distance between the marked line and the
histogram values between and is then calculated.
Figure3.2: (a) Gray Level Image
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Figure 3.2(b:) Gray Level Histogram
Figure 3.2 (c): Threshold Result

Figure. 3.2 The Proposed Approach for Background Identification by Thresholding
The intensity value, where the distance d reaches its maximum, defines the threshold
value. This algorithm is particularly effective when the histograms show some clear valleys
between an high peak and a weak peak, which are both present in the histograms, as it can be
seen in Figure. 3.2(b), generated respectively from the background and from the leukocytes
nucleus.Figure.3.2(c).shows how the result may not be accurate in all its parts, for example, it
has been detected as background even the center of the red blood cells. This does not
preclude the achievement of an effective background removal, since in the later stage, during
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leukocytes identification also the red blood cells will be removed. Background removal can
be performed later (as described in the proposed method) or directly in this phase, starting
from the original image of the blood sample in the RGB color space or from the gray level
image. In both cases the background removal can be performed by simple arithmetical
operations.
B. Leukocytes Identification
In many methods present in the literature the idea is to identify firstly the nucleus
which are more prominent than other components and then the entire membrane, for example
by region growing. In the proposed method instead, the membrane is detected firstly, inorder
to deal the subsequent separation of the adjacent cells more accurately. The white blood cells
identification wasmade possible thanks to the conversion into the CMYK color model. In
fact, we have observed that leukocytes are more contrasted in the Y component of CMYK
color model, this is because the yellow color is present in all the elements of the image except
in leukocytes, where it is practically absent.
Figure 3.3(a): RGB Image
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Figure3.3(b): Y Component Image

Figure 3.3 An Original Blood Sample Image And Its Y Component Image In The CMYK
color space
At this point a redistribution of image gray levels is necessary in order to make easier
the subsequent segmentation process. Then, an histogram equalization or a contrast stretching
can be used as this stage.
Figure 3.4(a): Histogram Equalization
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Figure 3.4(b): Contrast Stretching

Figure. 3.4 Histogram Equalization And Contrast Stretching
The segmentation is realized again using a threshold automatically calculated by the
triangle method. The weak peaks in the histograms represent again leukocytes, and the
highest peak is no longer related to the background (darker in the Y component), but to the
red blood cells. The complement image is then calculated in order to obtain white blood cells
on a dark background .
Figure.3.5(a): Threshold Result
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Figure 3.5(b): Complementary Image

Figure 3.5 the Image Result of Segmentation by Threshold and Its Complementary Image
C. Background Removal
The operation performed to identify more accurately the white blood cells is the same
previously seen for the background removal .
Figure 3.6(a): Background Removal
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Figure 3.6(b): Area Opening

Figure 3.6 the Image Result of Background Removal and the Enhancement by Area Opening
Obviously the background removal process does not produce a clean result in the
whole image. In order to clean up the image, the operation used, called area opening, allows
to delete all the objects with a size smaller than the structuring element. The structuring
element used has a circular shape and its size is calculated on the basis of the objects average
size in the image.
D. Identification of Grouped Leukocytes

Once obtained the image containing only the white blood cells it is possible to verify
if there are adjacent cells (or agglomerates of leukocytes) and, therefore, provide for their
separation. Several methods can be used to verify the presence of adjacent leukocytes [4]. In
this work we have decided to use the roundness value, calculated for each connected
component of the image. The connected components having a roundness value greater than a
certain predetermined threshold are classified as individual leukocytes and so they go directly
to the next step of the analysis process, while the connected components having a roundness
value smaller than the threshold are classified as grouped leukocytes and so they must deal
with the separation process.
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Figure 3.7(a): Single Leukocytes
Figure3.7(b): Grouped Leukocytes

Figure 3.7 The Images Result From The Identification Of Grouped Leukocytes
Note that one of the two images may in some cases be empty, if it is the second, it
means that the phase of separation of leukocytes will not take place.
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E. Separation of Grouped Leukocytes

