RESEARCH ARTICLE

A JH
Characteristics of a rapid, point-of-care lateral flow
immunoassay for the diagnosis of sickle cell disease
Patrick T. McGann,* Beverly A. Schaefer, Mary Paniagua, Thad A. Howard, and Russell E. Ware

Sickle cell disease (SCD) is a common and life-threatening hematological disorder, affecting approximately
400,000 newborns annually worldwide. Most SCD births occur in low-resource countries, particularly in sub-
Saharan Africa, where limited access to accurate diagnostics results in early mortality. We evaluated a
prototype immunoassay as a novel, rapid, and low-cost point-of-care (POC) diagnostic device (Sickle
SCANTM) designed to identify HbA, HbS, and HbC. A total of 139 blood samples were scored by three
masked observers and compared to results using capillary zone electrophoresis. The sensitivity (98.3–100%)
and specificity (92.5–100%) to detect the presence of HbA, HbS, and HbS were excellent. The test
demonstrated 98.4% sensitivity and 98.6% specificity for the diagnosis of HbSS disease and 100% sensitivity
and specificity for the diagnosis of HbSC disease. Most variant hemoglobins, including samples with high
concentrations of HbF, did not interfere with the ability to detect HbS or HbC. Additionally, HbS and HbC
were accurately detected at concentrations as low as 1–2%. Dried blood spot samples yielded clear positive
bands, without loss of sensitivity or specificity, and devices stored at 378C gave reliable results. These
analyses indicate that the Sickle SCAN POC device is simple, rapid, and robust with high sensitivity and
specificity for the detection of HbA, HbS, and HbC. The ability to obtain rapid and accurate results with both
liquid blood and dried blood spots, including those with newborn high-HbF phenotypes, suggests that this
POC device is suitable for large-scale screening and potentially for accurate diagnosis of SCD in limited
resource settings.
Am. J. Hematol. 91:205–210, 2016. V
C 2015 Wiley Periodicals, Inc.

䊏 Introduction
Inherited disorders of hemoglobin, primarily sickle cell disease (SCD) and the b-thalassemias, represent an enormous and unaddressed global
public health problem [1,2]. Conservative estimates suggest that approximately 400,000 infants are born with SCD each year worldwide [1,3–5].
The most common and severe form is homozygous hemoglobin SS (HbSS) disease, which represents over 75% of annual SCD births and occurs
most frequently in sub-Saharan Africa and India. The second most common form of SCD is compound heterozygous hemoglobin SC (HbSC) dis-
ease, which is found mostly in West Africa and the Americas. Over 90% of SCD births occur in low- and middle-income countries, and over 9
million women who are carriers for a significant hemoglobin disorder become pregnant each year [1,6]. In the vast majority of these cases, the
pregnant women and their partners are not aware of the type or significance of the hemoglobin variants that they carry, and outside of Europe
and the United States, neonatal screening programs are usually not available to identify affected infants.
The lack of appropriate and timely diagnosis of SCD has life-threatening consequences for affected infants and young children. In North America
and many European countries, neonatal screening for hemoglobinopathies provides early diagnosis of SCD, and allows provision of parental SCD
education and early initiation of life-saving preventive care [7,8]. Without the availability of early diagnosis, most children born with SCD in limited-
resource settings will die within the first several years of life, usually before a diagnosis is even made, presumably due to severe anemia or infection [9].
WHO estimates that SCD contributes the equivalent of 5% of all under-five mortality on the African continent and up to 16% in some high-
prevalence African countries [10]. The importance of early diagnosis of SCD is beginning to be recognized and several pilot newborn screening
programs have been developed across sub-Saharan Africa [11–16]. While most laboratory methods that diagnose SCD are accurate, such as hemoglo-
bin electrophoresis by isoelectric focusing (IEF), high-performance liquid chromatography (HPLC), and capillary zone electrophoresis (CZE), these
techniques require expensive equipment, reagents, and electricity, as well as specialized technology with training, and typically provide results several
days after blood collection. In limited-resource settings with unreliable telephone service and no definitive street addresses, it can be difficult to track
and inform families of infants with positive screening results [11]. For these reasons, the development and implementation of a simple, rapid, inexpen-
sive, and accurate point-of-care (POC) diagnostic tool for SCD is urgently needed. Within the United States, the National Institutes of Health has rec-
ognized the importance of POC diagnostics for SCD through a recent Request for Applications for the development of such assays [17]. Due to the

