IMPLEMENTATION OF IMAGE PROCESSING FOR PUS CELLS AND RED

BLOOD CELLS DETECTION AND COUNTING IN URINE SAMPLES

A Thesis Proposal

by


JAN MASON S. SANCHEZ
MARIEL LIAN M. SANTISTEBAN
KENNETH A. SUAREZ



Submitted to the Electronics and Communication Engineering Department of the
Gokongwei College of Engineering,
De La Salle University Science and Technology Complex
In partial fulfillment of the requirements for the degree



BACHELOR OF SCIENCE IN ELECTRONICS ENGINEERING


December 2013

TABLE OF CONTENTS
Page
Table of Contents ii
List of Figures iii
List of Tables iv
List of Equations v

Chapter

1 INTRODUCTION
1.1 Background of the Study 1
1.2 Statement of the Problem 4
1.3 Significance of the Study 6
1.4 Objectives of the Study 6
1.4.1 General Objective 7
1.4.2 Specific Objectives 7
1.5 Scope and Limitations of the Study 8

2 REVIEW OF RELATED LITERATURE
2.1 Urinalysis 10
2.2 Microscopy 12
2.3 Image Processing 15

3 THEORETICAL CONSIDERATIONS
3.1 Urinary Tract Infection 19
3.1.1 Causes of Infection 19
3.1.2 Risk Factors 20
3.1.3 Prevalence 20
3.1.4 How are UTIs diagnosed? 21
3.2 Microscopic Examination of Urine Sediment 21
3.2.1 Sediment Examination 23
3.2.2 Identification of WBC and RBC 24
3.3 Microscopy 25
3.3.1 Resolution 26
3.4 Image Processing 27
ii

3.4.1 Median Filter 28
3.4.2 Threshold Segmentation and Color Reversion 29
3.4.3 Power Law Transformation 30
3.4.4 Circle Hough Transform 30
3.4.5 K-Means Clustering Algorithm 31

4 DESIGN CONSIDERSTIONS
4.1 Proposed Design 32
4.1.1 Hardware Plan 32
4.1.2 Software Plan 34
4.1.2.1 Open Computer Vision Library (OpenCV) 34
4.1.2.2 C++ - Code::Blocks 35
4.1.2.3 MySQL 36
4.2 Design Flow 37
4.2.1 Sampling and Testing Phase 38
4.2.2 Image Capturing 39
4.2.3 Image Processing 39
4.2.4 User Interface and Database 39
4.3 Program Flow 40
4.3.1 Image Processing Flow 41

5 SUMMARY OF THE PROPOSAL 42

BIBLIOGRAPHY/REFERENCES 43

APPENDICES

Appendix A: Gantt Chart 48
Appendix B: Proposed Costing 49
iii

LIST OF FIGURES
Figure No. Description Page
Figure 2.1 Schematic Diagram of lens module 12
Figure 2.2 Schematic diagram of the lens-free holographic imaging platform 13
Figure 2.3 Image performance of lens-free microscope 14
Figure 2.4 Attachable Microscope with 60x Magnification 15
Figure 2.5 Actual Image of cells captured with camera 16
Figure 2.6 Threshold Image in Binary 16
Figure 2.7 Final Window Marked RBCs and unmarked WBCs 17
Figure 2.8 K-Means clustering and color thresholding of epithelial cells 18
Figure 2.9 K-Means clustering and color thresholding pus cells 18
Figure 3.1 Red blood cells 22
Figure 3.2 Pus cell (red arrow) and RBC (blue arrow) 22
Figure 3.3 Image productions in a Microscope 26
Figure 3.4 a sample object image 28
Figure 3.5 Result of Median Filter 28
Figure 3.6 Result of Threshold Segmentation 29
Figure 3.7 Result of Color Reversion 30
Figure 4.1 Basic Concept of the Proposed Design 38










iv

LIST OF TABLES
Figure No. Description Page
Table 3.1 Element count per milliliter calculation 23
Table 3.2 RBC and WBC characteristics 25
Table 3.3 Magnification 26

















v

LIST OF EQUATIONS
Equation No. Description Page
(1) Total Magnification 25
(2) Actual Limit of Resolution 27
(3) Normalization Coefficient 29
(4) Weight Coefficient 29
(5) Gamma Transform 30
(6) Hough Transform for x 30
(7) Hough Transform for y 30
(8) Direct K-means Clustering 31
(9) Quality of Clustering 31












vi

Chapter 1
INTRODUCTION
1.1 Background of the Study
Concerning the current situation, public health is one of the main concern of the
society. Various biochemical tests are typically done for continuous monitoring or for
diagnosis of certain diseases. Although there are different body fluids which can be
examined, blood and urine are the two of the most commonly analyzed specimens due to
its simplicity in sample acquisition. Urine is one of the most easily accessible biological
samples, and it provides a treasure trove of molecules essential for clinical diagnostics (Rai,
2010). This examination is thoroughly done through one of the most prominent clinical
laboratory examination, urinalysis.
Urinalysis is a diagnostic test which can provide useful relevant information in a wide
spectrum of clinical solution (Fogazzi & Garigali, 2003). A urinalysis requires three types
of examination: direct observation, dipstick analysis and microscopic analysis (Lockwood,
2011). Macroscopic, direct observation, deals with the physical features of the urine in
terms of color and odor. Dipstick analysis uses a strip containing multiple pads arranged
linearly and each of it contains different reagents depending on the reaction being tested
which certain findings can then be determined. Microscopic analysis uses an instrument in
the form of a microscope which explores urine sediments and its contributing factor to the
occurrence of various illnesses.

