Clinical Chemistry 59:10
1506–1513 (2013)
Simple Paper-Based Test for Measuring Blood Hemoglobin
Concentration in Resource-Limited Settings
Xiaoxi Yang,1 Nathaniel Z. Piety,1 Seth M. Vignes,1 Melody S. Benton,2 Julie Kanter,2,3
and Sergey S. Shevkoplyas1*
BACKGROUND: The measurement of hemoglobin con-
centration ([Hb]) is performed routinely as a part of a
complete blood cell count to evaluate the oxygen-
carrying capacity of blood. Devices currently available
to physicians and clinical laboratories for measuring
[Hb] are accurate, operate on small samples, and pro-
vide results rapidly, but may be prohibitively expensive
for resource-limited settings. The unavailability of ac-
curate but inexpensive diagnostic tools often precludes
proper diagnosis of anemia in low-income developing
countries. Therefore, we developed a simple paper-
based assay for measuring [Hb].
METHODS: A 20-␮L droplet of a mixture of blood and
Drabkin reagent was deposited onto patterned chro-
matography paper. The resulting blood stain was digi-
tized with a portable scanner and analyzed. The mean
color intensity of the blood stain was used to quantify
[Hb]. We compared the performance of the paper-
based Hb assay with a hematology analyzer (compari-
son method) using blood samples from 54 subjects.
RESULTS: The values of [Hb] measured by the paper-
based assay and the comparison method were highly
correlated (R2 ⫽ 0.9598); the standard deviation of the
difference between the two measurements was 0.62
g/dL. The assay was accurate within 1 g/dL 90.7% of the
time, overestimating [Hb] by ⱖ1 g/dL in 1.9% and
underestimating [Hb] by ⱖ1 g/dL in 7.4% of the
subjects.
CONCLUSIONS: This study demonstrates the feasibility of
the paper-based Hb assay. This simple, low-cost test
should be useful for diagnosing anemia in resource-
limited settings, particularly in the context of care for
malaria, HIV, and sickle cell disease patients in sub-
Saharan Africa.
© 2013 American Association for Clinical Chemistry
1
Department of Biomedical Engineering, Tulane University, New Orleans, LA; 2 Sickle
Cell Center of Southern Louisiana and 3 Department of Pediatrics, Section of
Hematology/Oncology, Tulane University School of Medicine, New Orleans, LA.
* Address correspondence to this author at: Tulane University, Department of
Biomedical Engineering, 500 Lindy Boggs Building, New Orleans, LA 70118.
E-mail shevkop@tulane.edu.
1506
Point-of-Care Testing
Hemoglobin (Hb) is the oxygen-transporting protein
contained within red blood cells (RBCs). The mass
concentration of Hb ([Hb]) is a measure of the oxygen-
carrying capacity of blood. The reference ranges of
[Hb] in blood of adults are 12–16 g/dL in women and
14 –18 g/dL in men; values ⬍5 g/dL or ⬎20 g/dL are
considered critical (1 ). A decreased [Hb], referred to as
anemia, may be an indication of chronic blood loss,
decreased RBC production, or abnormal destruction of
RBCs (hemolysis). Hemolytic anemia may be associ-
ated with inherited blood disorders such as sickle cell
disease, but can also be autoimmune in origin or result
from exposure to certain drugs, toxins, infections, or
transfusion of mismatched blood products (2 ). Other
common causes of anemia include dietary deficiencies
(3, 4 ), cirrhosis, and renal disease (2 ). Anemia reduces
the oxygen-carrying capacity of blood, straining the
cardiopulmonary system, which responds by increas-
ing the heart rate and stroke volume to maintain oxy-
gen delivery (5 ). Acute-onset anemia may be associ-
ated with fatigue, lethargy, and/or pallor (4 ). Patients
with chronic anemia and having [Hb] ⬍5 g/dL may
experience more serious complications such as angina,
congestive heart failure, heart attack, and stroke (5 ).
