IEEE TRANSACTIONS ON BIOMEDICAL ENGINEERING, VOL. 58, NO.

3, MARCH 2011 777

Point-of-Care Device for Quantication of
Bilirubin in Skin Tissue
Suresh K. Alla*, Adam Huddle, Joshua D. Butler, Peggy S. Bowman, Joseph F. Clark, and Fred R. Beyette Jr.
AbstractSteady state diffuse reectance spectroscopy is a non-
destructive method for obtaining biochemical and physiological
information from skin tissue. In medical conditions such as neona-
tal jaundice excess bilirubin in the blood stream diffuses into the
surrounding tissue leading to a yellowing of the skin. Diffuse re-
ectance measurement of the skin tissue can provide real time
assessment of the progression of a disease or a medical condition.
Here we present a noninvasive point-of-care system that utilizes
diffuse reectance spectroscopy to quantifying bilirubin from skin
reectance spectra. The device consists of an optical system inte-
grated with a signal processing algorithm. The device is then used
as a platform to study two different spectral databases. The rst
spectral database is a jaundice animal model in which the jaundice
reectance spectra are synthesized from normal skin. The second
spectral database is the spectral measurements collected on human
volunteers to quantify the different chromophores and other phys-
ical properties of the tissue such as Hematocrit, Hemoglobin, etc.
The initial trials from each of these spectral databases have laid
the foundation to verify the performance of this bilirubin quanti-
cation device.
Index TermsBilirubin quantication, hyperbilirubinemia,
Jaundice, skin reectance.
I. INTRODUCTION
S
TEADY state diffuse reectance spectroscopy is a nonde-
structive method for obtaining biochemical and physiolog-
ical information from tissue. When light strikes a substance, it
can be absorbed, transmitted, scattered or reected. Reectance
spectroscopy measures the reected portion of the light, which
can then be utilized to extract information regarding the absorb-
ing and scattering properties of the material.
Neonatal jaundice, clinically known as hyperbilirubinemia,
is associated with elevated serum bilirubin levels in the blood-
stream of infants. Hyperbilirubinemia is termed as severe when
the serum bilirubin concentration is greater than 12.9 mg/dL,
a condition that has been reported to occur in 10% of new-
borns [1]. As the bilirubin levels increase further, the unbound
Manuscript received July 23, 2010; revised October 14, 2010; accepted
November 3, 2010. Date of publication November 18, 2010; date of current
version February 18, 2011. Asterisk indicates corresponding author.
*S. K. Alla, J. D. Butler, P. S. Bowman, and F. R. Beyette, Jr., are with the
Department of Electrical and Computer Engineering, University of Cincinnati,
Cincinnati, OH 45221-0030 USA (e-mail: allas@mail.uc.edu).
A. Huddle is with the Department of Biomedical Engineering, University of
Cincinnati, Cincinnati, OH 45221-0048 USA.
J. F. Clark is with the Department of Neurology, University of Cincinnati,
Cincinnati, OH 45267-0536 USA.
Color versions of one or more of the gures in this paper are available online
at http://ieeexplore.ieee.org.
Digital Object Identier 10.1109/TBME.2010.2093132
bilirubin has a greater propensity to cross the blood brain bar-
rier, staining the basal ganglia and causing an irreversible brain
damage known as Kernicterus. The chances of developing ker-
nicterus are higher in sick and preterm newborns even at lower
concentrations of serum bilirubin. This is due to the imma-
ture development of the blood brain barrier [2]. Detection and
accurate quantication of the yellowness caused by bilirubin
deposition in skin tissue provides a window of opportunity to
prevent the onset of kernicterus by introducing proper interven-
tion procedures such as phototherapy and exchange transfusion.
In this letter, we discuss spectral analysis methods (forward
and inverse models) and provide the verication results, which
demonstrate the viability of our proposed noninvasive point-of-
care device for the quantication of bilirubin.
Test results from two different spectral databases are shown.
The rst is spectra from an animal model of jaundice. Jaundiced
reectance spectra were generated by developing a porcine skin
ap soaking method that yields synthesize jaundiced skin from
normal porcine skin samples. A signal processing algorithm
was then developed to simulate the reectance spectrum of the
porcine skinunder jaundice conditions usingananalytical model
of light transport in skin. The signal processing system coupled