Microscopic examination is an indispensable part of urinalysis; the identification of
casts, cells, crystals, and bacteria aids in the diagnosis of a variety of conditions
(Simerville, Maxted & Pahira, 2005). The presence and concentration of urine sediments
can result to various diseases such as urinary tract infection, renal diseases, bladder defects
and other illnesses that can be detected through a thorough examination which deals with
the scanning, low power and high power field lenses of the microscope. With lower power
total magnification 100 200x, distribution of elements can be assessed and rare particles
such as casts and epithelial cells can be noticed while the high power total magnification
(400x) are for corpuscular elements (Fialov & Vejraka, 2012/2013). Urine sediments
such as white blood cell (WBC), in the form of pus, and red blood cell (RBC) can be best
examine under high power field (HPF) lens. Having a cell count greater than the normal of
RBC and WBC, can be an indicator of having a urinary tract infection (UTI).
Knowing that UTI is a common and prevalent illness, it is an infection in the urinary
tract. Infections are caused by microbes including fungi, viruses, and bacteria. According
to JJustad (2010), it typically occurs when bacteria enter the urinary tract through the
urethra. The bacteria then multiplies in the bladder and can cause result in an infection.
This is most often experienced by people depending on the environment around them. The
detection of this illness is then established in the microscopic phase of urinalysis. The
bacteria that mainly causes this illness is the Escherichia coli (E. coli) and with the presence
of this kind of bacteria, pus forms thus leading to a bleeding in the urinary tract resulting
to an excretion of red blood cells.
2

Traditionally, this kind of tests are done in a clinical laboratory but with the current
evolution of technology and the availability of image processing, automated means of
examination becomes feasible. There are already several other studies which involves
testing urine samples using semi-automated analyzers with the technology of reflectance
photometer which dwells on dipstick type of analysis.
The current technologies that are available in the market utilizes reagent strips that
detect and quantify a number of analytes including bilirubin, protein, glucose and red blood
cells which uses a semi-automated machine. With the current evolution of modern
technology, a much more sophisticated way for testing is being discovered with the use of
the camera phones. As of 2013, uChek (urine analyzer) is the only available urine
application in the apple and android market which utilizes on reagent strips and image
processing.
There are already several other studies dedicated to microscopic image processing in
which it analyses blood samples using microscopic lens attached to a camera phone and an
image processing software. Most of the software programs produced by the studies that
were found by the researchers uses blob detection in Open Source Computer Vision Library
(OpenCV) which handles the detection of cells.
With the advent of camera phones, tablets and laptops, this examination can be
converted to a more convenient and portable way. Economically, the use of digital
microscopy, a clinical microscope and compact digital camera, is costly. Now, digital
3

microscopy can be reduced to a low-cost equipment yet performs the same quality and
functionality as the one stated above.
With the current trend of portable gadgets, a software program that could run among
all of those can be quite a tremendous solution to a much more accessible health care and
disease prevention for the use of anyone, may it be in secluded areas or people who are
busy enough not to be able to attend to their personal wellbeing.
As such, the researchers have come up with the concept of image processing, use it
for automation of urinalysis in terms of microscopic examination of red blood cells and
white blood cells and have a record storage system. The researchers intend to create a
convenient and portable way of testing with the use of the currently available technology
and to address the issue in terms of public health, the researchers plan on making a
prototype of a microscopic image processing software program that will analyze urine
samples using OpenCV incorporated in a C++ platform, Code::Blocks, and results will
then be stored in MySQL, database.
1.2 Statement of the Problem
According to the Philippine Health Statistics conducted by the Department of Health,
statistics show that in 2009, the number of cases involved with UTI reached 82,867 with a
rate of 91%, making the UTI seventh cause of morbidity in the Philippines (DOH, 2011).
With this statistical information, UTI is being considered as one of the most dominant case
of illness in the country. Given this situation, there is a need to address this issue and make
the most possible resolutions to its prime capability.
4

In medical practice, UTI is being detected by having the patient undergo a urinalysis
which is to be processed in a clinical laboratory. This kind of examination is mostly done
in urban areas where there exists medical facilities with the aide of clinical equipment and
professional medical personnel. People living in rural areas, most especially those who are
living in secluded places, health is always one of their primary concern. The researchers
thought that by having an accessible and convenient way to this kind of tests can help
alleviate this major concern.
With the rising need for a more convenient and efficient way in addressing issues
connected to public health, it is found by the researchers, upon writing, that there are few
studies existing that are aimed to develop new related applications that generate the use of
technology used as an early detection of this kind of illness.
Traditionally, the equipment being used to analyze UTI is a high power field
microscopic lens and to be able to further examine the sample, a high-cost camera is being
attached to it then sync it with a sophisticated software which is to be operated by a
knowledgeable individual from the medical field. Since the advent of modern technology,
this high-cost examination can somehow be reduced in a more economical means and this
is what the researchers are trying to resolve using low-cost testing instrument yet can
produce high quality and efficient results.
Existing systems that are available in the market is a portable kind which uses a semi-
automated urinalysis machine. It is a simple and compact machine that dwells on the
5

dipstick analysis of a urine sample. Urinalysis is composed of 3 phases of analysis, and the
microscopic part that is the most crucial yet the most important phase.
Recent studies show that with the emerging technology and the power of image
processing, the researchers have found an advantage on using and implementing it.
1.3 Significance of the Study
This study will address not only the issue about Urinary Tract Infection, being one of
the most dominant illness in the country, but also the cost of urine sediment microscopic
examination, mobility and most importantly accessibility of the software. In the
researchers proposed design, having the microscopic camera phone separated from the
processing software located on a computer or laptop will help diagnose errors through
image processing where the necessary parameters of the captured image will be adjusted
before the automated cell count.
The researchers proposed design will likely benefit people living in rural areas, most
especially those who are living in secluded places, who are in need of a microscopic urine
examination without having to spend much to afford and to travel a long way for an
examination in a clinical laboratory. Being able to have a portable device and software, it
can make a difference in public healthcare awareness for urinary tract infection. By being
able to detect RBCs and WBCs in microscopic level and by having a database of the
acquired result, easier diagnosis will conducted by medical professionals as results for early
prevention of UTI.

