SELECTION THE BEST FEATURES FOR LEUKOCYTES

CLASSIFICATION IN BLOOD SMEAR MICROSCOPIC IMAGES

Omid Sarrafzadeh*a, Hossein Rabbania, Ardeshir Talebib, Hossein Yousefi-Banaema

a
Dept. of Biomedical Engineering, Faculty of Advanced Medical Technology, Isfahan University of
Medical Sciences, Isfahan, Iran; bDept. of Pathology, School of Medicine, Isfahan University of
Medical Sciences, Isfahan, Iran

ABSTRACT

Automatic differential counting of leukocytes provides invaluable information to pathologist for diagnosis and treatment
of many diseases. The main objective of this paper is to detect leukocytes from a blood smear microscopic image and
classify them into their types: Neutrophil, Eosinophil, Basophil, Lymphocyte and Monocyte using features that
pathologists consider to differentiate leukocytes. Features contain color, geometric and texture features. Colors of
nucleus and cytoplasm vary among the leukocytes. Lymphocytes have single, large, round or oval and Monocytes have
singular convoluted shape nucleus. Nucleus of Eosinophils is divided into 2 segments and nucleus of Neutrophils into 2
to 5 segments. Lymphocytes often have no granules, Monocytes have tiny granules, Neutrophils have fine granules and
Eosinophils have large granules in cytoplasm. Six color features is extracted from both nucleus and cytoplasm, 6
geometric features only from nucleus and 6 statistical features and 7 moment invariants features only from cytoplasm of
leukocytes. These features are fed to support vector machine (SVM) classifiers with one to one architecture. The results
obtained by applying the proposed method on blood smear microscopic image of 10 patients including 149 white blood
cells (WBCs) indicate that correct rate for all classifiers are above 93% which is in a higher level in comparison with
previous literatures.
Keywords: Blood smear microscopic image, feature extraction, fuzzy C-means clustering, leukocytes (WBCs)
segmentation and counting, SVM classifier.

1. INTRODUCTION
Blood consists of three types of cells and cell fragments floating in a liquid called plasma. These cellular components
are: Red Blood Cells ("erythrocytes", "RBCs"), White Blood Cells ("leukocytes", "WBCs") and Platelets. WBCs play a
significant role in the diagnosis of different diseases such as leukemia and different types of infections [1], so extracting
information from them is valuable for hematologists. WBCs composition also reveals important diagnostic information
about patients. Substituting automatically detecting and counting of WBCs for manually locating and counting different
classes of WBCs is an important topic in the domain of cancer diagnosis [2]. Microscopic differential WBC count is still
performed by hematologists, being indispensable in diagnostics with malignance suspicious. This as a reference method
is slow and subjective and its reproducibility is poor, however its value for blood samples containing abnormal cells
remains indisputable. Therefore, automation of this task is very helpful for improving the hematological procedure and
accelerating diagnosis of many diseases [3]. WBCs contain nucleus and cytoplasm and there are five types of leukocytes
(WBCs) found in the blood: Neutrophils, Basophils, Eosinophils, Lymphocytes, and Monocytes. Each cell type has a
specific role to play in our body's immune system [4]. The texture, color, size and morphology of nucleus and cytoplasm
make differences among WBCs. The segmentation step is very crucial because the accuracy of the subsequent feature
extraction and classification depends on the correct segmentation of WBCs. It is also a difficult and challenging problem
due to the complex nature of the cells and uncertainty in the microscopic images. Therefore, this step is the most
important challenge in many works in the literatures and improvement of cell segmentation has been the most common
effort in many researches. Many blood smear image segmentation methods have been proposed in the area of general
segmentation of WBCs which are generally based on edge and border detection, region growing, filtering, mathematical
morphology, and watershed clustering [5-14]. Despite many beneficial explorations have been carried out in WBCs
segmentations, majority of them have some defects such as complexity of arithmetic, difficulty to ensure parameters and
 (a) (b) (c (d) (e)
Figure 1. Types of Leukocytes (WBCs) (a) Neutrophil, (b) Eosinophil, (c) Basophil, (d) Monocyte and (e) Lymphocyte.
so on. In this paper, the boundary of WBCs in the images is manually determined to decrease the effects of segmentation
errors, but the nucleus and cytoplasm of leukocytes are separated automatically by a fuzzy C-means clustering method.
Also, several researchers have previously proposed features to differentiate leukocytes [15-20]. Although many useful
researches have been done for classification of WBCs, many of them have some defects. Some methods extracted
features only from nucleus which lead to have less correct rate. In some works features are extracted from gray-level
images which do not use rich information that color images have. In some researches features are extracted from the
entire cell (not specific features from nucleus or cytoplasm) which usually causes more error in classification. Many
methods used singly geometric features or texture features. In this paper, first nucleus and cytoplasm are segmented
using fuzzy C-means (FCM) clustering and morphological operations in blood smear microscopic images. Then, proper
features are extracted from nucleus, cytoplasm and the entire cell. Finally, leukocytes are classified using proper features
by SVM classifiers. The outline of the article is as follows. Section two deals with methodology and presents the
proposed algorithm. Results and discussion are presented in section three.