Additional Supporting Information may be found in the online version of this article.
Division of Hematology, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio
Conflict of interest: Nothing to report.
*Correspondence to: Patrick T. McGann, MD MS; Division of Hematology, Cincinnati Children’s Hospital, 3333 Burnet Ave, MLC 11027 Cincinnati, OH
4522. E-mail: patrick.mcgann@cchmc.org
Received for publication: 22 September 2015; Revised: 30 October 2015; Accepted: 2 November 2015
Am. J. Hematol. 91:205–210, 2016.
Published online: 4 November 2015 in Wiley Online Library (wileyonlinelibrary.com).
DOI: 10.1002/ajh.24232

C 2015 Wiley Periodicals, Inc.

V

doi:10.1002/ajh.24232 American Journal of Hematology, Vol. 91, No. 2, February 2016 205
McGann et al.
importance of timely diagnosis and early intervention, the development
of a simple, robust, accurate, reliable, and affordable POC device could
be transformative for the identification and management of SCD in
low-resource settings such as sub-Saharan Africa.
Recently, there have been several reports of POC devices that quickly
establish the diagnosis of SCD based on differential characteristics
between HbAA and HbSS erythrocytes, including a paper-based test that
quantifies sickle hemoglobin and a density-based rapid test using aqueous
multiphase systems [18–21]. A third POC immunoassay test was recently
described, reporting near perfect sensitivity and specificity for the identifi-
cation of HbA, HbS, and HbC in normal, trait, and disease samples [22].
We designed and performed simultaneous experiments using the same
POC device, prior to knowledge of this publication; however, with several
important differences. Our work included rigorous experiments designed
to carefully evaluate multiple important performance aspects of the POC
assay from an unbiased perspective, with an overall objective of determin-
ing whether this test is likely to be an effective and reliable diagnostic or
screening test.
䊏 Methods
Description of the device. The Sickle SCANTM (Biomedomics, Inc., Research Tri-
angle Park, NC) device is a lateral flow chromographic qualitative immunoassay for
the rapid determination of the presence or absence of HbA, HbS, and HbC that is
intended to diagnose the common forms of SCD [22]. The testing cartridge has
four detection bands, including a distal control band that appears when the sample
has flowed to the end of the testing strip (Fig. 1). The presence of normal (HbA)
or variant (HbS or HbC) hemoglobins is indicated by a dark blue line in the spe-
cific region indicated on the device.
Study objectives. The main objectives of this study included a determination of
the sensitivity, specificity, accuracy, and ease of using the Sickle SCAN device to
identify the presence of HbA, HbS, and HbC using blood samples from pediatric
and adult patients with known or suspected hemoglobin disorders. All experiments
were performed using deidentified blood samples previously tested at the Erythro-
cyte Diagnostic Laboratory at Cincinnati Children’s Hospital Medical Center, using
a research protocol approved by the local Institutional Review Board. Additional
study objectives included (1) analysis of samples containing high fetal hemoglobin
(HbF) and other variant hemoglobins; (2) comparison of liquid blood samples to
dried blood spot (DBS) cards; and (3) reliability of the results after storing the
devices at warm temperatures.
Blood samples. This study included the analysis of the following types of blood
specimens: (1) venous samples collected in EDTA and stored at 48C until analysis;
(2) venous samples transferred to filter paper (Whatman FTATM Classic Cards, GE
Healthcare Bio-Sciences, Pittsburgh, PA, USA) to create DBS; and (3) artificial
“spiked” samples with low fixed percentages of HbA, HbS, and HbC. The majority
of samples included combinations of HbA, HbS, or HbC, but in order to identify
potential interference by hemoglobin variants, some samples included both com-
mon and uncommon variants that were available for testing. POC devices were
kept at room temperature, except for some experiments that involved storage at
378C for 30 days, to evaluate the effects of elevated temperature.
Sample analysis. Blood samples collected in EDTA were analyzed by CZE to
determine the types and percentages of normal and abnormal hemoglobins in each
sample; these results were used to identify samples for testing by the POC device
and served as the reference value. Variants detected by CZE were confirmed by a
second method (citrate agar electrophoresis or isoelectric focusing). Neither the
results nor the types of hemoglobins were discussed with the investigators evaluat-
ing the device, so that all scoring was performed by masked observers. These three
na€ıve observers read the instructions in the package insert but received no formal
training or hands-on experience with the test prior to the results presented here.
The Sickle SCAN kit includes tubes prefilled with 1.0 mL of buffer, which lyses
erythrocytes and releases hemoglobin. As per the manufacturer’s recommendation,
whole blood samples were tested by adding 5 mL of blood from the EDTA tube to
the prefilled buffer container, mixing by inverting the tube three times, discarding