6

1.4 Objectives of the Study
In this part of the study, the objectives serves as the tool and specific targets that are
systematically planned and achieved within the given time frame.
1.4.1 General Objective
This study aims to develop a software which detects the presence and cell
count concentration of red blood cell per high power field and pus per cubic
millimeter in urine samples for early signs of urinary tract infection using image
processing.
1.4.2 Specific Objective
Specifically, this aims to:
1.4.2.1 utilize a microscopic lens attached to a mobile phone camera for
sample image acquisition
1.4.2.2 design a graphical user interface for patient data encoding, sample
image display, processed image display and count results
1.4.2.3 create a database for patient record storage using MySQL (My
Sequel, Structured Query Language)
1.4.2.4 determine the presence and identify the concentration of red blood
cell and pus using the image processing features of Open Source
Computer Vision Library (Open CV)
1.4.2.5 obtain results which are within the 20% - 30% difference margin as
compared with clinical results.
7

1.5 Scope and Limitations of the Study
There are three methods of examining a urine sample: direct observation, dipstick
analysis and microscopic analysis. The study would only use one method, which is the
microscopic analysis, for counting cells in a urine sediment. The reason for choosing the
microscopic analysis method over the other two methods will be discussed in the
theoretical framework of the study. The microscopic analysis method is a traditional way
of examining a urine sediment in a microscopic level wherein the red blood cells and white
blood cells, in the form of pus, could be detected with a high power magnification of 40x,
using the microscope.
The testing and calibration will be done by comparing the cell count of RBCs and
WBCs that can be detected through a microscope which will be done manually by a
medical technologist as to the cell count which will be done automatically by the prototype
design on a same urine sediment. The researchers will use the results that will be done in a
clinical laboratory as the source of reference values of the prototype. The testing will
require at least 30 urine samples from various volunteers. There are specific requirements
for age, gender and condition. The age of the subjects will be tested and divided into two
groups: adolescent and young adult. The gender of the subjects should be treated
independently. The condition of the male subjects are at a condition wherein the subjects
are circumcised and the condition of the female subjects are at a condition wherein they
are not in the menopausal stage and is not within their menstrual period. The reasons for
specifying these requirements will be discussed in the theoretical framework of the study.
8

The design, construction, implementation and initial testing of the device will be
conducted within the premises of De La Salle University Science and Technology
Complex, specifically the science and robotics laboratories, which will be used for
utilization of the required equipment for the prototype. Further testing and calibration will
be conducted in a clinical laboratory.
















9

Chapter 2
REVIEW OF RELATED LITERATURE
This chapter serves as the backbone for the development of the study. This presents
important notes that need to be taken into consideration.
2.1 Urinalysis
Early detection of pus can only be obtained from microscopic urinalysis as Williams
(2013) elaborates, While test strip technology is what usually comes to mind when one
thinks of urinalysis, microscopic examination is a necessary partner. Chemistry alone
cannot detect casts, crystals, cells, and bacteria, which are critical for early diagnosis of
many disease states. This limitation on strip test urinalysis, urged us to focus on the
microscopic part of urine analysis to identify the said components of urine that contribute
to urinary tract infections attributed by the number of pus cells. Cell detection is key to our
proposed microscopic urinalysis, and this can only be brought about by close examination
of the urine specimens under a microscope.
Urinalysis is divided into two categories: the reagent strip test and the microscopic
examination. Our proposal aims to focus on the microscopic part of the urinalysis, to
determine whether the urine sample is a candidate for infections based on the studies
conducted by clinical physicians. While there are studies that implement microscopy in
smart phones, there is an existing technology for reagent strip analysis, one of them is: the
StripTest app a mobile application for fully-automated analysis of bio-chemical tests.
The app uses the camera phone to capture images of the colored strips, subsequently

analyzes these images using image processing techniques and makes a classification by
comparison with the reference colors provided by the manufacturer as said by Velikova,
Lucas, Smeets, and Terwisscha van Scheltinga (2012). The same concept applies to our
proposal, although instead of a reagent strip, the researchers will obtain a magnified image
of the urine specimen and use image processing to analyze the cells present from the
sample. The strip app also uses chemical reaction from the reagent strip as its basis for
analyzing the urine sample.
Another hand-held device for a colorimetric urine reader is the Healthy-100 proposed
by Lee et al. (2011), it analyzes assays in test strips with not only the ability of semi-
quantification but also the ability to quantify concentrations of urinary glucose and protein.
The system can analyze and exchange its assays results with off-site experts for clinical
evaluation. The system has an added feature that uses a mobile phone to send the results to
an expert and interprets it. The expert may reply and submit a diagnosis based on the
results.
The researchers came across another device that is available on the market: uChek.
According to Biosense Technologies (April, 2013), the uChek system works on the
principle of reflectance photometry which analyses the intensity and color of light reflected
from the reagent areas of the urinalysis reagent strip. It uses a light emitting diode (LED)
flash of the mobile phone being used as its light source and a complementary metal oxide
semiconductor (CMOS) image sensor module. It is a urinalysis made available for smart
phones on apple and android operating systems.
11

2.2 Microscopy
A mobile device-based digital microscopy was developed for mobile devices such as
smart phones and enables new possibilities for health, education and agricultural
applications that include the study of microscopic particles such as cells, tissue, grains and
others. A simple lens module attached to the camera of a smart phone with a high resolution
can easily magnify an image such as the one proposed by Sumriddetchkajorn,
Somboonkaew, and Chanhorm (2012) composed of a molded plastic convex lens with a
focal length of 4mm around a 20mm diameter plastic holder. The setup is shown in figure
2.1. The problem is that it has a certain range of magnification that does not meet the
requirements of our thesis proposal although the concept is practically the same.