Depending on the clinical status of the patient, a [Hb]
⬍6 – 8 g/dL is currently recommended as the threshold
trigger for initiating RBC transfusion (6 ). Increased
[Hb] may indicate erythrocytosis due to smoking (7 )
or in response to hypoxia at high altitudes (8 ), polycy-
themia vera (9 ), congenital heart disease (10 ), severe
chronic obstructive pulmonary disease (11 ), or severe
dehydration (12 ). The increase in blood viscosity asso-
ciated with an increased [Hb] may impair oxygen de-
livery by reducing the capillary perfusion, which may in
turn increase the risk of hypoxia, as well as thrombotic
and hemorrhagic complications (13 ).
Because of its importance, [Hb] is measured rou-
tinely as a part of a complete blood cell count. Clinical
Received February 7, 2013; accepted June 11, 2013.
Previously published online at DOI: 10.1373/clinchem.2013.204701
4
Nonstandard abbreviations: Hb, hemoglobin; RBC, red blood cell; POC,
point-of-care; HbCS, Hb color scale; ␮PAD, microfluidic paper-based ana-
lytical device; RGB, red/green/blue; LOD, limit of detection; LOQ, limit of
quantification.
Paper-Based Hb Test for Resource-Limited Settings
laboratories measure [Hb] with automated hematol-
ogy analyzers as well as spectrophotometers, blood gas
analyzers, and stand-alone CO-oximeters (14 ). The
need for rapid and accurate measurement of [Hb] at
the bedside has driven the development and imple-
mentation of [Hb] testing on the point-of-care (POC)
platform (15 ). Currently available POC devices are de-
signed to operate in close proximity to the patient, pro-
vide accurate results (within 1 g/dL of clinical labora-
tory analyzers), require small volumes of blood, and be
used by persons without expert training, making the
nearly instantaneous measurement of [Hb] in operat-
ing room or emergency care settings a reality (16 –18 ).
Notwithstanding their obvious advantages, the
relatively high per-test and analyzer costs make the use
of conventional POC devices for measuring [Hb] pro-
hibitively expensive in resource-limited settings of low-
income developing countries in sub-Saharan Africa, on
the Indian subcontinent, and throughout Southeast
Asia (19, 20 ). Currently available low-cost alternatives
including the “hematocrit/3” method (21 ) and the
WHO Hb color scale (HbCS) test (22 ) have several
drawbacks that limit their use in the field. In addition
to being somewhat inaccurate, hematocrit/3 relies on
knowledge of hematocrit and, therefore, requires a
centrifuge (21, 23 ). The HbCS test is highly sensitive to
the ambient lighting conditions, operator bias, and de-
viations from the recommended readout time (22, 24 ).
The lack of confidence in the quality of [Hb] measure-
ments may result in clinicians relying exclusively on
their clinical judgment to prescribe transfusions in
resource-limited settings (25 ). Therefore, our goal was
to develop a simple, low-cost, paper-based Hb assay
that addressed these limitations.
Methods
BLOOD SAMPLES
The study protocol was approved by Tulane University
Biomedical Institutional Review Board. After written
informed consent was obtained, blood samples were
collected into 4- or 9-mL Vacutainer tubes (K2EDTA,
BD) during routine blood draws (January–April 2013)
from patients of the Pediatric Hematology-Oncology
Clinic (Tulane University Hospital) and the Sickle Cell
Center of Southern Louisiana (New Orleans, LA). We
measured standard hematological parameters includ-
ing [Hb] using a Medonic M-Series hematology ana-
lyzer (Medonic M16C/M20C, Boule Medical). The Hb
content of blood samples was adjusted artificially to
prepare samples with [Hb] ranging from 2.5 to 25 g/dL
by adding autologous plasma (for lower [Hb]) or pel-
leted RBCs (for higher [Hb]) to the original sample.
FABRICATION OF THE MICROFLUIDIC PAPER-BASED
ANALYTICAL DEVICES
The microfluidic paper-based analytical devices
(␮PADs) were fabricated by a previously published
method (26, 27 ). Briefly, the pattern of the ␮PADs was
designed with illustration software (Canvas 11, ACD
Systems International). The devices were printed onto
sheets of chromatography paper (No. 1, Whatman)
with a solid-ink (wax) printer (Phaser 8560N, Xerox),
and then heated on a hot plate at 150 °C for 3 min.