with an optimization algorithm is able to track the changes oc-
curring in the different jaundiced skin tissue spectra. The system
is validated through the analysis of several porcine samples pre-
pared to have different bound bilirubin concentrations.
The second spectral database includes diffuse reectance
spectra collected from the skin of four human subjects. The
device is tested on the human skin diffuse reectance spectral
database by validating the model estimated results to the assay
results obtained from blood chemistry.
II. BIOMEDICAL OPTICAL SYSTEM
The principal hardware components of the device are a white-
light source optimized for the VIS-NIR (3601100 nm), two
SMA connectors used for routing light, a reectance probe that
both directs incident light to the skin surface and collects the re-
ected light fromthe skin surface, a USB4000 spectrometer, and
a laptop computer. Operationally, the light source is coupled to a
SMA connecter which routes the incident light to a reectance
probe. The reectance probe, which provides the mechanical
support and integrity, holds six light illumination bers, which
direct the light onto the skin surface and a detection ber, which
collects the reected light from the skin surface. The bifurcated
bundle assembly splits the bers from the reectance probe to
the light source and to the spectrometer. The spectrometer ac-
cepts light energy transmitted through optical ber and disperses
it via a xed grating across the linear CCDarray detector, which
0018-9294/$26.00 2011 IEEE
778 IEEE TRANSACTIONS ON BIOMEDICAL ENGINEERING, VOL. 58, NO. 3, MARCH 2011
Fig. 1. The system built to capture the reectance spectral data of human skin
and the interface between the spectral data.
resolves the intensity of reected light at every wavelength. The
spectrometer then converts the optical signal into a digital sig-
nal, which can be processed by a proprietary algorithm which
interprets the reected spectrum to predict the concentrations of
the different chromophores (ex. bilirubin) present in the skin.
Fig. 1 shows the system built to capture the reectance spectral
data of human skin and the interface between the spectral data
III. SIGNAL PROCESSING ALGORITHM
The signal processing system used to quantify the serum
bilirubin fromskin reectance spectra is comprised of two parts.
The rst part of the system generates the reectance spectra ex-
pected from the skin with varying absorption and scattering co-
efcients associated with different biological materials present
in different layers of skin. The process of building a spectrum
from the skin optical properties is dened as developing a for-
ward model.
In the second part of the signal processing system, the biliru-
bin concentration is estimated using the forward model along
with a nonlinear iterative method that adjusts the parameters
of the forward model until a best t is achieved between the
simulated and measured spectra. Once the best-t parameters
are obtained, they can be correlated with the experimental con-
ditions associated with the sample preparations.
Fig. 2 shows the owchart of the signal processing algorithm.
The R
sim
is the forward analytical model described in this sec-
tion. The input parameters to the analytical model are the optical
properties and concentration values of different chromophores
in the skin. The R
m
in the ow chart is the reectance spectra
measured using the hardware system dened in the previous
section.
The analytical model of skin reectance (R
sim
) is derived
using existing methods, which solve the diffusion approximation
problemthat has been extended to a two layer skin tissue system
comprised of epidermal and dermal skin layers [3], [4]. The
existing reectance model [5] assumes that the skin as is a
semi-innite turbid media
In a jaundiced individual, the bilirubin ows in the blood
stream bound to albumin. As the bilirubin levels rise beyond the
binding capacity of albumin, unbound bilirubin concentration
Fig. 2. A ow chart showing the engineering ow process in which the sim-
ulated spectrum (R
sim
) is compared with the measured spectrum (R
m
) and the
parameters are updated using the nonlinear least squares optimization solver to
nd the best t parameters.
rises in the blood stream. This unbound bilirubin diffuses out
of the blood stream and is deposited in the skin tissues where
it binds to collagen and fat. Based on the biophysical system,
a three layered analytical reectance model has been dened
using the reectance expression
Reectance = e
m
m
2l
e p i
e
c
e b