2. METHODOLOGY
First, we briefly introduce different types of WBCs and how to differentiate them in a blood smear microscopic image.
There are five different types of Leukocytes (WBCs). Leukocytes could generally be divided into two groups: 1)
Granulocytes which contain granules (inclusions in their cytoplasm) and usually have lobulated or segmented nucleus.
Neutrophils, Basophils and Eosinophils are in this group; 2) Agranulocytes (Mononuclear) which do not contain granules
and do not have lobulated or segmented nucleus (Mononuclear refers to having one nucleus). Monocytes and
Lymphocytes are in this group. Neutrophils account for the highest amount of WBCs (about 60%). Neutrophils’ nucleus
is divided into 2 to 5 segments and stains dark purple (multi-lobed nucleus). Cytoplasm is pale pink to tan with fine pink-
purple granules and have 12-16 micrometers in diameter. Eosinophils account for about 3% of WBCs in the blood.
Eosinophils’ nucleus is blue and divided into 2 segments. Cytoplasm is full of pale pink to tan with large orange and red
granules and have 14-16 micrometers in diameter. Basophils have lowest number of WBCs in blood (about 1%).
Basophils’ nucleus has 2 lobes that stains purple and is difficult to see and their cytoplasm is pale pink –tan but contains
large purple/blue-black granules obscure nucleus and have 14-16 micrometers in diameter. Monocytes comprise about
6% of WBCs in the blood. Monocytes have singular nucleus (convoluted shape); kidney shaped, bean shaped, or
horseshoe shaped with a deep indentation and stains a blue-gray color with “ground glass” cytoplasm with tiny granules
and Vacuoles (cavity filled with fluid in the cytoplasm) are sometimes presented. Monocytes are the largest normal
blood cells and have 14-20 micrometers in diameter. Lymphocytes are second most common WBCs in blood (about

