Forensic Science International 316 (2020) 110461

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Forensic Science International

journal homepage: www.elsevier.com/locate/forsciint

Study on development of forensic blood substitute:

Focusing on bloodstain pattern analysis
Sang-Yoon Leea,b,* , Young-Il Seoa , Byung-Sun Moona , Jin-Pyo Kima , Jae-Mo Goha ,
Nam-Kyu Parka , Se-Hyun Shinb
a
Department of Forensic Engineering, National Forensic Service, Wonju 26460, Republic of Korea
b
Department of Mechanical Engineering, Korea University, Seoul 02841, Republic of Korea

A R T I C L E I N F O A B S T R A C T

Article history: Bloodstain pattern analysis, one of the areas of forensic science, is performed to analyze the physical
Received 3 March 2020 characteristics of bloodstains, including their size, shape, and distribution, to reconstruct a crime scene. A
Received in revised form 11 August 2020 bloodstain pattern analyst should obtain through experiments and education the capabilities to both
Accepted 13 August 2020
understand the generation mechanisms of bloodstains and identify the characteristics of the bloodstains.
Available online 19 August 2020
Experiments and education about bloodstain pattern analysis are carried out by using human blood
taken from subjects, animal blood (porcine or bovine) supplied from butcheries, and blood substitute
Keywords:
products developed in other countries. However, these kinds of blood have many limitations in their
Bloodstain pattern analysis
Forensic blood substitute
application due to various problems.
Physical properties The blood substitute developed in the present study is more similar to human blood than other blood
Drip bloodstain substitute products developed in other countries with regard to the physical properties, including
Luminol viscosity, viscoelasticity, and surface tension, as well as the drip bloodstain patterns depending on the
Pattern transfer surface and coordinate characteristics of drip stains impact angle. The blood substitute developed in the
present study is more practical, because the materials that are used in its preparation are readily available
in the market and do not include chemicals that are harmful to the human body, and the blood substitute
has luminol reaction functionality and pattern transfer bloodstain (bloodstain fingerprint, bloodstain
footprint, etc.) dyeing functionality.
© 2020 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND
license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction have been introduced to identify the causes of the target

Bloodstain pattern analysis, one of the areas of forensic science,
is carried out to analyze the physical characteristics of bloodstains,
including their size, shape, and distribution, to reconstruct a crime
scene. Bloodstain pattern analysis enables one to not only
reconstruct the crime scene of a bloody murder but also determine
the authenticity of a suspect's statement and assume a suspect
depending on the case [1,2].
A bloodstain pattern analyst should obtain through education
the capabilities to understand the generation mechanisms of
bloodstains and to identify the characteristics of the bloodstains. A
bloodstain pattern analyst should find out, from an experiment
based on the identified characteristics, the assumption that can
best explain the crime scene among the many assumptions that
* Corresponding author at: Department of Forensic Engineering, National
Forensic Service, Wonju 26460, Republic of Korea.
E-mail address: sylee1221@korea.ac.kr (S.-Y. Lee).
bloodstain or the bloodstain pattern. Ultimately, the bloodstain
pattern analyst should be able to secure the probative power of the
evidence in court by acquiring scientific reliability of the evidence
through the reconstruction of the scene.
Experiments and education about bloodstain pattern analysis
are carried out by using the human blood taken from subjects,
animal blood (porcine or bovine) supplied from butcheries, and
blood substitute products developed in other countries. The
problems of human blood include the objection that the subjects
have against blood sampling, the possibility of offensive smell and
decomposition, the danger of biological infection, the difficulty of
massive blood supply when needed, and the difficulties involved in
the control of experimental conditions due to the change of the
physical properties caused by the use of an anticoagulant. Animal
blood also has problems such as the possibility of bad smell and
decomposition, the danger of biological infection, the difficulty of
supply due to the low marketability, and difficulties involved in the
control of experimental conditions due to the physical properties
that are different from those of the human blood. In addition, the

http://dx.doi.org/10.1016/j.forsciint.2020.110461
0379-0738/© 2020 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
2 S.-Y. Lee et al. / Forensic Science International 316 (2020) 110461

Fig. 1. Blood substitutes for reproducing bloodstain patterns and those for reproducing luminol reaction intensity, which were developed and sold by foreign companies.

