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HAYATI Journal of Biosciences March 2014

Available online at:


Vol. 21 No. 1, p 48-52 http://journal.ipb.ac.id/index.php/hayati
ISSN: 1978-3019 DOI: 10.4308/hjb.21.1.48

SHORT COMMUNICATION

Haemozoin Detection in Mouse Liver Histology Using Simple


Polarized Light Microscope
DWI RAMADHANI∗, SITI NURHAYATI, TUR RAHARDJO

Center for Technology of Radiation Safety and Metrology, National Nuclear Energy Agency of Indonesia
Jalan Lebak Bulus Raya No. 49, Kotak Pos 7043, Jakarta 12070, Indonesia

Received February 25, 2013/Accepted September 6, 2013

The presence of malarial pigment (haemozoin) due to Plasmodium infection is a common histopathological effect
in mouse liver. Previous research showed that by using a polarized light microscope, researchers were better able
to detect haemozoin in mouse liver histology section. Thus, the aim of this research was to compare the haemozoin
area observed by a conventional vs. simple polarized light microscope by using image processing analysis. A total of
40 images produced from both conventional light microscope and simple polarized light microscope were collected.
All images were analyzed using ImageJ 1.47 software to measure the haemozoin areas. Our results showed that non
birefringent haemozoin and birefringent haemozoin area was significantly different. This was because when using
conventional light microscope the brown area that contained images of non birefringent haemozoin images also
contained Kupffer cells which appeared as the same brown color as haemozoin. In contrast, haemozoin gave bright
effect and can be easily differentiated with Kupffer cells in the birefringent haemozoin images. This study concluded
that haemozoin detection in mouse liver histology using a simple polarized light microscope was more accurate
compared to that of conventional light microscope.

Keywords: haemozoin, liver histology, mouse, microscope, polarization


___________________________________________________________________________

INTRODUCTION accumulation, liver histology is reveals congestion


with brown or black pigmentation (Baheti et al.
Malaria is a serious global disease and a leading 2003).
cause of morbidity and mortality in tropical and Examination of haemozoin in liver histology is
subtropical countries. It affects between 350 and usually manually identified under microscope. It is
500 million people worldwide and causes more commonly observed in association with late stage
than one million deaths each year (Syaifudin et al. parasitic infection or as an indicator of previous
2011). Malaria is caused by protozoan parasites (including treated) infection. Because it contains
of the genus Plasmodium (Hisaeda et al. 2005) a birefringent (doubly refracting) substance,
and its common histopathological effect due to haemozoin is highly visible when viewed using
Plasmodium infection is the presence of haemozoin crossed polarized light (Lawrence & Olson 1986).
in the liver (Baheti et al. 2003). Haemozoin is a The haemozoin in human placental histological
heme polymer produced by the parasite as a result specimens using polarizing microscope was more
of hemoglobin breakdown inside the red blood sensitive than conventional light microscope
cells (RBC) of the host. The lysis of red blood cells (Romagosa et al. 2004). However, the haemozoin
during infection results in release of merozoites detection using polarized light microscope can
with this heme pigment, which are phagocytized produce false results. This is because dust and dirt
by circulating monocytes, neutrophils and resident on the slide may produce a birefringence similar
macrophages (Sullivan & Meshnick 1996; Egan to that of haemozoin. However such false positive
2003). The amount of haemozoin in tissues increases results can be avoided by double checking the
over the duration of infection, therefore the amount pigment containing cells for parasites by using light
of pigment correlates with the chronicity of the microscopy (Romagosa et al. 2004).
lesion (Silva et al. 2011). As a result of haemozoin Maude et al. (2009) devised a simple method
_________________ for adapting a conventional light microscope for

Corresponding author. Phone: +62-21-7513906/7659511, polarized light microscopy. They used a pair of
Fax: +62-21-7657950, E-mail: dhani02@btan.go.id gray or black polarizing sunglasses and a small
Vol. 21, 2014 Short Communication���������
�������
49