the first 3 drops of the lysate, and then applying 5 drops to the testing cartridge.
For DBS samples, a 3 mm punch from the card was dropped into the prefilled
buffer container, and then the same procedure was followed. Five minutes after
sample application, the three observers visually scored each sample independently
for the presence or absence of all 4 potential bands (HbA, HbS, HbC, and control).
Figure 2 illustrates the individual steps of the assay and the Supplemental Video
demonstrates the assay procedures and result interpretation in real time.
Study design. The study was designed to assess multiple characteristics of this
POC assay and to evaluate its potential utility as a screening and diagnostic test for
SCD in clinical settings. The specific objectives of this study included the following
206 American Journal of Hematology, Vol. 91, No. 2, February 2016
RESEARCH ARTICLE
determinations: (1) sensitivity and specificity of the POC test to detect HbA, HbS,
and HbC in both typical homozygous and heterozygous states; (2) accuracy in
establishing the diagnosis of sickle cell disease; (3) specificity of the assay for HbA,
HbS, and HbC by including samples containing many common and variant hemo-
globins that might cross-react in the immunoassay; (4) sensitivity to detect low
concentrations of HbA, HbS, and HbC using artificial “spiked” samples; (5) feasibil-
ity of testing DBS samples, analogous to heel stick samples from neonatal screening
or finger stick samples from older children and adults; (6) stability of the POC
reagents and cartridge at 378C to simulate the climate of sub-Saharan Africa; (7)
consistency of results interpretation over time; and (8) interobserver agreement of
scoring and interpretation using three observers.
Statistical analysis. All statistical analyses were performed using Stata 13.1 (Sta-
taCorp, College Station, TX). A P value of <0.05 was considered significant. For all
calculations, the presence or absence of HbA, HbS, and HbC as detected on the
POC assay was compared to the gold standard result obtained by CZE. Sensitivity
and specificity were calculated for each individual observer and also in aggregate,
such that the score by each observer was counted as a single experiment. The 95%
confidence intervals for the sensitivity and specificity were calculated using the
exact Clopper–Pearson method. Interobserver agreement was calculated using
Fleiss’ kappa statistic.
䊏 Results
Sensitivity and specificity for the detection of HbA,
HbS, and HbC
A total of 139 whole blood samples were systematically analyzed
with Sickle SCAN by three independent masked clinicians and com-
pared to CZE results as a reference. The samples had both normal
and abnormal hemoglobin patterns, including sickle cell trait (HbAS),
sickle cell disease (HbSS and HbSC), both homozygous and heterozy-
gous HbC (CC and AC), and several samples from transfused sickle
cell patients. As shown in Table I, the presence of HbA, HbS, and
HbC was easily detected in both heterozygous and homozygous sam-
ples. Both HbS and HbC were correctly identified by all 3 observers
every time they were present in a sample, except for one patient with
HbSb1-thalassemia, for which a single observer did not identify the
presence of HbA. The sensitivity (99.5%) and specificity (92.5%) for
the detection of HbS were excellent. There were no false-positive or
false-negative results for the detection of HbC, with sensitivity and
specificity of 100%. For HbA, the sensitivity (98.3%) and specificity
(94.0%) were also excellent. Table II details the sensitivity, specificity,
and positive and negative predictive values for the detection of each
hemoglobin band with the associated 95% confidence intervals. The
table provides data for each individual observer and also in aggregate.
Accuracy for the diagnosis of sickle cell disease
After determining the specificity and sensitivity to detect individual
hemoglobin bands (HbA, HbS, and HbC), the POC test was next
evaluated to establish the diagnosis of SCD, either homozygous HbSS
or compound heterozygous HbSC disease. For the accurate diagnosis
of homozygous HbSS disease, the test demonstrated a cumulative sen-
sitivity of 98.4% (95% CI 91.5–100%) with individual observer sensi-
tivity ranging from 95.2% to 100%. Cumulative specificity for the
diagnosis of HbSS disease was 98.6% (95% CI 96.7–99.5%) with indi-
vidual observer specificity ranging from 97.5% to 99.2% for each of
the three observers. For the diagnosis of HbSC disease, both sensitiv-
ity and specificity were 100%. There was only one “missed” HbSS
diagnosis, i.e., a false-negative result, by a single observer in the initial
batch of samples, when the observer questioned the presence of a
faint HbA band and scored the sample SA. The other two observers
accurately identified HbSS in all 21 samples (Table I). Supporting
Information, Figure 2 illustrates the aggregate results for the accurate
diagnosis of the most common abnormal hemoglobin patterns.
Test performance with variant hemoglobins
In addition to HbA, HbS, and HbC, samples included several com-
mon and uncommon variant hemoglobins, in order to determine the
doi:10.1002/ajh.24232
RESEARCH ARTICLE Sickle cell point-of-care assay

