Determines the Number of Pancreas Beta Cells with IHC

i

DETERMINES THE NUMBER OF PANCREAS BETA CELLS WITH

IHC (IMMUNOHISTOCHEMISTRY) METHOD USING IMAGE

PROCESSING AND CALCULATING METHOD OF MATLAB

Oleh:

YOSPINA RERU

NIM : 642011801

TUGAS AKHIR

Diajukan kepada Program Studi Fisika, Fakultas Sains dan Matematika

guna memenuhi sebagian dari persyaratan untuk mencapai gelar Sarjana Sains

Program Studi Fisika

FAKULTAS SAINS DAN MATEMATIKA

UNIVERSITAS KRISTEN SATYA WACANA

SALATIGA

2016

Proceedings of International Symposium on BNCT

The Application of Nuclear Technology to Support National Sustainable Development:

Health, Agriculture, Energy, Industry and Environment October 26–28, 2015, Satya Wacana Christian University, Indonesia

Original paper available at http://... pp. …–…

Determines the number of pancreas beta cells with IHC (Immunohitochemistry) method using image processing and calculating

method of matlab

Yospina Reru, Jodelin Muninggar, Suryasatrya Trihandaru.

Departement of Physics, Satya Wacana Christian University, Salatiga 50711, Indonesia

Email : [email protected]

Abstract

Cell nucleus is located in the cytoplasm. The cell nucleus of pancreatic beta cells

located in the islets of Langerhans. The pancreas consists of exocrine and endocrine

glands. Endocrine function produces a rich part of the insulin, glucagon, and

pancreatic polypeptide. Insulin is a hormone that work to increase cellular energy

reserve which will affect the working system of the pancreas. Through the image of the

IHC method (Immunohystochemistry), can determine the amount of the cell nucleus in

the cytoplasm. The purpose of this project is to determine the number of pancreatic beta

cell nucleus contained in the cytoplasm using matlab. The calculation method of the cell

nucleus consists of 4 parts : determine color boxplot and operating boxplot RGB color

to black and white, imfill and noise reduction of image and the last step is labeling and

counting cell. Result of calculating the number of cell nucleus manually is 73, while the

calculation using matlab is 64 cell nucleus. It shows the results of less than 10%, which

means that this method could be used in determining the amount of the cell nucleus with

some improvement. However calculation using this method still has the blind spot that

each cell nucleus that is very close to the nucleus of cells that would otherwise be

counted as one nucleus, the cell nucleus which is close to the boundary would not be

counted, and this calculation is also affected by noise in the picture.

Keywords : Cell nucleus, Cytoplasm, Pancreas, IHC, Matlab.

Introduction

Cell nucleus is the main part of a cell which act to response to environmental

stimulation. Cell nucleus controls the entire cell activities and located in the cytoplasm,

in this case is the pancreatic beta cells which is located in islands of Langerhans.

Pancreas consists of exocrine and endocrine glands. Region of the endocrine has

function to produce insulin, glucagon, and pancreas polypeptide which are closely

related to the energy regulation in the body (Junqueira and Carneiro, 1992).

The pancreas’s endocrine gland composed of the Langerhans islands which is a

cluster that spread along the pancreas exocrine glands. Endocrine unit is referred to as

the island of Langerhans has 4 kind of cell i.e. alpha cells, beta cells, delta cells, and the

p. … Yospina Reru, Jodelin Muninggar, Suryasatrya Trihandaru.

cells on the pancreas polypeptide (Seungbum et al., 2007). The beta cells of the

pancreas on animals and humans are responsible for producing insulin. Insulin is

released into the blood by pancreatic beta cells vesicle in response to increase blood

sugar levels. Amount of beta cells can be an indicator for the levels of insulin, so that

when beta cells are detected more show insulin production will be more. If there is a

damage to the beta cells, it will effect to insulin levels. Damage of pancreatic beta cells

causes the body can not produce insulin, causing increase of blood glucose levels

(occurs state of hyperglycemic). The condition of hyperglycemic can result in the

formation of reactive oxygen species (ROS). Excessive ROS can cause oxidative stress

and may effect damage of pancreatic beta cells (Robertson et al., 2003).

Insulin has an important role in glucose metabolism. Insulin would help out

glucose entry into cells to do metabolism. Insulin is a hormone that function to increase

the cellular energy reserves that will affect the work system of pancreas (Erwin et al.,

2013). Beta cells damage occurs in diabetes mellitus. Thus, the amount of insulin will

be reduced. (Groop, 1999, 2001; De Fronzo, 1983; Masharani and Karam, 2001).

Through the image of the IHC (Imunohistochemistry) method, we can determine

the total of cells nucleus in the cytoplasm. However image from IHC visible irregular

cell shape and cell membranes that limit it not clear. It is difficult to determine the

boundary of the cell and calculate cell nucleus (Pezoa et al., 2015).