QUANTIFICATION OF THE BLOOD STAIN COLOR
Blood samples were mixed at 1:10, 1:15, or 1:20 ratios
(vol/vol) with Drabkin reagent (Ricca Chemical Co.)
and incubated at room temperature (20 –25 °C) for 10
min. The components of Drabkin reagent lysed red
blood cells and reacted with all forms of Hb except
sulfhemoglobin present in the sample, converting
them to cyanmethemoglobin, a stable brownish-
colored compound. A 20-␮L drop of the mixture was
placed onto the center of each ␮PAD. The resulting
blood stain was allowed to dry for 25 min (28 ).
Quantification of [Hb] was accomplished by scan-
ning the sheets of chromatography paper containing
arrays of ␮PADs on a portable flatbed scanner (Ca-
noScan LiDE110, Canon USA) and then analyzing the
images automatically with a custom-coded algorithm
(MATLAB, The Math Works). The quantitative analy-
sis of blood stains was based on the red/green/blue
(RGB) color model values of the digitized images. The
color data in the red, green, and blue channels were
extracted from the RGB values of each pixel within a
blood stain. We defined the color intensity of each
channel as 255 – (R, G, or B); e.g., for red channel, color
intensity ⫽ 255 – R. The mean color intensity of the
blood stain for each color channel was calculated from
the color values of all pixels within the blood stain,
including the pixels corresponding to the darker ring
on the periphery of the stain. The correlation between
the mean color intensity and the actual [Hb] was eval-
uated for all color channels; the green channel showed
the best linear fit and was selected to quantify [Hb] in
the blood samples.
STATISTICAL ANALYSIS
Pearson correlation coefficient and regression were
calculated to directly compare data obtained from the
paper-based Hb assay and the Medonic M-Series he-
matology analyzer (comparison method). Three re-
search associates performed both types of measure-
ments on the same day. A Bland–Altman plot (29 ) was
also constructed, with consideration for limitations, to
visualize the comparison.
Clinical Chemistry 59:10 (2013) 1507

Fig. 1. Design and fabrication of the paper-based Hb
assay.
(A), The pattern of the ␮PAD was printed with a solid ink
(wax) printer on chromatography paper. (B), The printed
pattern was melted to create a 1-mm-wide hydrophobic
barrier spanning the entire thickness of the paper sub-
strate. (C), To perform the assay, a drop of blood mixed
with Drabkin reagent was placed onto the center of the
device. The lysate wicked laterally toward the periphery,
coloring the paper faint red (pink). (D), The image analysis
algorithm automatically detected the blood stain within
each ␮PAD (dashed circle) and extracted the RGB color
information for all pixels contained within the blood stain.
(E), The color intensity of the pixels (between dashed lines)
was used to calculate the mean color intensity (solid red
line) for each blood stain. The values of the mean color
intensity were used to calculate the [Hb] in the blood
samples. au, Arbitrary unit.
Results
DESIGN AND OPERATION OF THE PAPER-BASED Hb ASSAY
Figure 1 illustrates the design and operation of the
paper-based Hb assay. Each individual ␮PAD com-
prised a 2.8-cm-diameter circle (Fig. 1a) designed to
create a constant area for quantification with an image
analysis algorithm and prevent potential cross-
contamination of samples. Each sheet of chromatogra-
phy paper contained a 5-by-4 array of ␮PADs and in-
cluded an alignment mark to simplify automation of
the subsequent image analysis. We fabricated the
␮PAD arrays by printing their pattern with a solid-ink
(wax) printer (Fig. 1A). We then used a hot plate to
heat the printed chromatography paper above the
1508 Clinical Chemistry 59:10 (2013)
melting point of wax to allow the wax to reflow and
form hydrophobic barriers through the full thickness
of the paper (Fig. 1B). When designing the pattern of
the ␮PAD (Fig. 1A), we accounted for the natural wid-
ening of the printed lines (to about 1 mm thick) during
the melting process (Fig. 1B).
We performed this paper-based Hb assay by first
mixing a sample of whole blood with Drabkin reagent
and then depositing a 20-␮L droplet of the mixture
onto the ␮PAD and letting the blood stain develop
(Fig. 1C). The droplet spread radially from the center
through the paper substrate toward the periphery of
the ␮PAD due to wicking by capillary action (30, 31 ),
forming the characteristic blood stain pattern (Fig. 1D)
reminiscent of the stains produced by drying coffee
(32 ). We selected the volume of the droplet (20 ␮L)
and size of the ␮PAD (2.8 cm) so that the outermost
margin of the stain could not reach the periphery of the
␮PAD (Fig. 1D).