blood substitute products developed in other countries have
problems such as the economic burden due to their high price,
incomplete validation of the physical properties through experi-
mental comparison with the human blood, and difficulties of
preparation and sale due to the separate functionalities of the
pattern reproduction and luminol reaction.
Studies on forensic blood substitute have been steadily
conducted by Millington J. (2000) [3], Theresa Stotesbury. (2017)
[4,5], and Jingyao Li. (2018) [6]. Although various effective blood
substitute products have been developed by these studies, they
may not be practically applied because the details about the
materials and preparation methods are not specifically recited.
The present study was conducted to develop a forensic blood
substitute that is more similar to human blood and more practical
then the previous products and thus the blood substitute
developed here may be applied to the education of bloodstain
pattern analysis, relevant experiments, and various studies in
forensic science.
2. Materials and method
2.1. Human blood and blood substitute developed in other countries
The human blood (HB) used in the present study was the fresh
whole blood taken from a healthy male in his 30 s. For the
experiment, the HB injected to an EDTA tube was kept at 20℃ in a
“digital water bath (Lab Corporation, Korea)” and rolled at 30 rpm by
using a “digital roller (Thermo Scientific, USA)”. The blood substitute
products developed in other countries were the spatter blood samples
from “AFBS (Arrowhead, USA)”, “SFBS (Sirchie, USA)”, and “TFBS
(Tritech Forensics, USA)” [Fig. 1].
as much as possible. A long shelf life was sought by selecting
and mixing a preservative that does not disturb the realization
of the desired physical properties [Table 1].
Distilled water and Hyaluronate acid were added to the
prepared Amino acid solution at the predetermined ratios and
mixed with an agitator. Sodium chloride, Potassium ferricya-
nide, Hemoglobin from bovine blood, and Bovine serum
albumin were then added to the resulting mixture at the
predetermined ratios and mixed with an agitator. The resulting
mixture was subsequently kept at room temperature for about
one hour. When the supernatant layer of bubbles and
impurities is separated from the lower layer, the mixture
solution of the lower layer is separated for further use by using
a pipette or a fine sieve. To the separated mixture solution, R#
504 (tar color) and V# 401 (tar color) were added according to
the predetermined ratios, and the resulting mixture was mixed
by using an agitator and kept at room temperature for about
one hour. Again, when the supernatant layer of bubbles and
impurities separated from the lower layer, the mixture
solution of the lower layer was separated for further use by
using a pipette or a fine sieve. The completed NFBS was then
kept at room temperature. If the NFBS is stored in a refrigerator
for a long time, Phenoxyethanol is mixed at a predetermined
ratio.

2.2. Choice of materials and method of preparation

The materials for the preparation of the blood substitute

developed in the present study (NFBS) [Fig. 2] were selected by
considering the physical properties such as viscosity, visco-
elasticity, and surface tension, to make the properties similar
to those of the HB, as well as properties to add luminol reaction
functionality and pattern transfer bloodstain dyeing function-
ality. “Hyaluronate acid (WizGarden, Korea)” [7,8,9,10] was
included to adjust the viscosity and viscoelasticity, “Hemoglo-
bin from bovine blood (Sigma-Aldrich, USA)” [11], and
“Potassium ferricyanide(Daejung Chemicals, Korea)” [12] to
adjust the luminol reaction functionality, and an “Amino acid
solution” prepared according to Schwarz, L. (2009) [13] and
“Bovine serum albumin (Sigma-Aldrich, USA)” [14,15] to adjust
the pattern transfer bloodstain dyeing functionality. The
selected materials were mixed at appropriate composition
ratios to prepare the NFBS. In the choice of the materials,
chemicals that are harmful to the human body were excluded Fig. 2. Developed blood substitute (NFBS).
 S.-Y. Lee et al. / Forensic Science International 316 (2020) 110461 3

Table 1
Materials used in the preparation of NFBS and their composition ratios.

No. Material Artificial Blood Substitute

Content (g, %)

Type-1 Type-2
(Not Preservative)