Figure 2.1 Schematic Diagram of lens module (Sumriddetchkajorn et al. 2012)
Upon further research, the researchers came across a lens free type of magnification
that uses digital holography that utilizes an incoherent light source and an optoelectronic
sensor-array to record lens-free holograms of micro-objects within a sample as
12

demonstrated by Mudanyali and Ozcan Research Group (2011).They used a lens-free on-
chip microscopy platform based on incoherent digital in-line holography achieving sub-
cellular resolution (~1.5 m) over a wide field-of-view (FOV) of ~24 mm
2
. The schematic
is shown in figure 2.2 and image samples are shown in figure 2.3.


Figure 2.2 Schematic diagram of the lens-free holographic imaging platform (Mudanyali
and Ozcan Research Group 2011)
13
















Figure 2.3 Image performance of lens-free microscope (Mudanyali and Ozcan Research
Group 2011)
Although the system meets the requirements for magnification, the researchers cant
use this type of microscope since the researchers are not familiar with its use, and the
hardware required for this system is not readily available. On the other hand, the
researchers came across an apparatus that has met the requirements of the proposal which
14

is available at a reasonable cost. The one used in applications such as DERMA/care brought
about by Karargyris A., Karargyris, O., and Pantelopoulos (2011) where the attachable
microscope for smart phones is used to detect and monitor skin cancer, is the most qualified
apparatus for our proposed mobile application. The detachable microscope apparatus used
is show in figure 2.4.







Figure 2.4 a) Microscope with 60x zoom capability designed for
the iPhone 4/4S. b) Microscope mounted on the iPhone while
in use (LED light is on) (Karargyris et al. 2011)
2.3 Image Processing
There are many algorithms that can help the researchers in finding the cells or blob
images obtained from the microscope that can help us in the detection and counting of pus
cells. A blob detection algorithm is made by Gupta (2012), which consists of resizing,
flipping, changing the color space, comparing and separation, smoothing, and rendering,
will help identify cells by using this blob detection method. Although his method proved
useful in blob detection, the problem is that its input is a live feed. The researcher can still
apply his method but they will have to adjust it to be able to utilize images. As a sample,
Gupta uses a sample image of cells both WBCs and RBCs as captured and show in Figure
15

2.5. After setting parameters, a threshold image will be produced based on the set
parameters, the binary threshold image is show in Figure 2.6. Lastly, the detection of the
cells will be seen and marked where the system only recognizes RBCs based on its initial
settings as shown in Figure 2.7.







Figure 2.5 Actual Image of cells captured with camera (Gupta 2012)







Figure 2.6 Threshold Image in Binary (Gupta 2012)
16








Figure 2.7 Final Window Marked RBCs and unmarked WBCs (Gupta 2012)

There is another detection algorithm presented by Halim, Hamid, Mohamed, Ghazali,
Arshad and Naim (2012) for counting of pus cells and epithelial cells but for sputum, where
it uses K-Means clustering and color thresholding. Halim et al. (2012) used Matlab R2010a
in order to implement the algorithm: input, image enhancement, image segmentation,
image conversion and morphological operation, image analysis, criteria selection, object
summation and output with the results of epithelial cells shown in Figure 2.8 and pus cells
in Figure 2.9.





17







Figure 2.8 (a) Resized image, (b) Color thresholding image, (c) Binary image, (d) Image
after removing noise, final image, (e) Number of epithelial cell in image.
(Halim et al. 2012)









Figure 2.9 - (a) Resized image, (b) Color thresholding image (background), (c)
Subtraction image, (d) Contrast image, (e) Cluster image, (f) Grayscale image, (g) Image
after removing noise, (h) Image that consists pus cell, (i) Number of pus cell in image, (j)
Final image (pus cell is marked with blue colour). (Halim et al. 2012)

18

Chapter 3
THEORETICAL CONSIDERATIONS
This chapter contains the different theories that are taken into consideration to be able
to implement the study that the researchers proposed. This presents formulas that are
involved in the study.
3.1 Urinary Tract Infection
Understanding the Urinary Tract Infection is a key in this study since presence of
white blood cells also known as pus cells in the urine may indicate that a person may have
an infection in the urinary tract whereas presence of red blood cells may indicate.
Bacteria are the most common cause of UTIs. Normally, bacteria that enter the
urinary tract are rapidly removed by the body before they cause symptoms. However,
sometimes bacteria overcome the bodys natural defenses and cause infection (National
Kidney and Urologic Diseases Information Clearinghouse [NKUDIC], 2011).
3.1.1 Causes of Infection
Urinary tract infections typically occur when bacteria enter the urinary tract
through the urethra. The bacteria then multiply in the bladder and can result in an
infection. The most common organism is Escherichia coli but other bacteria can also
cause infections (JJustad, 2010).
The urinary tract has several systems to prevent infection. The points where
the ureters attach to the bladder act like one-way valves to prevent urine from backing
up toward the kidneys, and urination washes microbes out of the body. In men, the

