2015 IEEE 28th International Symposium on Computer-Based Medical Systems

Unsupervised Segmentation of Leukocytes Images Using Thresholding

Neighborhood Valley-Emphasis
Leandro Alves Neves
Department of Computer Science and Statistics
Sao Paulo State University, UNESP
Sao Jose do Rio Preto, Brazil
neves.leandro@gmail.com

Thana A. A. Tosta, Andressa Finzi de Abreu,

Bruno A. N. Travencolo and Marcelo Zanchetta do Nascimento
Department of Computer Science
Federal University of Uberlandia, UFU
Uberlandia, Brazil
{tosta.thaina, andressanzi, travencolo, marcelo.zanchetta}@gmail.com

II. M ATERIALS AND M ETHODS

AbstractBlood smear image analysis is essential to correlate the amount of leukocytes in these images with malignancies
such as the leukemias. Techniques of digital image processing
can be used to aid pathologists in this analysis, leading to
appropriate treatments for the patient. This paper presents an
unsupervised segmentation method for the nuclear structures
in leukocytes. Deconvolution was used to split the Giemsa stain
components and the regions of interest were selected using a
thresholding algorithm called Neighborhood Valley-emphasis.
A postprocessing approach based on morphological operators
was applied in these detected structures. The proposed algorithm was tested on 367 images containing leukocytes and
other blood structures. A performance analysis was conducted
through the Jaccard and accuracy metrics featuring results of
89.89% and 99.57%, respectively. Such results were compared
to other published articles and this was considered the most
promising method.

A. Dataset
A total of 367 images were used for evaluation of this
system. RGB images with 640 480 pixels and 100
magnication were obtained from peripheral blood samples
stained with Giemsa. The images are characterized by different illumination and contrast conditions, and differences in
shape, spatial distribution and size of the cells. This dataset
was obtained from [3] and contain images with their nuclear
regions marked manually by an expert.
B. Proposed Algorithm
1) Deconvolution: This technique separated two components of Giemsa stained images, methylene blue and
eosin, based on optical density, which is proportional to
the concentration of each component in specic cellular
structures [4][5]. Deconvolution uses these concentrations to
quantify the individual contribution of each stain component
on R, G and B channels through orthonormal transformations. Figures 1(a), 1(b) and 1(c) exhibit an image from
the dataset and its deconvolution results using the eosin and
methylene blue components, respectively.
2) Preprocessing: After deconvolution, the images were
processed using a median lter in order to remove noise
and standardize nuclear regions, due to differences in the
intensity values of these structures. This lter was chosen
for its efciency and low processing time. Resulting images
were submitted to linear contrast stretching to enhance the
contrast and make nuclear regions better represented by
assigning darker colors to them, making them closer to a
unimodal distribution. Figures 1(d) and 1(e) illustrate the
applications of median lter and linear contrast stretching,
respectively.
3) Segmentation: The Neighborhood Valley-emphasis
method [6], used in this work, automatically determine
a threshold value that separates regions of interest and
background. This method uses the variance between classes,
valley points and information from the neighborhood of

Keywords-Segmentation; Thresholding; White Blood Cells;

Leukocytes; Nucleus; Blood Smear Images and Deconvolution.

I. I NTRODUCTION
Leukocytes, also called white blood cells, are responsible
for body defense [1]. Qualitative and quantitative characterization of these structures in blood images can indicate
the presence of infections, inammation and diseases, such
as the leukemias [2]. However, blood cells identication is
a tedious, time-consuming and subjective task [2]. Digital
image processing techniques are used in order to aid specialists in this task. Among them, the segmentation is essential
to preserve most of the useful information and suppress
irrelevant data, and its results also serve as basis in further
steps [1].
Several studies in literature propose segmentation methods
for leukocytes identication. Among them, Madhloom et
al. presented a algorithm using a combination of contrast
enhancement, arithmetical operations, minimum lter and
Otsus thresholding [2]. Those techniques were also used
by Mohamed et al. [3] but with some modications, such
as the use of morphological operation of opening and the
removal of objects with small area.
2372-9198/15 $3.00 2015 IEEE
DOI 10.1109/CBMS.2015.27

93

(a)

(f)

(b)

(g)

(c)

(h)

(d)

(i)

technique was also effective, being based on color information while disregarding spatial distribution, size and shape
of the cells. However, a limitation of this algorithm is its
unsatisfactory results with weak boundaries.

(e)

IV. C ONCLUSION
Nuclei detection of leukocytes are sufcient for diagnosis of diseases and contribute for leukocytes classication,
making quantitative analysis even more accurate. This work
presented a method for the automatic segmentation of these
structures associating median lter, linear contrast stretching, Modied Valley-emphasis, removal of small areas and
morphological operations. Experimental results proved that
the proposed algorithm achieved better results using Jaccard
and Accuracy metrics compared to [3] and [2] methods.