1 Distilled Water 27.00 27.40

2 Amino acid solution L-Serine 45.00 5.1654 45.00 5.1654
Glycine 3.0992 3.0992
DL-Alanine 1.5496 1.5496
L-Lysine 2.0556 2.0556
L-Leucine 0.5165 0.5165
L-Threonine 0.7695 0.7695
L-Asparagin Anhydrous 0.7695 0.7695
(Asparagin acid)
L-Histidine 0.7695 0.7695
L-Valine 0.5165 0.5165
Sodium chloride 34.7873 34.7873
Magnesium chloride hexahydrate 0.0002 0.0002
Calcium chloride anhydrous 0.0008 0.0008
Zinc chloride 0.0001 0.0001
3 Bovine serum albumin 1.00 1.00
4 Hemoglobin from bovine blood 0.15 0.15
5 Potassium ferricyanide 2.30 2.30
6 Hyaluronate acid 22.00 22.00
7 Sodium chloride 0.90 0.90
8 R# 504 (Tar color) 1.00 1.00
9 V# 401 (Tar color) 0.25 0.25
10 Phenoxyethanol 0.40 　
Total 100.00 100.00

2.3. Measurement of physical properties
Through many major measurements related to the engineering
of viscosity, viscoelasticity, density, and surface tension, the
physical properties of the individual blood substitute and the
similarity between individual blood substitute and HB were
confirmed. The measurement was performed by using a “Viscom-
eter (DV-III ULTRA, Brookfield, USA)”, a “Rheometer (RS-6000,
Thermo Scientific, USA)”, a “Density Meter(DDM2910/2911,
Rudolph Research Analytical, USA), and a “Tensionmeter (K11,

Fig. 3. Drip stain generating system.

4 S.-Y. Lee et al. / Forensic Science International 316 (2020) 110461

Fig. 4. Viscosity of the individual blood samples.

Fig. 5. Viscoelasticity of the HB sample.

Fig. 6. Viscoelasticity of the AFBS sample.

S.-Y. Lee et al. / Forensic Science International 316 (2020) 110461 5

Fig. 7. Viscoelasticity of the SFBS sample.

Fig. 8. Viscoelasticity of the TFBS sample.

Fig. 9. Viscoelasticity of the NFBS sample.

6 S.-Y. Lee et al. / Forensic Science International 316 (2020) 110461

Table 2
Density and Surface tension data of the individual blood samples.

No. Blood Type Temperature [ C] Surface Detection Speed [mm/min] Density Surface Tension [mN/m]
[g/㎤]
1 HB 20 10 1066.03  1.92 47.65  1.25
2 AFBS 1021.80  1.84 38.99  1.16
3 SFBS 1016.14  1.83 45.50  1.41
4 TFBS 1026.74  1.85 44.23  1.61
5 NFBS 1031.71  1.86 46.93  1.35

Fig. 10. Density of the individual blood samples.

Fig. 11. Surface tension of the individual blood samples.

KRUSS, Germany & Theta Flex, Biolin Scientific, USA)”, applying the
same settings and conditions for all the individual blood samples.
2.4. Drip bloodstain pattern characteristics
The drip bloodstain pattern characteristics on different
surfaces were compared between the HB, AFBS, SFBS, TFBS,
and NFBS. To perform the test upon different surfaces, a sheet of
A4-sized paper (porous surface), a glass plate (nonporous
surface), and a stainless steel plate (nonporous surface) were
used. Each of the blood samples was dripped on the different
surfaces ten times under the same conditions of temperature
20℃, volume 20㎕, height 30 cm, and angle 90 to generate the drip
bloodstain.
 S.-Y. Lee et al. / Forensic Science International 316 (2020) 110461 7

Fig. 12. Droplets that have formed prior to release from the tip of a 1cc syringe for surface tension measurement.

Table 3
Drip bloodstain patterns of the individual blood samples on different surfaces.

No. Surface Blood Type

HB AFBS SFBS TFBS NFBS

1 Paper

2 Glass plate

3 Stainless steel plate

Fig. 13. Image J quantitative analysis of the drip bloodstain patterns on a porous surface(paper).
8 S.-Y. Lee et al. / Forensic Science International 316 (2020) 110461

Table 4
Image J quantitative analysis of the drip bloodstain patterns on a porous surface(paper).