Many approaches have been proposed to separate the adjacent cells, some of which
are included in the process of segmentation and other specifically dedicated to separate the
overlapping cells. For example some approaches used by Kovalev, Sinha and Ramakrishnan,
work on sub images extracted from the original image by cutting a square around the nucleus
previously segmented. So, assuming that each sub-image has a single white blood cell, a
clustering around the nucleus is performed, via the restrictions on the form and the color
information. The proposed approach is divided into two parts. In the first part it is used the
method proposed by Lind lad which uses the distance transform. The latter, applied to the
binary image, associates to each pixel its distance from the border. Applying the distance
transform of the watershed segmentation, it is possible to make a first roughly separation
between adjacent leukocytes.
The separation in this way tends to be inaccurate, as it uses the distance transform as a
form delimiter, but performs well only in the presence of leukocytes adjacent with a nearly
rounded shape, but it does not perform equally well in the presence of multiple complex
forms.
Figure.3.8: Two Original Blood Sample Sub-images And Their Respective Watershed
Results
For this reason it is necessary a second step to refine the contours extracted through
watershed transform. Then, althea pixels of the component under examination which are
located at a distance not greater than a predetermined value from the watershed line
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concerned, are taken in consideration. These pixels are then used to derive the deepest
concavity for which the line of exact separation will have to pass. Therefore, by exploiting
the information of the points of concavity and the information related to the points of
maximum image in gray tones, it is possible to obtain a cutting line that best fits the contour
of the leukocytes, as we can see in Figure. 11.
Figure.3.9: Local Maxima Image And Final Separation Results
F. Image Cleaning
The image cleaning requires the removal of all the leukocytes located along the
border of the image and of all abnormal components (that are not leukocytes), in order to
avoid errors in the later stages of the analysis process. The cleaning of the image border is a
simple operation, while the removal of abnormal components is a more complex process.
To do this it is necessary to first determine the number of leukocytes present in the
image. For each of them it is then calculated the size of the area and the size of the convex
area. The size of the area is used to calculate the mean area, necessary to determine and
eliminate the components with irregular dimensions. For example, a very small area might
indicate the presence of artifacts not removed adequately, on the other side, a very large area
may indicate the presence of adjacent leukocytes not separated adequately.
Area and convex area are then used in combination for the calculation of the solidity
value. All objects with a solidity value less thana predetermined threshold are discarded. In
fact, even in thiscase, a value of solidity less than the threshold value indicates the presence
of artifacts not removed adequately.
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Figure 3.10(a): Border Cleaning
Figure. 3.10(b:) Abnormal Components Removal

Figure. 3.10 The Final Results Of The Border Cleaning And Of The Abnormal Components
Removal
G.Nucleus and Cytoplasm Selection

Once the leukocytes have been identified, it is possible to move to the second segmentation
level that provides the selection of nucleus and cytoplasm.
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Figure.3.11: Gray level And Binary Sub-image Of Individual Leukocytes. Border Cleaning
From Binary Image. Gray level Sub-image With
Superimposed Border. Gray level Sub-image With External Leukocyte Image Cropped
This step can be simplified performing an image crop using the bounding box size
that is the smallest rectangle that completely contains the connected component, with the aim
to have a single leukocyte for sub-image. A border cleaning operation is again necessary in
order to preserve only the white blood cell in question.
Since by definition, leukocytes nucleus is internal to the membrane, it is possible to
perform further simplification, through the crop of the entire portion of the image outside the
leukocyte in question . This procedure allows a more robust nucleus selection, because it
excludes completely artifacts from the selection. Nucleus selection approach takes advantage
from Csekes observations, who found that white blood cells nuclei are more in contrast in
the green component of the RGB color space.
Threshold operation using Otsu in this color space, however, does not produce clean
results, especially with the presence of granulocytes, whose granules are selected erroneously
as part of the nucleus.
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Figure. 3.12: Images Of The G Component In The RGB space And a* Component In The
CIE Lab Space. Binary Image After Nucleus Selection. Binary Image After Cytoplasm
Selection
For this the binary image obtained from the green component, is combined with the
binary image, obtained from the a* component of the CIEL a, b color space, again through a
threshold operation.
The mask obtained makes it possible to extract clearly the leukocytes nucleus. At the
end, to obtain the cytoplasm you just have to perform a subtraction operation between the
binary image containing the whole leukocyte and the image containing only the nucleus.
Finally, the nuclei and cytoplasm of leukocytes is identified .Then after performing the
identification of leukocytes by using thresholding, and region growing like morphological
operations, then the counting of leukocytes is done .
Here when compared to existing methodologies, proposed method identifies and counts the
no. of leukocytes is done efficiently.
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CHAPTER4
RESULTS AND APPLICATIONS
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4.1 RESULTS
Figure.4.1: A Sample Picture Of Blood Cells
In this picture , the white blood cells count is 3. In this the count is very less
in the taken blood sample
Figure.4.2: Second Sample Picture Of Blood Cells