(j)

Figure 1.
Obtained results using the image BloodImage 00052 (a)
from the dataset by application of: (b) eosin component deconvolution,
(c) methylene blue component deconvolution, (d) median lter, (e) linear
contrast stretching, (f) histogram of the Figure (e), (g) Neighborhood
Valley-emphasis segmentation, (h) postprocessing step, (i) a mask from
the automatic segmentation and (j) manual marking by a expert.

V. ACKNOWLEDGMENTS
T.A.A.T and A.F.A thank to CAPES and B.A.N.T. thanks
to FAPEMIG (Rede RED-00011-14 and APQ-01345-13) for
nancial support.

intensity levels from the histogram to determine the threshold value. This technique was chosen because most of
the database images has unimodal histograms and some
of them has more than one valley region, as illustrated by
Figure 1(f), for which this method is indicated. The red line
represents the threshold value chosen by this technique. The
Figure 1(g) shows the result of applying this segmentation
on the image 1(e).
4) Postprocessing: In order to obtain images closer to
those marked by the expert, regions with less than 2000
pixels, dened experimentally, were removed. The morphological operations of opening and closing were applied to
open spaces between near objects and eliminate small gaps.
A disk shape was chosen for opening and a square for
closing, both with size 5 and set empirically. Figure 1(h)
displays the processing output.

R EFERENCES
[1] R. Adollah, M. Y. Mashor, N. F. Mohd Nasir, H. Rosline,
H. Mahsin and H. Adilah, 2008. Blood Cell Image Segmentation: A Review, 4th Kuala Lumpur International Conference
on Biomedical Engineering 2008. Springer, 141 144.
[2] H. T. Madhloom, S. A. Kareem, H. Arifn, A. A. Zaidan,
H. O. Alanazi and B. B. Zaidan, 2010. An Automated White
Blood Cell Nucleus Localization and Segmentation Using
Image Arithmetic and Automatic Threshold, J. Applied Sci;
10: 959 966.
[3] M. Mohamed, B. Far and A. Guaily, 2012. An Efcient
Technique for White Blood Cells Nuclei Automatic Segmentation, IEEE International Conference on Systems, Man, and
Cybernetics (SMC). IEEE, 220 225.

C. Evaluation methods

[4] A. C. Ruifrok and D. A. Johnston, 2001. Quantication of Histochemical Staining by Color Deconvolution, Analytical and
quantitative cytology and histology/the International Academy
of Cytology [and] American Society of Cytology. 23: 291
299.

Two metrics were used to evaluate the segmentation.

Jaccards Similarity Coefcient measures the similarity between two segmentations by dividing the size of its objects
intersection by the size of their union. The second is the
Accuracy and divides the amount of pixels which receive
the same classication on both segmentations, by the total
amount of pixels in the image [7].

[5] J. C. Junqueira and J. Carneiro, 2004. Histologia Basica,

chapter Celulas do Sangue, Guanabara Koogan, 10 edition.
pages 223 225.

III. R ESULTS AND D ISCUSSION

[6] J. Fan and B. Lei, 2012. A Modied Valley-Emphasis Method

for Automatic Thresholding, Pattern Recognition Letters. Elsevier, 33: 703 708.

Figure 1(i) presents the result of applying the mask

obtained by automatic segmentation on original image and
Figure 1(j) the nuclear region marked by a specialist. Using
367 images with one or more nuclear regions, the proposed
method achieved results of 89.89% and 99.57% with Jaccard
and Accuray metrics, respectively, whereas the obtained
by [3] were 73.07% and 98.15%, and by [2], 54.41%
and 95.49%. The use of deconvolution allows considerable
information gain because it preserves nuclear regions, and
eliminates other irrelevant structures. Furthermore, deconvolution reduces color variations that could be represented due
to different tissue preparation [8]. Use of the thresholding

[7] S. Ghosea, A. Olivera, R. Marta, X. Lladoa, J. C. Vilanovac,

J. Freixeneta, J. Mitraa, D. Sidibeb and F. Meriaudeaub,
2012. A Survey of Prostate Segmentation Methodologies in
Ultrasound, Magnetic Resonance and Computed Tomography
Images, Computer Methods and Programs in Biomedicine.
Elsevier, 108: 262 287.
[8] F. Dong, H. Irshad, E-Y. Oh, M. F. Lerwill, E. F. Brachtel,
et al., 2014 Computational Pathology to Discriminate Benign
from Malignant Intraductal Proliferations of the Breast, PLoS
ONE 9(12): e114885.

94