prostate gland produces secretions that slow bacterial growth. In both sexes, immune
defenses also prevent infection. But despite these safeguards, infections still occur.
Certain bacteria have a strong ability to attach themselves to the lining of the urinary
tract (NKUDIC, 2011).
3.1.2 Risk Factors
Everyone has some risk of having UTI but some of us are more prone of
getting the infection than others. The level of risk varies depending on the age, gender
and condition of a person.
Females are more prone to urinary tract infections than are males. This is
because the urethra is shorter which cuts down the distance that bacteria have to travel
to reach the bladder. As males age, they often have enlargement of the prostate gland.
This causes an obstruction to the flow of urine. In males who are not circumcised,
there are more bacteria living closer to the opening of the urethra which increases
their risk for developing an infection. After menopause, females are also more prone
to infections due to lack of estrogen (JJustad, 2010).
3.1.3 Prevalence
Knowing the degree of widespread of the Urinary Tract infection is also an
important key in this study since the percentage of population affected with this
infection is significantly large.
In the community setting, the rate of UTI accounts for 24% of all diagnosed
infections, followed by respiratory tract infection (Gusewelle, 2011).
20

UTIs are a very common type of infection particularly in women. It is
estimated in the Philippines that one woman in three will have a UTI before the age
of 24, and that half of all women will have at least one UTI during their lifetime. This
is less common in men. It is estimated that every year in the Philippines, in otherwise
healthy men, only one in every 2,000 will develop a UTI (Yoro et al., 2010).
3.1.4 How are UTIs diagnosed?
According to the National Kidney and Urologic Diseases Information
Clearinghouse (2011), to find out whether a person has a UTI, the health care provider
will ask about urinary symptoms and then test a sample of urine for the presence of
bacteria and white blood cells, which are produced by the body to fight infection.
Because bacteria can be found in the urine of healthy individuals, a UTI is diagnosed
based both on symptoms and a laboratory test.
3.2 Microscopic Examination of Urine Sediment
The presence of various cells could be found in a urine sediment including the red
blood cells and white blood cells. A fine focus adjustment on the microscope is essential
to differentiate pus cells from RBCs. The researchers have found that the microscopy
technology on a smartphone device is relevant in the biomedical field. Since dipstick
analysis using a smartphone is already being implemented, the researchers decided to go
with the microscopic analysis using a camera phone with an aid of a computer.
The microscopic examination was traditionally performed on all urine specimens.
Today, many laboratories perform a urine microscopic only if preliminary evaluation
21

indicates the need for microscopic examination. Such laboratories must have criteria
determining the specimens on which urine microscopic examinations will be performed.
The microscopic exam is often important in detecting and evaluating renal and urinary tract
disorders as well as other systemic diseases (Smith & Foster, 2003).
According to Smith and Foster (2003), there are many red and white cells in this high
power field. There are a group of five red cells that can be identified on the lower right-
hand side of the field as shown in figure 3.1. It is important to use the fine focus adjustment
on the microscope when trying to differentiate RBCs from WBCs.





Figure 3.1 Red blood cells (Smith & Foster, 2003)






Figure 3.2 Pus cell (red arrow) and RBC (blue arrow) (Smith & Foster, 2003)
22

3.2.1 Sediment Examination
According to the Cornell University, for this examination, the standard
volume of urine is centrifuged in a low speed centrifuge. The supernatant is decanted
and the urine is gently re-suspended in a standard volume (0.5 ml) of urine
supernatant. A drop of the re-suspended urine is placed on a slide, cover slipped and
examined under a light microscope using the 10x and 40x magnification objectives.
Subdued lighting is necessary to increase refractivity of the unstained urine elements.
Table 3.1 Element count per milliliter calculation
(Clinical and Laboratory Standards Institute [NCCLS], 2001)
Diameter of the high-
power field
0.35 mm
Area of the high-power
field
0.096 mm
2

Area under the cover slip 484 mm
2




484
0.096
= 5040
Measure 0.020 mL of
sediment onto the slide
1.2 mL urine
5040 high power fields
1.2 mL urine
4000 high power fields/mL
23

Table 3.1 shows an example of how to calculate elements count per milliliter.
White blood cells and red blood cells are reported semi-quantitatively as number seen
per high power field. The interpretation for WBC is less than 5 WBC/HPF is
commonly accepted as normal. Greater numbers generally indicate the presence of
an inflammatory process somewhere along the course of the urinary tract. The
interpretation for RBC is up to 5 RBC/HPF generally are acceptable for normal urine.
Increased red cells in urine is termed hematuria, which can be due to hemorrhage,
inflammation, necrosis, trauma, or neoplasia somewhere along the urinary tract
(Cornell University).
3.2.2 Identification of WBC and RBC
In regular unstained urine sediments, WBC are small cells, usually 1.5-2x
larger than a RBC, are regularly round, colorless and have a slightly grainy
appearance. Like erythrocytes, WBC may lyse in very dilute or highly alkaline urine.
The type of WBC cannot be determined from a regular urine wet preparation, but
they are usually neutrophils. The grainy nature of WBC helps to distinguish WBCs
from RBCs which are smoother. WBC are also colorless whereas RBC are slightly
red-tinged from hemoglobin. Identification of the segmented nuclear shape of a
neutrophil is helpful, but this feature is often in apparent, particularly if the specimen
is not fresh (Cornell University).
Table 3.2 shows the characteristics of the cellular elements that can be seen
in urine.
24

(1)
Table 3.2 RBC and WBC characteristics (Smith and Foster, 2003)
Cell Significance
White Blood Cells Bacterial infection.
Red Blood Cells
More than an occasional RBC can be
significant. They are often associated with
damage to the glomerular membrane of
vascular injury within the genitourinary
tract.