TABLE 1. Leukocytes (WBCs) specification

~% in Size
WBCs Nucleus Cytoplasm
blood (μm)
Divided into 2 to 5 segments and stains dark
Neutrophils 60% Pale pink to tan with fine pink-purple granules. 12-16
purple (multi-lobed nucleus).
Full of pale pink to tan with large orange and
Eosinophils 3% Blue and is divided into 2 segments. 14-16
red granules.
Have 2 lobes that stains purple and is Pale pink–tan but contains large purple/blue-
Basophils 1% 14-16
difficult to see. black granules obscure nucleus.
Have singular nucleus (convoluted shape); Stains a blue-gray color with “ground glass”
Monocytes 6% kidney shaped, bean shaped, or horseshoe cytoplasm with tiny granules, Vacuoles are 14-20
shaped with a deep indentation. sometimes present.
Have large, round or oval, dark staining Little to no cytoplasm with pale blue in color.
Lymphocytes 30% 8-15
nucleus. Occasional purple-reddish granules.
30%). Lymphocytes have large, round or oval, dark staining nucleus and little to no cytoplasm with pale blue in color.
Lymphocytes occasionally have purple-reddish granules and have 8-15 micrometers in diameter. Table 1 contains brief
specification of WBCs and figure 1 shows different types of leukocytes. Blood smear of 10 patients have been used in
this work. Digital microscopic images of WBCs are captured with Canon V1 camera mounted on Canon optical
microscope. The data are stored in jpg format with 3872×2592 pixels and 300 dpi resolution. The images are in sRGB
color space. Border of WBCs is manually determined to overcome the error of segmentation, but nucleus and cytoplasm
are separated using fuzzy C-means and morphological operations which is presented in the following.
2.1 Nucleus segmentation
The algorithm for nucleus segmentation is briefly illustrated in figure 2. The input microscopic image stores in three
Red, Green and Blue (RGB) channels. The median filter is applied on each R, G and B band separately to reduce noise
and also not to lose edges. Since the red, green and blue components are highly correlated, it is difficult to execute some
image processing algorithms. So, the image is converted from RGB color space to L*a*b* color space. The L*a*b*
color space (also known as CIE L*a*b* or CIELAB) is colorimetric, perceptually uniform and device independent. The
L*a*b* color space is an excellent decoupler of intensity (represented by lightness L*) and color (represented by a* for
red minus green and b* for green minus blue) [21]. The sRGB and L*a*b* images of WBCs with their subspaces are
shown in figure 3. The colors in a* and b* subspaces are classified using fuzzy C-means clustering to cluster the cells
into two clusters. Since the nucleus is darker than cytoplasm, the cluster with minimum mean of 3 R, G and B colors is
consider as nucleus cluster and the other one as cytoplasm cluster. Some morphological operations are performed on the
nucleus cluster to eliminate probable false segments. First, the morphological open operation (erosion followed by
dilation) is done to smoothes the contour of nuclei, breaks narrow isthmuses and eliminate thin protrusions. For opening
operation, a flat disk-shaped structuring element with radius 2
is used. After opening, the morphological hole filling
operation is applied to fill inside the segmented regions. Input sRGB Image
Finally, the nucleus segmented part subtract from the entire
cell to obtain cytoplasm part. Figure 4 shows the result of
applying the algorithm on some images in our data base. Apply median filter to R,G,B bands
2.2 Feature extraction
Features that are extracted categorize in color features, Convert to L*a*b* color space
geometrical features and texture features which are introduced
each one in the following. It should be mentioned that we had
few basophils in our data set and then we disregard classifying Select a* & b* components as
basophils. features
2.2.1 Color features
As can be seen in table 1, types of WBCs have less color
Apply Fuzzy C-means
difference in nucleus but more in cytoplasm, so a measure of clustering
color from both nucleus and cytoplasm could be valuable.
RGB color space is not proper for extracting color features
because the red, green and blue components are highly
correlated and also is not well suited for describing colors in Apply morphological
terms that are practical for human interpretation. HIS (Hue, operations on nucleus cluster
Saturation, Intensity) color space corresponds closely with the
way human describe and interpret color. The HIS model also
has the advantage that it decouples the color and gray-scale Subtract nucleus and extract
information in an image [21]. From table 1 it is clear that color cytoplasm
of nucleus in WBCs doesn’t vary so much and usually contain
single chromatic colors, but color of cytoplasm in WBCs vary
especially in agranulocytes due to the existence of granules. So Retrieve the nucleus and
the median of hue and saturation of both nucleus and cytoplasm segments from
cytoplasm and standard derivation of hue and saturation of just original RGB image
cytoplasm are considered as color features. So, 6 color features
are considered. Figure 2. Steps to segment nucleuses
 Figure 3. From top to bottom: Neutrophil, Eosinophil, Lymphocyte and Monocyte. From left to
right: sRGB, Red band, Green band, Blue band, L*a*b*, L* band, a* band and b* band.
2.2.2 Geometric features
From specification of leukocytes in table 1, there are noticeable differences for nucleus geometry of leukocytes, so
geometric features should be extracted from nucleus not cytoplasm and are considered as follow. The number of
segments of nucleus, compactness, eccentricity and solidity of nucleus which are defined as follow (for nucleus with
more than one segment, these features are calculated for the biggest segment). The normalized shape compactness
measure to be unity for a circle is given by (1):

P2
C  (1)
4 A
where C is the value of shape compactness, A is the shape area and P is the shape perimeter [22]. Eccentricity is used as a
scalar that specifies the eccentricity of the ellipse that has the same second-moments as the region. In this definition,
eccentricity is the ratio of the distance between the foci of the ellipse and its major axis length. Solidity is a scalar

Figure 4. From top to bottom: Neutrophil, Eosinophil, Lymphocyte and Monocyte. From left to
right one after the other: WBC and segmentation of nucleus and cytoplasm.
specifying the proportion of the pixels in the convex hull that are also in the region and is computed by equation (2).