e b
2l
e b

d
s
s
1
+ s
2

d
a

.
The rst exponential term takes into account the effect of
melanin present in the epidermal layer. As light traverses twice
through the melanin layer a factor of 2 is introduced in this term.
The second exponential term takes into account the effect of ex-
tra vascular bilirubin. The third term in the expression is used
to estimate the back-scattered light from the dermal layer of the
skin tissue. The parameters in the rst exponential term: m,
m
,
l
epi
are melanin volume fraction (skin tone), absorption coef-
cient of single melanosome, and epidermal layer thickness. The
second exponential term parameters: c
eb
,
eb
, l
eb
are the extra
vascular bilirubin concentration, molar extinction coefcient of
bilirubin, and the path length which the light travels (i.e., the
thickness of the extra vascular bilirubin layer). The parameters

d
s
and
d
a
are the scattering and absorption coefcients of der-
mal layer. The parameters s1 and s2 take into account the effects
probe geometry.
From systems analysis perspective, an inverse model of the
system can be used to extract parameter from measured data. In
this work, the parameters of interest are the physiologic factors
that contribute to the optical properties of the tissue. A nonlin-
ear least squares solver has been adopted as an inverse model
for this work. Sequential quadratic program (SQP) is one of the
most popular and robust algorithms for nonlinear continuous
optimization. In this research, we have used the NLSSOL (non-
linear least squares solver) routine available in the optimization
software TOMLAB interfaced with MATLAB [6]. The primary
goal of this solver is to minimize the error function (differ-
ence between measured and simulated spectra) by updating the
IEEE TRANSACTIONS ON BIOMEDICAL ENGINEERING, VOL. 58, NO. 3, MARCH 2011 779
Fig. 3. The pre-, post-spectra data of pig skin samples (pig2 spectral database)
soaked in bilirubin solutions.
parameters of the analytical algorithm to nd a best match to
measured spectrum. Preliminary studies of the signal process-
ing algorithm on simulated spectra using the forward analytical
model have been shown to estimate the bilirubin concentration
with an average error of less than 15% [7].
IV. JAUNDICE ANIMAL MODEL
Due to its similarity with human skinin aspects, such as lay-
ered architecture and optical properties, porcine skin tissue can
be used as an animal model for skin reectance studies. To
model a jaundice condition with normal skin, we require that all
skin tissue absorbance parameters (e.g., melanin concentration,
hemoglobin concentration, epidermis thickness, and scattering
parameters) be held constant except the bilirubin concentration.
To accomplish this reectance spectrum of skin tissue on the
pig forehead was captured before the skin sample was biopsied.
Bilirubin solutions of different concentrations are then prepared
and added to tissue culture plates. The biopsied skin samples
were placed in the tissue culture dishes and left oating in the
bilirubin solutions. The tissue culture plates are sealed with
paralm and placed on a gentle shaker platform for 4 h.
This process replicates the in vivo condition that leads to
jaundice. After the 4 h soaking period, the tissue samples are
removed from the tissue culture plates and placed back on
their respective pig foreheads to capture a post soak spectral
scan. Post soak spectral scans of different tissue samples resem-
ble the different jaundice levels of skin tissue. Tissue samples
which were exposed to low, medium, and high concentrations of
bilirubin, showed the anticipated increase in the bound bilirubin
concentration, as shown in Fig. 3.
Finally, three spectral databases of pre and post spectral scans
have been collected on three different pigs to study the per-
formance of the algorithm. The positive correlation of R
2
=
0.95,0.96,0.96 between the model estimated and soaked biliru-
bin concentration in the three spectral databases forms the basis
of justication that the signal processing model is able to track
the bilirubin concentration changes in jaundice skin samples.
The next step in the process is validating the model is by
comparing the bilirubin concentration values estimated from
the model to the bilirubin concentration calculated from the
assay process of the skin samples. The assay is performed by
rst freezing and then pulverizing a small portion of the soaked
TABLE I
MODEL ESTIMATED VALUES AND THE ASSAY CALCULATED VALUES FOR THE
SKIN SAMPLES (29, 13. 3)
TABLE II
SHOWS THE COMPARISON BETWEEN VALUES QUANTIFIED FROM THE SIGNAL
PROCESSING ALGORITHM TO THE VALUES CALCULATED FROM THE BLOOD
SAMPLE USING BIOCHEMISTRY
tissue. Once pulverized, the skin tissue can be homogenized
into a solution, which can then be assayed for quantication of