We used a portable flatbed scanner to digitize the
sheets of chromatography paper carrying ␮PADs with
developed blood stains. We analyzed the images to de-
termine the mean color intensity for the blood stain
contained within each ␮PAD using a simple image
processing algorithm developed specifically for this
purpose (Fig. 1, D and E). The algorithm used the
alignment mark on the sheets of paper to determine the
location of each ␮PAD (Fig. 1D). The algorithm ap-
plied a circular binary mask to the image to select the
area within the hydrophobic border of the ␮PAD, and
then isolated the pixels of the blood stain within the
circular region by removing pixels below a threshold
value on the basis of the difference in color intensity
between the paper substrate and the blood stain (Fig.
1D, dashed circle). Finally, the algorithm used the color
information from all pixels within the blood stain to
calculate the mean color intensity of the blood stain
(Fig. 1E, red solid line). We used the mean color inten-
sity of the stain (Fig. 1E) and a calibration curve (Fig.
2B) to measure [Hb] in the blood sample.
All operations of the paper-based Hb assay, in-
cluding sample preparation, placement of the sample
onto the ␮PAD, drying of the blood stain, image scan-
ning, and the automated image analysis, could be com-
pleted within 35 min.
EFFECT OF DILUTION ON THE MEAN COLOR INTENSITY OF THE
BLOOD STAIN
Figure 2 illustrates the effect of dilution on the mean
color intensity of the stains produced by blood on pa-
per. To perform these experiments, we prepared a se-
ries of calibration samples with their [Hb] adjusted to
2.5, 5, 10, 15, 20, and 25 g/dL (as described in Methods)
using blood from 3 consenting volunteers. We then
mixed a small volume of each calibration sample with
Paper-Based Hb Test for Resource-Limited Settings

Fig. 2. Dependence of the mean color intensity of the blood stain on the dilution ratio.
(A), Whole blood samples were diluted or concentrated with plasma or pelleted RBCs to achieve [Hb] ranging from 2.5 to 25
g/dL. A 20-␮L drop of blood mixed with Drabkin reagent at 1:10, 1:15, and 1:20 ratios (vol/vol) was placed at the center of
each ␮PAD. (B–D), Mean color intensities for the red (B), green (C), and blue (D) channels of the blood stain in a ␮PAD produced
by each sample at each dilution ratio. Data shown as mean (SD) (n ⫽ 3). au, arbitrary unit.

Drabkin reagent at 1:10, 1:15, and 1:20 ratios (vol/vol),
waited 10 min, and placed a 20-␮L droplet of each mix-
ture onto paper.
Droplets with markedly different Hb content, due
to either a difference in [Hb] or dilution, produced
blood stains with different color intensities that were
easily distinguishable visually. As expected, the blood
stain color intensity increased with increasing [Hb] for
each mixing ratio (dilution) and decreased with in-
creasing dilution for each [Hb] value (Fig. 2A). The
mean color intensity of each blood stain correlated very
strongly with its [Hb] (Fig. 2B–D). The combination of
1:15 mixing ratio and green channel showed the best
linear fit (mean color intensity ⫽ 2.0986 ⫻ [Hb] ⫹
3.7226, R2 ⫽ 0.996). Therefore, we used the 1:15 mix-
ing ratio to prepare samples in all further experiments,
the mean color intensity in green channel, and the lin-
ear equation produced by this graph (Fig. 2C) as the
calibration curve for estimating the [Hb] of the sample
on the basis of the mean color intensity of its blood
stain.
To determine the limit of detection (LOD) and the
limit of quantification (LOQ) of the assay, we varied
[Hb] of blood samples from 0.5 to 25 g/dL by diluting
whole blood with autologous plasma or concentrating
whole blood with packed RBCs (as described in Meth-
ods). We defined the LOD as the lowest [Hb] at which
the blood stain could be identified and measured using
our image processing algorithm, and the LOQ as the
lowest [Hb] at which the disagreement with the com-
parison method (Medonic hematology analyzer) was
within 1 g/dL. We found that the LOD for our paper-
based Hb assay was 1 g/dL and the LOQ was 2.5 g/dL.