Figure 1. POC device and results for common hemoglobin patterns. The Sickle SCAN POC device is an immunoassay that utilizes lateral-flow technology to
identify the presence of HbA, HbS, and HbC. The sample is applied on the pad at the bottom of the device, and the liquid flows toward the final control
band (Ctrl), with individual bands for HbC, HbS, and HbA spaced equally along the testing strip. The six common combinations of HbA, HbS, and HbC are
illustrated.

Figure 2. Steps of the point-of-care assay. The Sickle SCAN assay is a rapid and point-of-care device performed easily in several minutes. First, 5 mL of
blood (either from the finger as depicted in Panel A or pipetted from a previously collected sample) is added to 1.0 mL of prefilled buffer solution (Panels B
and C). The tube containing the sample and buffer solution is mixed well by inverting three times (Panel D) and after discarding 3 drops, 5 drops of the
lysate are added to the application pad on the Sickle SCAN cartridge (Panel E). The result is often visible within 1 min, but the final result is scored after 5
min with a darkened blue band indicating the presence of each hemoglobin (Panel F).

specificity of the HbS and HbC band detection. Variant hemoglobins was present at >50% in 24 samples (including 11 newborn samples
included Hb Bart’s, HbD-Los Angeles, HbE, HbG-Philadelphia, Hb with >80% HbF), with accurate scoring for the presence of HbA,
Hope, Hb Lepore, Hb I-Texas, Hb Lepore, and HbO (Table I). HbF HbS, and HbC in all cases, with no obvious interference from HbF.

doi:10.1002/ajh.24232 American Journal of Hematology, Vol. 91, No. 2, February 2016 207
McGann et al. RESEARCH ARTICLE

TABLE I. Performance of point-of-care Sickle SCAN test

No. of samples No. of samples incorrectly scored

No. of Result by Expected Sickle correctly scored by at least 1 observer
Sample type Samples reference method SCAN pattern by 3 observers (incorrect scored result)