bilirubin. The assay calculated values listed in Table I show a
good correlation with the model estimated values. The deviation
in the absolute values is associated with the different volume
perspectives for the two methods (tissue volume versus solution
volume).
As the jaundice skin has a yellowish appearance, and based
on the jaundice physiology, the intensity of the yellowness of
the skin increases as the more bilirubin is present in the skin.
The pig skin tissue samples soaked in higher concentrations of
bilirubin solution, had showed a strong yellowish signal. The
linear increase in yellow color intensity of the post-soaked skin
samples strongly correlated to the amount of bilirubin present in
the skin sample. The interpretation of the yellow color intensity
along with the spectral processing algorithm would provide a
real-time quantication of the jaundice progression.
V. HUMAN VOLUNTEER SPECTRAL DATA
As a step further toward building a robust signal process-
ing algorithm, four volunteer subjects were recruited to cap-
ture diffuse reectance spectral scans of human skin tissue on
the ventral side of forearm. After the spectral scans were col-
lected, a small amount of blood was drawn to perform bio-
chemistry assays on the blood samples to quantify bilirubin,
hemoglobin, and hematocrit. The output parameters, such as
hemoglobin, bilirubin, and hematocrit numbers generated from
the signal processing algorithm, are compared with the numbers
780 IEEE TRANSACTIONS ON BIOMEDICAL ENGINEERING, VOL. 58, NO. 3, MARCH 2011
Fig. 4. (a) Reectance spectral prole of forehead and nger site of light
pigmented. (Caucasian skin). (b) Reectance spectral prole of forehead and
nger site of lightly dark pigmented individual (Asian skin).
calculated from biochemistry analysis conducted on the blood
drawn from the volunteers is shown in Table II.
The performance of the model on initial sample size has given
a clear understanding of the system behavior, with a high posi-
tive correlation in the prediction of hemoglobin and hematocrit
value.
The spectral data collected on different skin sites of an indi-
vidual and different colored individuals have been analyzed, as
shown in Fig. 4(a) and (b).
The performance of the algorithm is justied by the higher
numbers for the blood-volume fraction and oxygen saturation
on the nger site compared to the forehead. The spectral data
collected on the nger site of a dark skinned individual has
hemoglobin signal bands that are clearly visible, primarily due
to the absence of melanin on the nger tissue site (vascular side).
The spectral data collected on the forehead has the melanin con-
tent from the epidermal layer masking the hemoglobin bands.
As most of the spectral information used to quantify the biliru-
bin is collected from the surface to a depth of around 12 mm,
which is usually the epidermis and dermal layer. With the prin-
ciple of blanching the skin the light can go further deep and
collect the spectral information from the subcutaneous fat layer
too.
The in vivo human data results collected off of the different
individuals was used as a starting step to train the algorithm
for varying optical properties. The poor correlation observed in
the bilirubin device measurement and standard assay could be
strongly improved by collecting more spectral data on healthy
and jaundice individuals. As more spectral data is fed into the
feedback mode of the signal processing algorithm inputs from
the standard assay can form the basis to correct for the missing
parameters in the model. This preliminary human data and the
pig skin jaundice model studies has established the fact that
the device has passed the concept and development phase and
could be headed into the clinical trial phase. The point of care
device with the preliminary results through the signal processing
algorithm does give us the proof that multiple parameters, such
as hemoglobin, hematocrit, and oxygen saturation in addition to
bilirubin, could be quantied using a single device.
VI. CONCLUSION
In this work, a noninvasive point-of-care device for the quan-
tication of bilirubin is developed and evaluated. The device
works by analyzing diffuse reectance spectra collected from
optical reectance from skin tissue. The system is comprised of
hardware components and a signal processing algorithm. The
signal processing algorithm allows the device to quantify the
different chromophores present in the skin tissue. Initial assess-
ment of the device has been completed using a porcine model
for jaundice that involves soaking biopsied pig skin in varying
concentrations of bilirubin solution. Analysis of these skin sam-
ples shows that the device is able to track the changes associated
with the absorption of unbound bilirubin in skin tissue.
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