RELATIVE ACCURACY AND PRECISION OF THE PAPER-BASED Hb
ASSAY
We tested the relative accuracy of the paper-based Hb
assay by comparing the values of [Hb] measured using

Clinical Chemistry 59:10 (2013) 1509


Fig. 3. Comparison of the [Hb] measurements per-
formed with the paper-based Hb assay and the Me-
donic hematology analyzer (n ⴝ 54 subjects).
(A), Linear regression analysis of the correlation between
the [Hb] values determined by a hematology analyzer
(actual [Hb]) and by the paper-based Hb assay (estimated
[Hb]). The diagonal (red dashed line) represents a 1:1
correspondence between the actual and the estimated [Hb]
values. (B), Bland–Altman plots of the agreement between
the paper-based Hb assay and the hematology analyzer.
The solid red line is the difference between the 2 methods
(mean bias); the dashed red lines are limits of agreement
(within 2 SD). The mean [Hb] was 11.50 (2.86) g/dL for the
paper-based Hb assay and 11.43 (3.05) g/dL for the Me-
donic hematology analyzer.
our assay and the Medonic M-Series hematology ana-
lyzer for blood samples from 54 subjects. Subjects in-
cluded individuals with Hb AA, Hb AS, Hb SC, and Hb
SS genotypes; the presence of Hb S in the blood samples
did not affect the [Hb] measurements (data not
shown). Figure 3 illustrates the results of this side-by-
side comparison. The data points representing these
measurements were distributed in close proximity to
1510 Clinical Chemistry 59:10 (2013)
the diagonal line (Fig. 3A, red dashed line), indicating a
good agreement between the [Hb] estimated from the
mean color intensity of blood stains in our paper-based
Hb assay and the actual [Hb] obtained from the stan-
dard analysis (Fig. 3A). The linear least-squares regres-
sion analysis (Fig. 3A, black solid line) showed a high
degree of correlation between the 2 measurements (y ⫽
1.05x – 0.58, R2 ⫽ 0.9598).
We also constructed the Bland–Altman plot (29 )
to evaluate the agreement between the paper-based Hb
assay and the Medonic hematology analyzer (Fig. 3b).
The standard deviation (SD) of the difference be-
tween the comparison method (hematology ana-
lyzer) and the paper-based Hb assay was 0.62 g/dL.
The limits of agreement between the 2 methods were
⫺1.30 and 1.18 g/dL. The paper-based Hb assay agreed
with the hematology analyzer within 1 g/dL 90.7% of
the time, overestimating [Hb] by ⱖ1 g/dL in 1.9% of
subjects and underestimating [Hb] by ⱖ1 g/dL in 7.4%
of subjects (Fig. 3B).
In a separate experiment, we tested the precision of
the paper-based Hb assay by measuring repeatedly
(n ⫽ 5) the [Hb] for a series of blood samples obtained
from the same subject with the actual [Hb] of each
sample adjusted (as described in Methods) to 2.5, 5, 10,
15, 20, and 25 g/dL. The SD for the [Hb] measurements
performed with different droplets from the same blood
sample was consistently ⬍0.5 g/dL for all values of the
true [Hb] tested: 0.21 g/dL (CV 8.3%) for the sample
with true [Hb] of 2.5 g/dL; 0.19 g/dL (CV 3.9%) for 5
g/dL; 0.11 g/dL (CV 1.1%) for 10 g/dL; 0.21 g/dL (CV
1.4%) for 15 g/dL; 0.34 g/dL (CV 1.7%) for 20 g/dL;
and 0.78 g/dL (CV 3.1%) for 25 g/dL. For comparison,
the [Hb] measurement range for the Medonic hema-
tology analyzer was 0 –35 g/dL, with CV ⬍1.0% (values
provided by the manufacturer).