In this microscopic image ,the white blood cell count is medium and the count
is 55.
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Figure. 4.3: Third Sample Picture Of Blood Cells
In this third sample of blood sample of image, the white blood cell count is
123.
4.2 APPLICATIONS
1.WBC count is useful in detecting and treating leukemia.
2.WBC count places a major role in detecting bone marrow diseases.
3.WBC count is the basic measure to be calculated to detect any disease attacked on
human body.
4.Leukocytes count is useful in detecting zica virus.
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CHAPTER 5
CONCLUSION
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CONCLUSION
In this work it has been proposed an innovative method for a completely automatic
identification of leukocytes from microscopic images, in order to provide an automated
procedure as support for medical activity. The results obtained show that the proposed
method is able to identify in a robust way the white blood cells present in the image,
separating the agglomerates of cells and selecting the nucleus and the cytoplasm.
Further developments of the proposed method could affect the phase of separation of
adjacent leukocytes, which, despite producing robust results, require a better tool than the
current one, for both the identification of groups of leukocytes (even artifacts and strips of
dye present in the image are identified as adjacent white blood cells), and for the separation
itself, which generates incorrect results with the presence of holes to the whole leukocyte.
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BIBLIOGRAPHY
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Am-sterdam The Netherlands. 1998.
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16) G. A. Daland and T. H. Ham, A Color Atlasof Morphologic Hematology, Cambridge,
Mass.:Harvard University Press. 1967.
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17) R. S. Ledley, High-speed automatic analysis ofbiomedical pictures, Science. October
(1964) 216-223.
18) P. G. Stein, L. E. Lipkin, and H. M. Shapiro,Spectra II: general-purpose microscope
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24) J. Hilditch and D. Rutovitz, Chromosome recognition, Ann. N.Y. Acad. Sci. vol. 157
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25) PramitGhosh, DebotoshBhattacharjee, MitaNasipuri and Dipak Kumar Basu,
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29) J. W. Bacus, \An automated classi_cation of the peripheral blood leukocytes by means
of digital image processing," Ph.D. dissertation, Univ. Illinois, Chicago, 1970.
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30) [2] C. W. Barnett, \The unavoidable error in the di_erential count of the leukocytes of
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31) J. W. Butler, M. K. Butler, and A. Stroud, Automatic classi_cations of chromosomesII," in 1964 Proc. Rochester Conf. Data Acquisition and Processing in Biology and
Medicine. New York:Pergamon, 1965.
32) J. W. Butler, \Automatic analysis of bone au- toradiographs," in Pictorial Pattern
Recognition, G. C. Cheng et. al., Eds. Washington, D.C.: Thompson Book Co., pp.
75-85, 1968.
33) T. D. Caspersson, G. Lomakka, G. Svenson, and R. Saftstrom, \A versatile ultramicro
spectrograph for multiple-line and surface scanning high resolution measurements
employing auto mated data analysis," Expt. Cell Res., vol. 3, pp40-51, 1955.
34) H. J. Conn, Biological Stains, 7th ed. Baltimore, Md.: Williams and Wilkens, 1961.
35) G. A. Daland and T. H. Ham, A Color Atlas of Morphologic Hematology. Cambridge,
Mass.: Harvard University Press, 1967.
36) rationalization of scintillation scan reading," Methods Inform. Med., vol. 6, no. 1, pp.
24-27, 1967.
37) J. Hilditch and D. Rutovitz, \Chromosome recognition," Ann. N.Y. Acad. Sci., vol.
157, pp.339-364, 1969.
38) A. Hughes, A History of Cytology. New York: Aberlard-Schuman, 1959.
39) M. Ingram, P. E. Norgren, and K. Preston, Jr., Automatic di_erentiation of white
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Berkeley, Calif.: Univ Calif. Press, 1968.
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216-223, Oct.1964.
43) M. L. Mendelsohn, B. H. Mayall, J. M. S. Pre- witt, R. C. Bostrom, and W. G.
Holcomb, \Digital transformations and computer analysis of microscopic images," in
Advances in Optical and Electron Microscopy, V. E. Coslett, Ed. New York:
Academic Press, 1968, pp. 77-150.
44) N. J. Nilsson, Learning Machines. New York: McGraw-Hill, 1965.
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45) J. M. S. Prewitt and M. L. Mendelsohn, \A general approach to image analysis by
parameter extraction," in Proc. Computers in Radiology,Chicago, 1966.
46) P. G. Stein, L. E. Lipkin, and H. M. Shapiro,\Spectra II: general-purpose microscope
input for a computer," Science, vol. 166, pp. 328- 333, 1969.
47) G. L. Wied et al., \Taxonomic intracellular analytic system (TICAS) for cell
identi_cation, Acta.Cytol., vol. 12, pp. 180-204, 1968.
48) F. Winsberg et al., \Detection of radiographic abnormalities in mammograms by
means of optical scanning and computer analysis," Radiology,vol. 89, pp. 211-215,
1967.
49) I. T. Young, \Automated leukocyte recognition," Ph.D. dissertation, Massachusetts
Inst. Technol., Cambridge, 1969.
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BIODATA
1.
2.
3.
4.
Name
Regd. No
Fathers Name
Address
:
:
:
:
Phone
Email
:
:
Name
Regd. No
Fathers Name
Address
:
:
:
:
Phone
Email
:
:
Name
Regd. No
Fathers Name
Address
:
:
:
:
M.MADHAVARAO
12JE1A0431
M.VENKATESWARLU
Kunduruvaripalem(V), Muppalla (M)
Guntur (DT), A.P.
Phone
9000143614
Email
madhavmg143@gmail.com
Name
Regd. No
Fathers Name
Address
:
:
:
:
Phone
Email
:
:
V.NARENDRA BABU
12JE1A0450
V.NAGESWARA RAO
Karumanchi(V),Savalyapuram (M)
Guntur (DT), A.P.
9160022847
v50narendra@gmail.com
Department of ECE
J.MANO JYOTIRMAI
12JE1A0434
J.NAGESWARA RAO
Gurijepalli (v), Santamaguluru(M)
Prakasam (DT), A.P.
9666014683
manojyoti434@gmail.com
K.DIVYA
12JE1A0414
K.SRINIVASARAO
Boppudi(V), Chilakaluripet (M)
Guntur (DT), A.P.
9618894627
divyakatru414@gmail.com
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White Blood Cells Identification And Counting From Microscopic Blood Image