3.3 Microscopy
Since, the researchers have decided to use the microscopic analysis method, where a
microscope will be mechanically attached to camera phone device with the magnifying
effect will be multiplied by arranging several lenses one behind another. According to
Zeiss, a microscope magnifies in two steps: The objective produces a magnified image of
the object in the so-called intermediate image plane, and the eyepiece or ocular magnifies
the intermediate image in the same way as a magnifier (Dr. H. G. Kapitza, 1997). The total
magnification can be calculated using the formula:
Total magnification = Objective magnification Eyepiece magnification
Figure 3.3 shows the basic setup of the components and image production in a
microscope. The condenser lens is used to focus light from a source to the specimen. As it
enters the objective lens, a real image is produced where it is magnified. To multiply the
magnification, additional lenses should be placed. The final component is the ocular lens
which will also magnify the image that is seen by the eye.
25


Figure 3.3 Image production in a Microscope (Kapitza, 1997)
Table 3.3 differentiates the three types of magnification: Scanning, Low and High.
Magnification represents the objective lens. The total magnification represents equation
(1).
Table 3.3 Magnification
(http://www.biologycorner.com/worksheets/microscope_use.html#.UoYTN_lRq2O)
Magnification Ocular Lens Total Magnification
Scanning 4x 10x 40x
Low Power 10x 10x 100x
High Power 40x 10x 400x

3.3.1 Resolution
As the magnification increases, the images clarity becomes difficult to
maintain. According to Leboffe and Pierce (2011), clarity of an image is called
26

(2)
resolution. The limit of resolution (or re-solving power) is an actual measurement of
how far apart two points must be in order for the microscope to view them as being
separate. Notice that resolution improves as resolving power is made smaller.
For a specific microscope, the actual limit of resolution can be calculated using the
formula
=

where: D = minimum distance at which two points can be resolved (nm)
= wavelength of light used
NACondenser and NAObjective = numerical aperture of the lens
Numerical aperture is a measure of a lens's ability to "capture" light coming
from the specimen and use it to make the image. As with magnification, it is marked
on the lens. Using immersion oil between the specimen and the objective lens
increases its numerical aperture and in turn, makes its limit of resolution smaller. The
result is better resolution (Leboffe & Pierce, 2011).
3.4 Image Processing
The researchers will undertake image processing that aims to determine the cell
concentration of white blood cells and red blood cells. The scale of image processing of
the study will be implemented using Open Source Computer Vision Library (Open CV).
This part of the study will show the basic concepts of image processing of the object image
27

that will undergo different processes such as elimination of image noise, threshold
segmentation, color reversion and circle detection and count.
3.4.1 Median Filter





Figure 3.4 a sample object image
Median filter is a nonlinear signal processing method. Median filter was
proposed and applied in one-dimensional signal. And then it was used in two-
dimensional image signal processing. Median filter can overcome the image detail
illegibility of linear filter such as minimum square root filter, mean filter and so on,
under certain extent. It is very efficient for filtering impulse interfere and image scan
noise. Median filter is convenient to be used because of no use for image statistic
character in practical operation course (Hudai et al., 2009).





Figure 3.5 Result of Median Filter (Hudai et al., 2009)
28

(3)
(4)
3.4.2 Threshold Segmentation and Color Reversion
The basic concepts of this approach are these: Using edge measurement, the
initial threshold can be obtained. Also, the gray-scale i of different pixels in the
image contribute dissimilarly to the optimal threshold. The more | i |, the less
weight. We regard the reciprocal of | i | as a weighting factor to calculate the
thresholds of the whole binary image (Hudai et al., 2009). Normalization coefficient
and weight coefficient of each pixel can be calculated using the following formulas
= 1/|i |

=0

= /|i |
Where i = pixel value of different edge spot
m = maximum grey value
The optimal threshold will be obtained under application of the statistic
speciality, after disposing of different pixels according to weighted measures. We
consider it as the optimal likelihood threshold for processing the object image into
a binary one (Hudai et al., 2009). Fig. 3.6 illustrates the results.




Figure 3.6 Result of Threshold Segmentation (Hudai et al., 2009)
29

(6)
(7)
(5)

Figure 3.7 Result of Color Reversion (Hudai et al., 2009)
3.4.3 Power Law Transformation
Power Law Transformation or simply gamma transforms are used to help map
dark intensities into a broader assortment of intensity values (Gonzales & Woods,
2008). Gamma defines the relationship between a pixels numerical value and its
actual luminance which is an important factor to microscopic samples. This is very
important in the matter of the amount of light the image had to contain so as to apply
the cell detection algorithm. The gamma transform can be obtained by:

Where C and r are +ve constants.
3.4.4 Circle Hough Transform
The Hough transform can be used to determine the parameters of a circle
when a number of points that fall on the perimeter are known (Rhody, 2005). A circle
with radius R and center (a, b) can be described with the parametric equations
= +()
= +()
30