additional piece of polarizing material that had were made in 7 μm thickness using hematoxylin
been cut out of from a plastic polarizing lens or eosin (HE) stain and were obtained from the
a polarizing test strip. The researchers placed a Nuclear Biomedical Laboratory in the Center for
black polarizing sunglass on the top of light source Technology of Radiation Safety and Metrology,
of microscope and a piece of polarizing material National Nuclear Energy Agency of Indonesia. Mice
placed on the top of thick blood smear slide from were inoculated with the irradiated Plasmodium
a patient with severe malaria. This apparatus was berghei of an ANKA strain at a dose of 150 Gy and
able to can successfully identify haemozoin inside were kept for 24 days before proceeding to liver
the slide (Maude et al. 2009). Here in this study, we histological study.
adapted those techniques using a conventional light Image Acquisition. A Nikon Biophot microscope
microscope for polarized light microscope to detect attached to Nikon D3000 digital single lens reflects
haemozoin area in the mouse (Mus musculus sp.) (DSLR) camera system was used to capture the
liver section histology. The aim of this research was images of the blood smear slides. Images were
to compare the measurements of haemozoin area captured at a resolution of 1936 x 1296 pixel and
obtained using a conventional light microscope vs. saved as JPEG files. The first image was captured
a simple polarized light microscope by using image using the adapted polarized light microscope,
processing analysis. whereas the second image was captured using
the same setting but after removal of the linear
MATERIALS AND METHODS polarizing sheet and CPL filter. A total of 20
images were collected using simple polarized and
Polarized Light Microscope. A Nikon Biophot conventional light microscope.
microscope was used as a conventional light Image Analysis. A macro program was developed
microscope. The equipments required for adapting in ImageJ 1.47 for measuring the birefringent
Nikon Biophot to become a polarized light haemozoin area in mouse liver histology. The
microscope were a linear polarizing sheet (45 x 35 algorithm of macro program can be divided into the
mm) (Polar Pro) and commercial circular polarizing following several sequential steps (Figure 2). The
filter (CPL) 58 mm (Hoya Pro1) for Digital Single first step was to split the images channels into green,
Lens Reflex (DSLR) Camera. The CPL filter 58 mm blue and red channels. The second step was to invert
was placed on the top of the microscope light source the green channel image to detect the haemozoin
and the linear polarizing sheet was placed on the top area using thresholding methods. The last step
of liver histology slide. The slides were examined was to measure the haemozoin area and determine
under 40x objective lens with maximum brightness the outlined haemozoin area in the images. The
(Figure 1). The CPL filter was rotated in a clockwise haemozoin area in images captured using the
direction until it reached the position where the conventional light microscope was measured by
object appeared to be most dark. The difference using an ImageJ 1.47 plugin previously developed
between light and dark was approximately in 45° (unpublished data). To obtain the measurement of
of rotation. The angle at which the object appears the areas presented in micrometer (µm) the Set
darkest was noted (Kramer et al. 2001). Scale command in ImageJ must be employed.
Mouse (Mus musculus sp.) Liver Histology. First an image of a microscope stage micrometer
Mouse (Mus musculus sp.) liver histology slides under the same microscope magnification that

Figure 1. CPL filter 58 mm on top of the microscope light source (blue arrow) (left) and a linear polarizing sheet on the top of a
liver histology slide (blue arrow) (right).
50 RAMADHANI ET AL. HAYATI J Biosci

used to capture the haemozoin must be obtained. µm2, respectively. Scatter plots of the data points
Then with the straight line selection tool, we drew showed that the linear relation between birefringent
a line that corresponded to known distance in the and non birefringent haemozoin area was quite
microscope stage micrometer image. Subsequently, low (r =0.0785; Figure 3). Statistical analysis using
the known distance and unit of measurement was t-test showed that there was a significant different
entered in the Set scale command dialog. ImageJ between the area of birefringent and non birefringent
will automatically fill in the Distance in Pixels field haemozoin (P = 0.011).
based on the length of the line (Papadopolus et al.
2007). 14000

Birefringent haemozoin area (um²)


y = 0.738x + 2002
Statistical Analysis. Birefringent and non 12000 R² = 0.078
birefringent haemozoin areas from 20 images 10000
were compared using t-test analysis. Significant
8000
level used in this research is 0.05 (5%), differences
6000
with p-value less than 0.05 were considered to be
4000
significant.
2000

RESULTS 0
0 2000 4000 6000
Non birefringent haemozoin area (um²)
Birefringent and Non Birefringent Haemozoin
Figure 3. Scatter plots comparing birefringent and non birefringent
Area Measurement. The average area of haemozoin area.
birefringent and non birefringent haemozoin
observed in our samples (of 20 images) were A B
2843.47 ± 779.49 µm2 and 4101.151 ± 2053.75

Original image Duplicated


image

Figure 4. Haemozoin that contain Kupffer cell image in liver


histology slides (A) obtained from conventional
Enhance contrast microscope (blue circle) and (B) from simple
polarized microscope (blue circle).

