The Simple Method for Immunohistochemistry (IHC) Analyzing of

Intensity using Corel Photo-Paint® with CMYK Split-channel

Fajar Shodiq Permata, Dyah Ayu Oktavianie Ardhiana Pratama and Albiruni Haryo

Faculty of Veterinary Medicine, Universitas Brawijaya, Puncak Dieng Eksklusif Kalisongo Dau, Malang, Indonesia

Keywords: Corel Photo Paint, Image Processing, Immunohistochemistry, Intensity, Quantitive Method.

Abstract: Immunohistochemistry (IHC) is a popular method to detect protein in the tissue. But, determining protein

level in tissue is using ELISA which needs more cost. Although, the intensity of IHC color relates to an

amount in tissue. Therefore, image processing to measure the intensity of IHC quantitively was developed

using much software. The common software used in Indonesia is Corel Photo-Paint® (CCP). This article

described the simple method to measure the IHC intensity using CCP based on Cyan-Magenta-Yellow-

Black (CMYK) Split channel. The conclusions were CCP could be used for image processing software to

measure the IHC intensity. The higher intensity has fewer grey level, the lower intensity has a higher grey

level

1 INTRODUCTION

Immunohistochemistry (IHC) is a popular technique

to detect a specific protein in the tissue. The IHC

was conducted by pathologists or histologist to

determine the expression of the protein (Kaliyappan

et al. 2012). The disadvantage of IHC is that can not

measure the protein in the tissue so that it has to use

another technique such as Enzyme-linked

Immunosorbent Assay (ELISA) (Drijvers et al.

2017). It will make more cost due to the use of

ELISA. However, if there has more contained

protein in the tissue will have more intensity in IHC

expression. Therefore, the amount of protein inside

the tissue relates to increasing the intensity of IHC

expression. Many software such as Image J (Jensen

2013), QuPath (Bankhead et al. 2017), ImunoRatio

(Tuominen et al. 2010), and IHC profiler (Varghese

et al. 2014) is developed to be image processing of

tissue pictures to quantitive measurements. These

software result in the number based on IHC pictures

then continue to statistic analysis. The quantitive of

IHC expression could be area percentages, number

of expressed cells, and intensity level. The image

processing of IHC expression would be reduced

more cost to check other parameter associated

protein in tissue(Kaczmarek, Górna, and Majewski

2004).Goldy chocolate of IHC expression in the

picture is composed of Magenta and Yellow color.

Yellow and Magenta color could be split from a

picture using CMYK split-channel (Pham et al.

2007) into a grey mode. The intensity is measured

based on the grey level using a histogram. Grey

level is described from 0 (black) until 255 (white)

(Padmavathi and Thangadurai 2016).

Corel Photo Paint® is a popular software for

image processing in Indonesia accompanied by

Corel Draw®. Corel Photo Paint® has the ability to

Cyan-Magenta-Yellow-Black (CMYK) split-channel

and measuring grey level with Histogram. Thus, this

article explained the simple method using Corel

Photo Paint® with CMYK split-channel for intensity

measurement of IHC expression in tissue pictures.

2 MATERIALS AND METHODS

2.1 Material

Materials for this research were Corel Photo-Paint

(CCP) ® Software version X7 and one photo of IHC

result of Positive Dopamine expression in neurons

of the brain.

2.2 Method

1. Open the Corel Photo Paint® Software (Figure 1)

Permata, F., Pratama, D. and Haryo, A.

The Simple Method for Immunohistochemistry (IHC) Analyzing of Intensity using Corel Photo-Paint

 with CMYK Split-channel.

DOI: 10.5220/0009587000530057

In Proceedings of the 6th International Conference on Advanced Molecular Bioscience and Biomedical Engineering (ICAMBBE 2019) - Bio-Prospecting Natural Biological Compounds for

Seeds Vaccine and Drug Discovery, pages 53-57

ISBN: 978-989-758-483-1

Figure 1: Opening Corel Photo Paint®

2. Click Open in Menu, please choose the file of the

figure 2

Figure 2: Opening the picture file

3. Click Image in Menu > Split Channel > CMYK.

The result would be as extraction into 4 pictures in

Cyan, Magenta, Yellow and Black picture in grey

mode (Figure 3-6).