(8)
(9)
When the angle sweeps through the full 360 degree range the points (x, y)
trace the perimeter of a circle. If an image contains many points, some of which fall
on perimeters of circles, then the job of the search program is to nd parameter triplets
(a, b, R) to describe each circle. The fact that the parameter space is 3D makes a direct
implementation of the Hough technique more expensive in computer memory and
time (Rhody, 2005).
3.4.4 K-Means Clustering Algorithm
The researchers will apply K-means clustering algorithm for estimating the
mean of a set of K-groups. According to Alsabti et al., the k-means method has been
shown to be effective in producing good clustering results for many practical
applications. A direct algorithm of k-means method requires time proportional to the
product of number of patterns and number of clusters per iteration. The number of
clusters is assumed to be fixed in k-means clustering. Let the prototypes
(
1
, ,

) be initialized to one of the input patterns (

1
, ,

, {1, , }, {1, , }
Equation shows a high level description of the direct k-means clustering
algorithm.

is the
th
cluster whose value is a disjoint subset of input pattern. The
quality of clustering is determined by the following error function
= |

|
2

=1

31

Chapter 4
DESIGN CONSIDERATIONS
This chapter mainly contains various components of the prototype of the study that are
needed to be considered. This consists of the flowcharts, system diagrams, hardware and
software plans.
4.1 Proposed Design
The proposed prototype that is all about the detection and cell count of RBCs and
WBCs in a urine sample is then divided into two sub-categories: the hardware and software
plans. The hardware plan is composed of the physical instruments and equipment that will
be used in order to attain the goal of the study. For the most crucial part of the study, it
evolves around the software plan which comprises of the image processing software, the
environment to be used and the database for record keeping in which all of these will be
incorporated.
4.1.1 Hardware Plan
This part of the study will focus on the hardware plan of the proposed design
by the researchers. Essential components of the prototype and their functions will be
discussed in the succeeding segments.
The design will require a microscopic lens which will be mechanically attached
to the camera unit of the smartphone. This integrated device will serve as the image
capturing operation of the urine sediment. The researchers forethought of acquiring
the microscopic lens online.

The attachable microscope must have at least 40X objective magnification,
while the camera phone must be at least five (5) mega pixels.
The researchers found an affordable microscope designed specifically for the
iPhone 5/5s. This microscopic lens will convert the smartphone into a portable
microscope with a magnification of 60x 100x. It has a built-in LED light for quality
image output. It also has a on and off switch, focus control and zoom control features
that would certainly help to improve the researchers proposed design. The portable
microscope would cost around 22.99 U.S dollars with exclusion of the shipping
payment, 60 100x zoomable microscope for iPhone5/iPhone 5s.
The setup of the study will deeply focus on the software environment. The
hardware will only deliver the required object image that will undergo various image
processing techniques such as Median filter, threshold segmentation, color reversion,
and circle and blob detection.
The proposed design will also require a laptop or a computer unit. This will
serve as an image processing unit, data base, and output of the results. The
transportation of the object image from the smartphone to the laptop will use a cloud
storage like Dropbox that has a function that will automatically sync the captured
image from the smartphone online and will update the Dropbox folder from the laptop
device. An alternative method is to connect directly the smartphone to the laptop
using a universal serial bus (USB) cable.

33

4.1.2 Software Plan
In this section, it will comprise of the plan on how the software of this study is
to be implemented. The image to be processed is then acquired through the camera
phone, as stated in the hardware plan, which is then sync with the computer or laptop
which contains the software prototype. The primary software for the image
processing part of the study is OpenCV while for the graphical user interface and
environment it will then be handled by Code::Blocks using C++ language. The
database will be done using MySQL.
4.1.2.1 Open Computer Vision Library (OpenCV)
There are a lot of available image processing software available in the
market. One of the most commonly used is Matlab and OpenCV. Based on a
study conducted by Matsuka, Hudec and Benco (2012), the comparison of CPU
time consumption for image processing algorithm in Matlab and OpenCV,
results show that OpenCV is faster than Matlab in selected algorithms from 4
to 30 times and sometimes up to 100 times.
OpenCV is an open source computer vision library which contains more
than 500 optimized algorithms suited for image and video analysis. Since it is
an open source software, it is more economical and efficient to use for this kind
of implementation. The image being captured is then synced on this image
processing software.
34

For this study, the use of blob detection is needed to be able to identify
the cell structures within the captured sample. It will be used in terms of
utilizing on the detailed features of the cells. Certain formulas are entered in the
program to be able to utilize the shape detection function of the software. The
formulas to be used are to be found in the theoretical part of this study. After
detecting the blobs in the acquired sample, it will then be filtered according to
the distinct features of a red blood cell and pus. As the sample is being filtered,
cell count will come into picture. The cell count will serve as a counter for every
cell that will have the same distinct features as mentioned. Analysis of the
sample will be done based on the result of the automated cell count compared
to the normal cell count.
4.1.2.2 C++ (Code::Blocks)
An appropriate environment, where the process will reside, should be
taken into good consideration. To address the current trend and needs, a stand-
alone software is the most viable thing to offer for this study. OpenCV can be
incorporated to different environment, it can be through Java, Phython, C++,
etc.
OpenCV is highly recommended based on established studies and
known professionals. It runs smoothly in a C++ environment. The researchers
had decided to use Code::Blocks since it is a free C++ IDE, it is designed to be
extensible and fully configurable. Built around a plugin framework, it can be
35