A
S  (2)
CA
where S is the value of solidity, A is the shape area and CA (Convex Area) is the area of convex image. As we see from
table 1, the size of WBCs is a crucial parameter, so area of cell and nucleus to cytoplasm area ratio are considered as
further geometric features. Thus 6 geometric features are extracted just from nucleus (except for area of cell and nucleus
to cytoplasm area ratio which used information of both nucleus and cytoplasm). It should be mentioned that the main
geometric features (the number of segments of nucleus, compactness, eccentricity and solidity) are extract just from
nucleus.
2.2.3 Texture features
From table 1 we see that cytoplasm’s texture varies among the cytoplasm of WBCs, so texture features are extracted only
from cytoplasm of leukocytes as follows. Statistical measures of texture such as mean (a measure of average intensity),
standard derivation (a measure of average contrast), smoothness, third moment (the skewness of a histogram), uniformity
and entropy (measure of randomness) [21] are computed. Therefore 6 statistical features are extracted. The expressions
for the nth moment about the mean and other statistical measures are defined in equations (3) through (8):
L 1
n    z i  m  p  z i 
n
(3)
i 0

Standard derivation :   2  z  (4)

Smoothness : R  1 1 1   2   (5)

L 1
3    z i  m  p  z i 
3
Skewness : (6)
i 0

L 1
Uniformity : U   p 2 z i  (7)
i 0

L 1
Entropy : e   p  z i  log 2 p  z i  (8)
i 0

Also the moment invariants are computed as follow: The 2D moment of order (p+q) of a digital image f(x,y) is defined
by equation (9).

m pq   x p y q f  x , y  for p,q = 0,1,2,… (9)

x y

where the summations are over the values of the spatial coordinates x and y spanning the image. The corresponding
central moment is defined by equation (10).

 p ,q    x  x  y  y  f x , y 
p q
(10)
x y

m10 m
where x  and y  01
m 00 m 00
The normalized central moment of order (p+q) is defined in equation (11).
  pq
 pq  
for p,q = 0,1,2,… (11)
00
p q
where  1 for p+q = 2,3,… .
2
A set of 2-D moment invariants that are insensitive to translation, scale change, mirroring, and rotation can be derived
from the equations (12) through (18):

1  20  02 (12)

2  20 02   4112

2
(13)

3  30  312    321 03 

2 2
(14)

4  30  12   21  03 

2 2
(15)

5  30  312 30  12  30  12   3 21  03   

2 2

 
(16)
 321 03 21  03  3 30  12   21  03  
2 2

6  20 02  30  12   21  03    411 30  12 21  03 
2 2
(17)
 

7   321  03 30  12  30  12   3 21  03   

2 2

 
(18)
 312 30 21  03  3 30  12   21  03  
2 2

So 7 moment invariants   [1 ,...,7 ] features are extracted. Finally, 25 features are used in order to classify
leukocytes into four groups: Neutrophil, Eosinophil,
Lymphocyte and Monocyte.
2.3 Classification
Figure 5. Selected categories for WBCs
The main task of the classification part is to classify the
WBCs into the category they belong to. Four categories of
WBCs were selected for recognition in this work (see Neutrophil
SVM1
figure 5). Four Support Vector Machine (SVM)-based
classifiers with one to one architecture are used for
classification as shown in figure 6. In this architecture, new
type of WBCs (Basophil) needs to be trained individually SVM2 Eosinophil
All data

without any effect on prior classifiers. SVM classifiers are

independent, which means each one is trained individually
and there are only two possible outputs for each classifier.
SVM3 Lymphocyte
For example, SVM1 is learned to detect Neutrophil and
reject other WBCs. All data are fed to each SVM
separately. For cross-validation of each classifier,
randomly half of the data is used for training and the SVM4 Monocyte
remaining is kept for testing. A linear kernel function is
used to map the training data into kernel space and a
Sequential Minimal Optimization (SMO) method is used to Figure 6. Classification based on one to one architecture.
 TABLE 2. Results of classification for the proposed features

No. of Correct
WBCs Sensitivity Specificity
cells rate
Neutrophils 38 % 98.5 % 98.6 % 98.2
Eosinophils 42 % 99.9 % 99.9 % 99.9
Monocytes 36 % 93.7 % 94.6 % 90.9
Lymphocytes 33 % 98.8 % 99.9 % 94.8
Total 149 % 97.73 % 98.25 % 95.95

find the separating hyperplane. Correct-rate, sensitivity and specificity are used to evaluate performance of classifiers.