A B

Red channel Blue channel Green channel

Figure 5. Image of liver histology slides obtained from (A)


Invert green simple polarized microscope: dust and dirt produced
channel image
a birefringence similar to haemozoin (blue circle),
(B) conventional microscope: no haemozoin in this
area (blue circle),.

Create selection and


measured black area

Combined image

Figure 2. ImageJ Macro flowchart for measure birefringent Figure 6. Haemozoin in unstained mouse liver histology (red
haemozoin area. circle) (Deroost et al. 2012).
Vol. 21, 2014 Short Communication���������
�������
51

DISCUSSION haemozoin detection can be false positive because


dust and dirt on the slide sometimes produced a
A significant difference between birefringent and birefringence effect similar to haemozoin. This
non birefringent haemozoin area was observed in this phenomenon can be avoided by checking the
research. Results also showed that the average area haemozoin area using light microscopy; as shown
of birefringent haemozoin measured in our images in Figure 5A, dust produced a birefringence similar
was lower than that of non birefringent haemozoin to haemozoin. Also, in comparison with images
areas. This might be because in the non birefringent obtained using conventional light microscope, there
haemozoin images the area occupied by brown color was no brown pigmentation in the area contaminated
also contained the Kupffer cells (Figure 4A), while with dust (Figure 5B). Acid formalin as a fixative
in the haemozoin images obtained from simple for liver histology section also can produce
polarized microscope only the haemozoin that gave formalin pigment. This pigment has physical
a birefringence effect (Figure 4B). Haemozoin is a and histochemical properties similar to malarial
disposal product formed from the digestion of red pigment and under polarization microscopy shows
blood cells by malaria parasites, and it is observed as a marked birefringent similar to that of malarial
as black or brownish granules under conventional pigment (Romagosa et al. 2004).
light microscope (Soniran et al. 2012). Therefore, it Another technique to measure the haemozoin
was difficult to differentiate haemozoin that attached area from mouse liver histology section is by using
to Kupffer cells with Kupffer cells itself due to the densitometry analysis in unstained mouse liver
same brown color. Kupffer cells are macrophages histology sections that will produced brown color
cells that adhere to the endothelial lining and have haemozoin (Figure 6) (Deroost et al. 2012). Frita
variable shapes, which probably meaning that they et al. (2012) also used non birefringent haemozoin
are motile and are capable of migrating. Kupffer images obtained from conventional light microscope
cells are preferentially located in the periportal and also used ImageJ software to measure the
sinusoids of the liver (Wisse et al. 1996). percentage of the liver histology section total area
In agreement with Romagosa et al. (2004), in in the images.
this study the use of polarization microscopy in We concluded that haemozoin detection in
this study significantly increased the detection of mouse liver histology can be conducted by using a
malarial pigment (Romagosa et al. 2004). The simple polarized light microscope and this technique
difficulty in localizing malarial parasites also is was more effective compared to conventional light
a major problem both in blood smears (Collier & microscope. Further, the use of neutral buffered
Longmore 1983) and in histological studies (Ismail formalin in place of acid formalin as a fixative will
et al. 2000). The use of polarization microscopy be considered in order to prevent the formation of
allows for the detection of haemozoin by scanning formalin pigment that can give a birefringent effect
the slide at low magnification (200x) and is also under the polarized microscope Romagosa et al.
proven to be a quick, sensitive and specific method (2004).
to identify the haemozoin in placenta histology
(Romagosa et al. 2004). Other methods that allow ACKNOWLEDGEMENT
for the recognition of the pigment and parasites
have been used in histological studies, such as The authors are greatly obliged to Mukh Syaifudin,
modified fluorescence microscopy. Nevertheless, Center for Technology of Radiation Safety and
fluorescence microscopes are less available and Metrology, National Nuclear Energy Agency of
more expensive than light microscopes and the Indonesia for the critical reading of the manuscript.
technique has not been validated (Romagosa et al. The valuable technical assistance of Devita Tetriana
2004). Polarization microscopes have the additional is gratefully acknowledged.
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