Surface Blood Type Drip bloodstain Image J analysis

Paper HB

AFBS

SFBS

TFBS

NFBS
 S.-Y. Lee et al. / Forensic Science International 316 (2020) 110461
Fig. 14. Image J quantitative analysis of the drip bloodstain patterns on a nonporous surface, (left: glass, right: stainless steel).
2.5. Coordinate characteristics of drip stains impact angle
The coordinate characteristics of drip stains impact angle on
paper were compared between the HB, AFBS, SFBS, TFBS, and
NFBS. Drip bloodstains were generated by using a home-built
drip bloodstain generator comprising a “Digital pipette (FP Novus
5-50㎕, Thermo Scientific, USA)”, a “Pipette tip (DIAMONDⓇ Tip
D200, France)”, a “Copy stand (PROSPOT, CS Studio Light,
Korea)”, a “Magic arm (Manfrotto, Italy)”, and an “Angle vise
(Bluetec, Korea)” (Fig. 3). With an amount of 20㎕ and at a
temperature of 37℃, a height of 30 cm, and angles of 20 , 30 , 40 ,
50 , 60 , 70 . A total of 1,000 tests were conducted, forty times for
each blood sample and angle. The obtained experimental results
were analyzed using HemoSpat software (Queen‘s Univ, Canada).
3. Results
3.1. Comparison of physical properties
3.1.1. Rheological characteristics
The rheological change of the viscosity with respect to the shear
rate was measured by using a viscometer in the cone&plate mode,
at room temperature of 20℃, an initial rate of 1/s, a final rate of 2000/
s, at a point per decade of 20 and a measurement time of three
minutes. The results showed that the rheological characteristics of the
viscosity of the NFBS were most similar tendency to the HB (Fig. 4).
The rheological behavior of the viscoelasticity with respect to the
frequency was measured at room temperature of 20℃, a strain of
800%, an initial frequency of 1/s, a final frequency of 100 rad/s, and a
point per decade of 10. The results showed that the rheological
characteristics of the viscoelasticity of the NFBS was most similar
tendency to the HB. However, AFBS, SFBS, and TFBS showed
different tendency results(Figs. 5–9).
3.1.2. Density and Surface tension characteristics
The Density was measured ten times for each sample at room
temperature of 20℃. The results showed that the Density of
NFBS (1031.71  1.86) was most similar to the HB (1066.03 
9
1.92), however, all blood substitute showed a difference in density
from HB. The surface tension was measured ten times for each
sample at room temperature of 20℃ and a detection speed of 10
mm/min. The results showed that the surface tension of NFBS
(46.93  1.35) was most similar to the HB (47.65  1.25) (Table 2)
(Figs. 10–12).
3.2. Comparison of drip bloodstain pattern of individual blood samples
on different surfaces
The experimental results showed that the drip bloodstain
pattern of all the blood samples was a circle of less than 1㎜
diameter on the paper surface (porous surface). The HB and NFBS
samples showed a similar drip bloodstain pattern of a circular shape
having a diameter of less than 1㎜, while the AFBS, SFBS, and TFBS
samples showed a spread drip bloodstain pattern similar to a circle
having a diameter of 1 to 1.5㎜ (Table 3). A quantitative analysis of the
drip bloodstains of the individual blood samples on the paper,
performed by using the “Image J software program (Wayne Rasband,
USA)”, showed that the result of the NFBS (47.56  1.45) was the
most similar to the HB (67.36  1.05). The analysis was performed by
converting images of the individual blood samples by using split
channels, analyzing the converted images as 8-bit data values (0-
255), and comparing the resulting data values (Fig. 13, Table 4) [16].
A quantitative analysis of the diameter of the bloodstains of the
individual samples, formed by dripping the blood samples on a glass
plate and a stainless steel plate, showed that the diameter of the
NFBS (9.74  0.76, 9.68  0.54) was the most similar to the HB
(9.58  0.93, 9.72  0.81) (Fig. 14).
3.3. Comparison of the coordinate characteristics of drip stains impact
angle
As a result of the experiment, AFBS, SFBS, TFBS, and NFBS
simulated impact angles at 50 , 60 , and 70 showed similar results
to those of HB. However, with acute angles of 208, 308, and 408,
NFBS showed similar results in comparison with HB than AFBS,
SFBS, and TFBS (Tables 5,6) (Figs. 15–18).
10 S.-Y. Lee et al. / Forensic Science International 316 (2020) 110461

Table 5
Drip stain patterns for each blood type and angle.

No. Impact angle Blood Type

HB AFBS SFBS TFBS NFBS

1 20

2 30

3 40

4 50

5 60

6 70
 S.-Y. Lee et al. / Forensic Science International 316 (2020) 110461 11

Table 6
Data on the coordinate characteristics of drip stains impact angle for each blood sample.