3. RESULTS AND DISCUSSION

The total numbers of images which are used in this research are mentioned in Table 2. Twenty five features are extracted
from each WBC in order to classify cells into four groups. Each classifier is trained for 100 times and in each training
procedure, randomly half of the data is considered as training set and the classifier is evaluated by residual half of the
data as testing set. The results of evaluation are presented by three parameters. Correct rate (Correctly Classified
Samples / Classified Samples), Sensitivity (Correctly Classified Positive Samples / True Positive Samples) and
Specificity (Correctly Classified Negative Samples / True Negative Samples) for each classifier are shown in table 2.
Our work is compared with two recent similar works. Features introduced in [20] are considered and we name them
Veluchamy features. Veluchamy features are 27: Geometrical features (Area, Perimeter, Circularity, Form-factor, K (No
of pixels)); First order statistical features (Mean, Dispersion, Variance, Average Energy, Skewness, Kurtosis, Median,
Mode); Second order statistical features (Energy, Inertia, Entropy, Homogeneity, Max probability, Inverse difference,
Correlation) and algebraic moment invariants ( [1 ,..., 7 ] ). Our algorithm has been implemented with no changes
except for feautures. We have extracted Veluchamy features from the entire cell as Veluchamy et al did. The results of
classification for Veluchamy features are shown in table 3. Other features introduced in [13] are considered for
comparison and is named Mohapatra features. Mohapatra features are: Fractal dimension; Contour signature; Shape
features (Area, Compactness, Solidity, Eccentricity, Elongation, Form-factor); Color features (the mean color values in
RGB and HSV color spaces); Texture features (Homogeneity, Energy, Correlation, Entropy). We have extracted
Mohapatra features from the nucleus as Mohapatra et al did. The results of classification for Mohapatra features are
shown in table 4. By comparing table 2 through 4, it is seen that proposed features yield better performance than two
recent works. In this paper we introduced appropriate features based on those features that pathologists consider to
differentiate leukocytes. These appropriate features are: 6 color features from both nucleus and cytoplasm (the median of
hue and saturation of both nucleus and cytoplasm and standard derivation of hue and saturation of cytoplasm); 6
geometric features (the number of segments of nucleus, compactness, eccentricity and solidity) from only nucleus
(except nucleus to cytoplasm area ratio and area of the entire cell); 6 statistical features (average intensity, average
contrast, smoothness, skewness, uniformity and entropy) from only cytoplasm; 7 moment invariants features from only
cytoplasm of each WBC. It should be mentioned that in this paper, the features are wisely extracted from both nucleus
and cytoplasm, not only from nucleus or cytoplasm. As segmentation is a crucial step in automatic leukocytes detection
systems, accurate segmentation of both nucleus and cytoplasm is needed for using our proposed features. With this point
of view, our results show that proper features as pathologists consider could give better performance and it is seen from
table 2 that correct rate for all leukocytes are above 93% which is a high and acceptable rate.
TABLE 3. Results of classification for Veluchamy features TABLE 4. Results of classification for Mohapatra features

No. of Correct No. of Correct

WBCs Sensitivity Specificity WBCs Sensitivity Specificity
cells rate cells rate
Neutrophils 38 % 97.2 % 97.9 % 95.1 Neutrophils 38 % 90.9 % 91.7 % 88.7
Eosinophils 42 % 97.8 % 85.4 % 94.1 Eosinophils 42 % 82.5 % 79.2 % 90.9
Monocytes 36 % 87.3 % 86.6 % 89.4 Monocytes 36 % 86.2 % 85.9 % 87.1
Lymphocytes 33 % 93.8 % 96.2 % 85.3 Lymphocytes 33 % 93.9 % 95.3 % 89.2
Total 149 % 94.03 % 91.53 % 90.98 Total 149 % 88.38 % 88.03 % 88.98
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