EFFECT OF DROPLET VOLUME VARIATION AND LONG-TERM
STABILITY OF THE PAPER-BASED Hb ASSAY
We measured the effect of variation in the volume of
the sample droplet, which may occur during the use of
the assay in the field, on the [Hb] measurement for
samples with true [Hb] of 5, 10, 15, and 20 g/dL. A 20%
variation in the volume of the sample droplet (16 –24
␮L) resulted in a ⱕ0.21 g/dL SD (CV 4.3%) of the
estimated [Hb] for samples with true [Hb] ⫽ 5 g/dL;
ⱕ0.66 g/dL (CV 5.6%) for 10 g/dL; ⱕ0.67 g/dL (CV
4.2%) for 15 g/dL; and ⱕ0.93 g/dL (CV 4.4%) for
20 g/dL (see Supplemental Fig. 1A, which accompanies
the online version of this article at http://www.
clinchem.org/content/vol59/issue10). As expected, the
size of the blood stain depended on the droplet volume.
For droplets of the same volume, but with different
true [Hb] values (5, 10, 15, and 20 g/dL) the normal-
ized size of the blood stains produced by the droplets
Paper-Based Hb Test for Resource-Limited Settings
had an SD of ⱕ0.03 (CV 4.7%) for 16 ␮L; ⱕ0.03 (CV
3.9%) for 18 ␮L; ⱕ0.03 (CV 4.5%) for 20 ␮L; ⱕ0.04
(CV 4.1%) for 22 ␮L; and ⱕ0.03 (CV 3.7%) for 24 ␮L
(see online Supplemental Fig. 1B).
Finally, we tested the long-term stability of the
paper-based Hb assay measurements by scanning the
sheets of paper containing the blood stains 4 times dur-
ing the first 20 min, while the stain was still drying, and
13 times over the next 24 h, for samples with true [Hb]
of 5, 10, 15, and 20 g/dL. The color of the dried blood
stain remained stable after the initial drying period.
We therefore were able to scan and analyze the blood
stains at any point over the next 24 h without any
significant change in the [Hb] measurement (see on-
line Supplemental Fig. 2). During this study, the
room temperature in our laboratory varied from
18 °C to 30 °C, and humidity varied from 20% to
80%. We did not systematically test the effect of
room temperature or humidity variations on the
performance of the assay.
Discussion
The measurement of blood [Hb] is one of the most
frequently performed laboratory tests within a wide
range of medical settings, from acute trauma care to
chronic disease management (1 ). While the primary
reason for initial [Hb] screening is to diagnose (or rule
out) anemia, frequent monitoring of [Hb] is often re-
quired in the context of many conditions to quantify
blood loss, track disease progression, assess treatment
efficacy, or monitor patients during procedures. When
reliable diagnostic devices are available, they are often
not used because they are considered to be prohibi-
tively expensive for the resource-limited settings of de-
veloping countries (33 ). For example, in Kenyan dis-
trict hospitals, nearly 15% of children with clinical
histories indicative of malaria or anemia did not have
their [Hb] tested (20, 34 ). In this context, a reliable,
low-cost assay for measuring [Hb] would improve
the diagnosis of anemia and could help minimize
waste of limited resources on unneeded treatments
and reduce the risk of complications associated with
misdiagnosis (20, 25, 33 ). The urgent need for such
an assay in sub-Saharan Africa (in areas endemic
with sickle cell disease and malaria) was the primary
motivation for this study.
Our paper-based Hb assay measures [Hb] by sim-
ply quantifying the appearance of a stain produced by
the blood sample on paper. Perhaps the most widely
used alternative low-cost method for estimating [Hb]
in resource-limited settings is the WHO HbCS. To per-
form this test, the user must visually compare the color
of a paper strip stained with blood to a standard at a
certain time after the blood was applied to the strip.
Unlike the HbCS, our paper-based Hb assay is not af-
fected by changes in ambient light because the flatbed
scanner itself provides consistent illumination, and the
color can be read at any time within a 24-h period after
collection of the blood stain. The result is not affected
by operator bias because the measurement is per-
formed completely automatically.
Very little training is required to perform our
paper-based Hb assay. All a user would need to do is to
mix a finger-prick volume of blood with Drabkin re-
agent at the appropriate ratio and deposit a droplet of
this mixture on paper. The complexity of these opera-
tions is about the same as that of the sample prepara-
tion steps required from consumers for performing at-
home rapid diagnostic tests for sperm count (35 ) or Hb
A1c (36 ). All subsequent operations of the paper-based
Hb assay are completely automated and user-
independent, including the scanning and analysis of
the stains. Notwithstanding its simplicity, our paper-
based Hb assay demonstrated excellent comparison
(SD ⫽ 0.62 g/dL, n ⫽ 54) with the Medonic hematol-
ogy analyzer. The performance of our assay also com-
pares favorably with conventional POC devices
(14, 19, 37 ), particularly in the context of our target
applications: (a) providing the measurement of [Hb]
when no other methods are available, and (b) enabling
public health screening in resource-limited settings
where the lowest cost, rather than the highest accuracy,
is most important.