No. Blood Type a McDonnell Result value

formula
208 308 408 508 608 708
1 HB W/L (Mean) 0.32 0.48 0.63 0.74 0.86 0.93
W/L (Std) 0.15 0.18 0.14 0.15 0.11 0.15
angle (Mean) 18.8 28.8 39 48.2 58.8 68.9
angle (Std) 2.8 3.5 3.3 3.0 3.3 3.8
2 AFBS W/L (Mean) 0.29 0.42 0.56 0.75 0.87 0.95
W/L (Std) 0.1 0.11 0.15 0.13 0.14 0.16
angle (Mean) 14.9 24.9 34.4 48.4 61.3 71.3
angle (Std) 2.8 2.3 2.7 3.2 2.7 4.2
3 SFBS W/L (Mean) 0.26 0.43 0.57 0.76 0.87 0.92
W/L (Std) 0.1 0.11 0.17 0.12 0.20 0.17
angle (Mean) 15.1 25.8 34.5 49.3 60.6 69.4
angle (Std) 3.0 2.7 3.3 3.9 2.8 4.2
4 TFBS W/L (Mean) 0.26 0.4 0.59 0.72 0.85 0.93
W/L (Std) 0.1 0.14 0.18 0.12 0.23 0.19
angle (Mean) 14.2 23.6 36.1 46.2 58.8 69
angle (Std) 2.1 3.4 1.9 2.7 3.2 3.3
5 NFBS W/L (Mean) 0.31 0.48 0.62 0.75 0.86 0.93
W/L (Std) 0.09 0.17 0.15 0.18 0.16 0.15
angle (Mean) 18 28 39 48.9 58.8 68.1
angle (Std) 1.7 3.7 2.6 2.4 3.9 3.3

Fig. 15. Compares the coordinate characteristics of drip stain's impact angle Fig. 17. Compares the coordinate characteristics of drip stain's impact angle
between the AFBS and HB. between the TFBS and HB.

Fig. 16. Compares the coordinate characteristics of drip stain's impact angle Fig. 18. Compares the coordinate characteristics of drip stain's impact angle
between the SFBS and HB. between the NFBS and HB.
12 S.-Y. Lee et al. / Forensic Science International 316 (2020) 110461
4. Conclusions
The physical properties of the NFBS, including the viscosity,
viscoelasticity, and surface tension, and its drip bloodstain pattern on
different surfaces and coordinate characteristics of drip stains
impact angle were more similar to those of the HB in comparison
with the blood substitute products developed in other countries. In
addition, the NFBS developed in the present study is more practical,
because the materials that are used in the preparation of the NFBS are
readily available in the market and do not include chemicals that are
harmful to the human body, and the NFBS has luminol reaction
functionality and pattern transfer bloodstain (bloodstain fingerprint,
bloodstain footprint, etc.) dyeing functionality.
Although the results of viscosity and viscoelasticity are similar in
general to those of other blood substitute, it is difficult to say that they
are very similar to HB, and verification by measurement will be
necessary even under low conditions of less than 1 second. For the
similarity of the density results, precision should be complemented
by mixing erythrocyte substitutes at an appropriate prices that meet
the practical purposes, quantitative analysis through the expansion
of the number of HB samples in consideration of the physical
characterisitics of each person's blood, and reliability through
changes in physical properties due to the use of anticoagulants.
Further studies will be conducted to measure the physical
properties of the NFBS more precisely, to analyze the luminol
reaction functionality and the pattern transfer bloodstain dyeing
functionality comparatively and quantitatively, and to examine the
compatibility with the various reagents and kits used in the DNA
analysis so that the NFBS may be applied to various fields of
forensic science. In addition, the method of preparing the NFBS will
be shared with competent authorities including the National Police
Agency and the Ministry of National Defense so that it may be
independently prepared and used according to need.
CRediT authorship contribution statement
Sang-Yoon Lee: Conceptualization, Methodology. Young-Il
Seo: Formal analysis, Investigation. Byung-Sun Moon: Formal
analysis, Investigation. Jin-Pyo Kim: Software, Validation. Jae-Mo
Goh: Writing - original draft. Nam-Kyu Park: Writing - review &
editing, Funding acquisition. Se-Hyun Shin: Supervision, Funding
acquisition.
Declaration of Competing Interest
To the best of our know ledge, the named authors have no
conflict of interest, financial or otherwise.
Acknowledgments
This study was supported by the Scientific Investigation and
Appraisal Technique R&D Program of the National Forensic Service
(NFS) of the Ministry of the Interior and Safety (MOIS) of Korea, and
by the Engineering Research Center (ERC) of the Basic Research
Program funded by the National Research Foundation of Korea
(NRF).
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