extended with plugins which is viable in incorporating it with OpenCV and
MySQL.
The graphical user interface will serve as the bridge communication
between the user and the image processing software unit. The researchers aim
to create a user-friendly environment in which anyone could easily use it and
be able to perform the needed analysis. This will contain the basic input
information such as name, age, gender and the image of the urine sample to be
analyzed. There would also be initial restrictions due to the proper analysis of
the samples. The result would contain the pre-processing, processing and post-
processing images of the sample and the cell count of RBCs and WBCs. It will
be projected in the main window of the software prototype after the testing had
been made.
4.1.2.3 MySQL
There are so many available databases which can be used for the sake
of record keeping but the researchers had decided to use MySQL. MySQL is
being considered as the worlds most popular open source database. As being
stated, being an open source sets a huge advantage for it since it is economical
to use yet can handle the necessary work load. It is also easy to operate and it
can be interfaced with the C++ environment.
The results of the entire process is then saved and recorded in a
database system so as to be beneficial for a later use. By having a database
36

system with the prototype, it enables the person to keep a record of their results
and can be used to routinely monitor their own cell count.
Through the database, it is then needed for the researchers to create
sufficient tables with their data field and should be all linked so that it can easily
provide the relative data needed to be stored. The database will consist of: the
name of the patient, age, gender and the acquired result if the process.
4.2 Design Flow
Based on the initial study done by the researchers, they have found that to be able to
apply the proposed prototype, composed of the hardware and software, they divided the
process into two phases: capturing the samples and image processing. In the sampling
phase, they will use or obtain samples from the mid-stream specimen of the urine. The
specimen will be captured using the microscopic lens attached to a camera phone synced
to a laptop that contains the software prototype. In the image processing phase, the software
will process the image and will analyze it according to the researched evaluations for
urinary tract infections and the results gathered will be sent to a medical database for
storage and final diagnosis brought about by a physician. The algorithm of the proposed
prototype approach is shown in figure 4.1.
37

Figure 4.1 Basic Concept of the Proposed Design
4.2.1 Sampling and Testing Phase
In the sampling phase, which consists of the laboratory test procedure dealing
with the image acquisition of the samples, urine samples coming from volunteers
within the age range of 13 to 19 (adolescence) and 20 to 40 (young adults) will be
tested accordingly using at least 30 samples and will be analyzed if the number of
pus and red blood cells in these samples are enough to be considered critical for the
standard cell count. A MSU (mid-stream specimen of urine) standard will be used as
samples. MSU is preferred to reduce the possible contamination of the sample from
debris in the urethra, which may give rise to inaccurate results as mentioned by
Stegall (2007).
The testing phase would be divided into 2 categories: initial testing and
prototype testing. The initial testing would consist of 15 samples, having a variety of
good and bad samples. This will be used to be the basis of the image processing
system of the prototype. The prototype testing would serve as the prototypes test of
ability, effectivity and efficiency which is then conducted with at least 30 samples.
Capture an
image of the
urine sediment
with a x40 total
magnification
Image
processing
using Circle
Hough
Transform
Display cell
count,
processed
image and save
to data base
38

The researchers will obtain the samples from a clinical laboratory with
approval of the medical facility. The samples are in need of careful handling so the
help of a trained physician in urinalysis will be present in obtaining the samples that
the group needs.
4.2.2 Image Capturing
After obtaining the samples for examination, the researchers will use the
equipment stated under the hardware plan. The microscope is attached to the camera
of the casing of the phone to prevent damage on the surface of the device.
4.2.3 Image Processing
Upon obtaining the image from the camera phone, the image will now
undergo a series of image processing, in order to analyze what are the shape and size
of the following sediments that are shown in the image. The researchers intend to use
algorithms that are within Open CV such as blob detection, cell counting, summation,
thresholding and set parameters. Since RBCs and WBCs are similar to circular in
shape, image processing using circle Hough Transform will be used to identify which
parts of the image are considered to be cells.
4.2.4 User Interface and Database
After the image processing step, the researchers will have made a graphical
user interface (GUI) that will help identify the input, data and results properly. Since
OpenCV is versatile with most know GUI associated programming languages like
Java, C++, Phyton, etc., then the proposed user interface will be made using C++ as
39

prescribed in the software plan. The interface should contain the requirements for
necessary inputs such as the name, age, gender and most importantly the image of
the urine sample. Once the data is collected, it will undergo the image processing
phase and will output results as well as save the information in a database based on
MySQL.
40

4.3 Program Flow


















4.3.1 Image Processing Flow




















41

Chapter 5
SUMMARY OF THE PROPOSAL
The proposed thesis has provided the necessary information that can help in making
the software feasible. Certain scopes and limitations have been pointed out such that there
is room for improvement on the study that was not met by the researchers. The methods
for implementation were brought about by the theories that relate to the whole process for
the automation of the microscopic part of urinalysis. The quality and proposed standard
was considered in the basis of the results, which was determined by the most recent studies
related to the know standards for determining the risk of having infections brought about
by urinalysis.












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47

APPENDICES
Appendix A: Gantt Chart



















48

Appendix B: Proposed Costing
EQUIPMENT
(MODEL)
QUANTITY PRICE
UF003124(Microscope
Lens)
1
$22.99
= 1003.28
*Lenovo Ideapad
G480(Laptop)
1 14,995.00
*Apple iPhone
5(Smartphone)
1
$664.99
= 29,020.16
*Open CV 1 Free (Open Source)
Visual Studio
Professional
1 75,825.91
My SQL 1 Free (Open Source)
Clinical equipment - Sponsored
Transportation - 10,000.00
Supplies - 5,000.00
*Already available Total: 135,844.35

Price Reference
http://www.usbfever.com/index_eproduct_view.php?products_id=2878
http://www.pccorner.com.ph/products.do?action=showproductdetail&typeid=23&categor
yid=56&productid=6111
http://www.amazon.com/Apple-iPhone-16GB-White-Unlocked/dp/B0097CZJEO
http://www.visualstudio.com/products/how-to-buy-vs
49