We used a USB-powered flatbed scanner (Canon
CanoScan LiDE110, $44 on amazon.com as of April 21,
2013) and a laptop computer to digitize and analyze the
blood stains. Any computer that has a functional USB
port and complies with the system requirements of the
scanner could in principle be used, including older sal-
vaged or refurbished models. Importantly, the scanner
and laptop could be used for many other purposes, not
exclusively for performing our paper-based Hb assay.
The primary reason for using these multipurpose,
consumer-grade electronic devices as the capital equip-
ment part of a low-cost diagnostic assay is the ability to
tap into the already well-established market of con-
sumer electronics. These devices are much more avail-
able in resource-limited settings than specialized med-
ical equipment such as a spectrophotometer and can be
used for performing the [Hb] measurements without
any modification of their original function. This ap-
proach represents an important advantage of our work
with respect to recent studies that follow the conven-
tional paradigm of POC development and propose to
measure [Hb] by use of a new, potentially low-cost
electronic device (38, 39 ). Such a device, designed spe-
cifically for measuring [Hb], would have to be sepa-
rately developed, manufactured, distributed, cali-
brated, and maintained. The use of these POC devices,
Clinical Chemistry 59:10 (2013) 1511
particularly the maintenance and/or replacement of
broken devices, requires a level of medical and engi-
neering infrastructure that is largely unavailable in
resource-limited settings (40 ).
Another important advantage of our approach is
that the use of existing scanners and laptops, which
may already be present in even the most remote facili-
ties because they were purchased by consumers for
their personal use, could allow for an effectively “zero-
cost” analyzer, compared with analyzer costs for con-
ventional POC devices ranging from $800 to $15 000.
In addition to the prohibitively high cost of the analyz-
ers, conventional POC devices often require proprie-
tary disposable supplies with per-test costs ranging
from $0.02 to $3.67 (19 ). In contrast, the components
required to perform our paper-based [Hb] assay are
available generically at an estimated combined cost of
⬍$0.007 (0.7 US cents) per test.
In summary, we developed and validated a paper-
based, point-of-care Hb assay for the low-cost assess-
ment of [Hb]. This assay represents a major step to-
ward effective intraoperative care as well as [Hb]
testing at the bedside or in urgent care settings where
traditional methods of the clinical laboratory are un-
available or prohibitively expensive. The paper-
based Hb assay will be useful for diagnosing anemia
in resource-limited settings of low-income develop-
ing countries (e.g., sub-Saharan Africa), particularly
in the context of malaria, HIV, and sickle cell
disease.
Author Contributions: All authors confirmed they have contributed to
the intellectual content of this paper and have met the following 3 re-
quirements: (a) significant contributions to the conception and design,
acquisition of data, or analysis and interpretation of data; (b) drafting
or revising the article for intellectual content; and (c) final approval of
the published article.
Authors’ Disclosures or Potential Conflicts of Interest: Upon man-
uscript submission, all authors completed the author disclosure form.
Disclosures and/or potential conflicts of interest:
Employment or Leadership: None declared.
Consultant or Advisory Role: J. Kanter, ApoPharma, Adventrix
Pharmaceuticals.
Stock Ownership: None declared.
Honoraria: None declared.
Research Funding: This work was supported in part by a 2012 NIH
Director’s Transformative Research Award (NHLBI R01HL117329,
PI: S.S. Shevkoplyas).
Expert Testimony: None declared.
Patents: X. Yang, 61/692,994, 61/558,009; N.Z. Piety, 61/692,994; J.
Kanter, 61/692,994; S.S. Shevkoplyas, 61/692,994, 61/558,009.
Role of Sponsor: The funding organizations played no role in the
design of study, choice of enrolled patients, review and interpretation
of data, or preparation or approval of manuscript.

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