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Figure1: A Digital Image

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Figure 1.1: View Of Image As Pixels And Intensities

Figure 1.2: Color Representation Of Pixels

1.1.1 IMAGE FILE SIZES

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1.1.2IMAGE FILE FORMATS

Figure1.3: Raster And Vector Formats Of an Image

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1.1.4 VECTOR FORMATS

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1.2 IMAGE PROCESSING

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FUNDAMENTAL STEPS IN DIGITAL IMAGE PROCESSING

Figure 1.4: Fundamental Steps In Digital Image Processing

1.2.1 IMAGE ACQUISATION

Figure 1.5: Devices Used For Acquisition Of Images

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Figure 1.6: Image Of A Scanner

1.2.2 IMAGE ENHANCEMENT

Figure 1.7; Original And Enhanced Image

1.2.3 IMAGE RESTORATION

Figure 1.8: View Of Restorated Image

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1.2.4 COLOUR IMAGE PROCESSING

Figure1.9: View Of Colour Image Processing

1.2.5 WAVELETS AND MULTI RESOLUTION FACTORING

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Figure 1.10: Multi Resolution Of An Image

1.2.7 MORPHOLOGICAL PROCESSING

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Figure 1.11: Morphological Processed Image

Figure1.12: Segmentation Of An Image

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1.2.9 REPRESENTATION AND DESCRIPTION

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1.3 COMPONENTS OF AN IMAGE PROCESSING SYSTEM

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SPECIALISED IMAGE PROCESSING HARDWARE

IMAGE PROCESSING SOFTWARE

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1.4 AREA OF RESEARCH

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Figure. 1.14: Original Blood Sample Image

In human blood is very common the presence of lymphocytes, with a percentage of

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Figure.1.15: Comparison Between Different Types Of White Blood Cells: Neutrophil,

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2.2. A FAST SEGMENTATION SCHEME FOR WHITE BLOOD CELL IMAGES

2.3. THE ACUTE LYMPHOBLASTIC LEUKEMIA IMAGE DATABASE FOR

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2.5. A THRESHOLD SELECTION METHOD FROM GRAY-LEVEL HISTOGRAMS