In determining clinical disease using the IHC method, required marker protein in

the cell nucleus, cytoplasm or membrane. The response expression of the cell determine

the result of this response will identify the presence of protein. Cell response against the

IHC method will be indicated by the expression of the color. Protein detection method

using marker (biomarker), also called IHC techniques. The underlying principle of this

technique is the staining of biopsy sample/tissue with specific antibodies for molecular

markers. IHC techniques is a method of staining on the tissue with the visualization of

antibody-antigen reaction using a secondary antibody conjugated to an enzyme, such as

peroxidase enzyme that catalyzes a reaction produces a brown color (Varghese et al.,

2014). Method of staining can show 4 types of beta cells on the island of Langerhans

cells i.e. alpha cells, beta cells, delta cells, and PP (pancreas polypeptide) (Ganong,

1995; Paulsen, 2000).

To help clarify the boundaries between cells tissues, image processing is needed.

Image processing allows to identify and obtain a clearer and accurate visualization of

Determines the number of pancreas beta cells with IHC (Immunohitochemistry) method

using image processing and calculating method of matlab. p. …

the tissue or cell (Ermatita et al. 2010). MATLAB was used in this study.

This project report to help determination the boundary between cells that makes

it easy to determine the amount of pancreatic beta cells nucleus on IHC slides using

MATLAB.

Materials and methods

Step 1. Color Boxplot Boxplot used to see the distribution of the data which can compare a lot of data

(more than 1 data). Usually to change the format of RGB to grayscale to see the

intensity difference, carried out the operation:

(1)

with:

(2)

(3)

Bloxplot is basis of the calculation with matrix and vector, if the data in the

matrix then there is one box per column and if the data in the vector, the data is only one

box. Bloxplot function to find a differentiator of color. In this section are differentiated

is gray, blue, and brown.

This section, format of two colors (black and white) can be made by selecting

the limit value of the intensity of use thresholding. For this case, it has always sought

pancreatic cells are in the middle of the image of the brown, so that the process of

conversion from RGB to grayscale is not done but required certain limits that should be

seen by a matrix R, G, and B separately. It is necessary for the calculation of the color

of each matrix. This can be done by using a boxplot. Boxplot will be obtained from the

limit value for conversion RGB into a two color.

Step 2. Imfill Color

This step including to the region where the filling is based on a number of

dilation, complementation and intersection. Toolbox provided is imfill. BW2 = imfill

(x) is an operation that is appropriate in this case because it displays the image on the

screen and lets us determine the area to fill with selecting points interactively using the

mouse. The area chosen is then replaced with a filling background black color so as to

distinguish between the nucleus and the cytoplasm more clearly.

Step 3. Noise Reduction This section to remove or reduce noise in the image of step before. Matlab used

black white area open (bwareaopen) which will remove all components connected to the

pixels that are less than the binary image, producing a binary image of the other.

Step 4. Labeling and Calculate cell nucleus


Last step of this project is providing labeling and count the number in each cell

nucleus using regionprops in matlab. To determine the number of objects contained in

the image detected, utilizing regionprops function by looping the number of objects that

have been known.

Results and Discussion

IHC methods of insulin antibody used to detect the presence of insulin in the

pancreatic beta cell cytoplasm. Dark color (brown) in the cytoplasm appears on beta

cells showed the presence of insulin (Ridwan et al., 2012) (figure 1). The more a lot of

beta cell cytoplasm brown and calculated the cell nucleus, it can be estimated insulin

levels in these cells. Number of beta cell nucleus can be an indicator for the level of

insulin produced.

Figure 1. One of original images of the pancreatic beta IHC slides.

Process of digital image processing is obtained boxplot different RGB colors.

Significant differences were found in section B (figure 2). This means that the basic

color combination of digital image (RGB) can be operated IHC slides into black and

white, making it easier to classify between the nucleus and the cytoplasm. White color

outside the nucleus of the cell (cytoplasm) and background black color outside the

cytoplasm, are made equal to one color. Thus, what appears is the black color of the cell

nucleus in the cytoplasm (figure 3).



Figure 2. RGB of gray, blue, and brown.

After that, the results of the black color made white through infill on black, so black

color (the cell nucleus) that are in the white area could be clearer and specific (figure

4).

Figure 3. Operation RGB color to black and white with

(a) black in white as in the cell nucleus, (b) beyond the

black and white as a background and (c) white as the

cytoplasm.

Figure 4. Output imfill black to white

Result of the calculation shows the cell nucleus results may varies. Number of cell

nucleus in the calculation using MATLAB is 64, can be seen in figure 5. While the

calculation using the manual method is 73 (figure 6).


Figure 5. Output of using matlab calculation, 64 cell

nucleus.

Figure 6. Output of manual calculation, 73 cell nucleus.

Blind spot in this project is the nucleus of the cell that is located close to the

boundary would not be detected / counted and the cell nucleus that is very close to the

core that would otherwise be counted as one cell nucleus. In addition, the number of cell

nuclei are counted affected by noise settings, causing the calculation of the number of

cell nuclei also changed.

Conclusion and Remarks

In a related issue, calculation result is less than 10%, which means that the

method in this project could be used to determine the number of pancreatic beta cell

nucleus.

Acknowledgment

Author would like to thank lecturer of Faculty Science and Mathematics, Satya

Wacana Christian University, Mr. Suryasatriya Trihandaru and Mrs. Jodelin Muninggar.



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Determines the Number of Pancreas Beta Cells with IHC