Figure 3. The cyan channel result in grey mode

Figure 4. The magenta channel result in grey mode

Figure 5. The yellow channel result in grey mode

Figure 6. The black channel result in grey mode

4. Combining the Magenta channel and Yellow

channel with click Image > Calculation (Fig 7).

Choose the magenta channel in source 1 and the

yellow channel in source 2 and then click OK.

ICAMBBE 2019 - 6th ICAMBBE (International Conference on Advance Molecular Bioscience Biomedical Engineering) 2019

Figure 7. Calculation image as combining the Yellow

channel and the magenta channel

5. The outcome was grey mode picture combination

the yellow and the magenta channel (Figure 8)

Figure. 8. Picture as the result of calculation processing

combining the Yellow and the Magenta channel

6. Click Rectangle Mask (in the Left Side) >

Normal (upper side) and change to Fixed Sized

(Figure 9)

Figure 9. Rectangle mask (blue arrow) and Fixed sized

(red arrow)

7. Please determine the size example 100x100, and

please choose the area or cell which want to be

calculated its intensity (Figure 10)

Figure 10. Determining the size 100x100 (blue arrow), and

choose the area or the cell which want to be measured the

intensity (green arrow)

8. Please click Image > Histogram to know the mean

grey level (Figure 11). Please record the mean grey

level into the data

Figure 11. The calculation of IHC intensity of cells based

on the mean grey level (blue circle).

2.2.1 Measurement the Neuron and the

Dendrit Intensity of IHC expression

As many as 10 areas randomly were calculated

either of neuron and dendrite by the method for

intensity measurement.

2.2.2 Data Analysis

Data were analyzed using an independent T-test with

significance 95% (p<0.05) comparing the intensity

IHC expression of the neuron and that of dendrites.

3 RESULTS AND DISCUSSIONS

3.1 Results

3.1.1 Comparison of the Intensity of IHC

Expression between the Neuron and

the Dendrites

Based on the measurement, there was significantly

different (p<0.05) of intensity between neuron and

The Simple Method for Immunohistochemistry (IHC) Analyzing of Intensity using Corel Photo-Paint

 with CMYK Split-channel

dendrite associated IHC expression (Table 1, Figure

12).

Table 1: The comparison of the intensity of IHC

expression between neuron and dendrite

Group Grey level p-value T-test

euron 26,13±9,05 0.000

Dendrite 52,96±8,09

otes: p-value showed under 0.05 indicate

significant difference between group

Figure 12: The graph of the mean of grey level between

neuron and dendrite for IHC expression of intensity.

3.2 Discussions

The software for image processing of the IHC

picture was developed in many forms. CPP® is the

popular software used in Indonesia for photo editing

besides Adobe Photoshop®. This article was showed

another function of CPP® to image processing relate

to medical or biological aspects for examining IHC

pictures.

Analyzing IHC picture give benefits such as

reduce the cost of other technique to tissue protein

measurement. This article explained the simple way

to measure the intensity of IHC expression

(Kaczmarek, Górna, and Majewski 2004).

This method was similar to Pham et al. 2007,

using the CMYK method but this method was

simpler to apply. The differences between

expression in the neuron (high expression) and the

dendrite (less expression) were significantly

different (p<0.05, Table 1). The higher expression

showed the lower grey level and the less expression

showed the higher grey level. It was indicated that

the CPP® could be used as a software to IHC

expression analysis for intensity. This article was

also be a module to operate CPP® to analyze IHC

picture to intensify expression.

4 CONCLUSIONS

The conclusions of this method were :

• Corel Photo Paint® could be used easily to

measure the intensity of IHC expression using

CMYK Split channel

• higher intensity is the fewer grey level, the less

intensity is the higher grey level

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The Simple Method for Immunohistochemistry (IHC) Analyzing of Intensity using Corel Photo-Paint

 with CMYK Split-channel