Normal 38 A or FA A 36 2 (A, S)
Sickle Cell Disease 38
Hb SS 21 S or FS S 20 1 (A, S)
Hb SC 11 SC or FSC S, C 11 0
Hb Sb1-thalassemia 1 SA A, S 0 1 (S only)
Hb SD 1 SD S 0 1 (A, S)
On transfusions 5 SA or SDA A, S 4 1 (S only)
Sickle cell trait 34 AS or FAS A, S 32 2 (S only)
Hb C disease 9
Hb CC 5 C C 5 0
Hb C trait 4 AC A, C 4 0
Hb variants 19
Hb Bart’s 4 A, Bart’s A 4 0
Hb G-Philadelphia 3 AG A 1 2 (A, S)
Hb D Trait 3 AD A 3 0
Hb E Trait 2 AE A 2 0
Hb Lepore 1 A, Lepore A 0 1 (A, S)
Hb Hope 1 A, Hope A 1 0
Hb I-Texas 1 A, I A 0 1 (A, S)
Hb O 1 A, O A 1 0
Hb EE 1 FE No bands 0 1 (A, S)
Thalassemia major 2 AF A 2 0
(on transfusions)
Total 139 126 13

TABLE II. Sensitivity and Specificity for the Detection of HbA, HbS, and HbC

Sensitivity (%) Specificity (%) PPV (%) NPV (%)

HbA Observer 1 99.0 92.3 97.1 97.3

Observer 2 97.0 94.9 98.0 92.5
Observer 3 99.0 94.9 98.0 97.4
Cumulative: 98.3 94.0 97.7 95.7
(95% CI) (96.2–99.5) (88.1–97.6) (95.3–99.1) (90.1–98.6)
HbS Observer 1 100 92.4 93.6 100
Observer 2 98.6 92.4 93.5 98.4
Observer 3 100 92.5 93.5 100
Cumulative: 99.5 92.5 93.5 99.5
(95% CI) (97.5–100) (87.9–95.7) (89.6–96.3) (97.0–100)
HbC Observer 1 100 100 100 100
Observer 2 100 100 100 100
Observer 3 100 100 100 100
Cumulative: 100 100 100 100
(95% CI) (92.1–100) (99.0–100) (92.1–100) (99–100)

The majority of samples were scored correctly without interference
from other variant hemoglobins, but six samples were incorrectly
scored by at least one observer (Table I). Two samples (Hb FE and
Hb SDF) were incorrectly scored by all three observers since HbA
was incorrectly identified, while two observers labeled a positive HbS
band for another sample with reported HbAF 1 G-Philadelphia pat-
tern. Other hemoglobin variants that produced incorrect results
included Hb I-Texas and Hb Lepore, with a “false-positive” identifica-
tion of a very faint HbS band when HbS was not present in the
sample.
The likely reason for these errors was related to the fact that prior
to adding the liquid sample, a faint blue line is present at each band
location on the test strip (Supporting Information, Fig. 1A) and as
the sample flows across the strip, the line usually fades if the hemo-
globin type is not present and the faint blue line becomes nearly
invisible, although a na€ıve observer may still visualize a very faint
band when HbS is not present (Supporting Information, Fig. 1B).
When hemoglobin S is actually present, the band is quite intense, a
technical aspect of the test that was learned quickly after hands-on
experience.
Sensitivity for low concentrations of HbA, HbS, and
HbC
Spiked samples containing titrated amounts of HbA, HbS, or HbC
were included in the blinded testing. Hemoglobin S was detected
down to concentrations of <1% by one observer and reliably to
approximately 3–4% by all three observers. Hemoglobin C was reli-
ably detected down to concentrations of <1% by two observers and
to approximately 3–4% by all three observers. These results indicate
excellent sensitivity for detecting both abnormal hemoglobins critical
for the diagnosis of SCD. HbA, which produces a less-intense band,
was reliably detected by all three observers down to concentrations of
approximately 5–10%.

208 American Journal of Hematology, Vol. 91, No. 2, February 2016 doi:10.1002/ajh.24232
RESEARCH ARTICLE
Feasibility of dried blood spots
To evaluate the potential utility for analyzing DBS in a newborn
screening setting, whole blood was spotted onto DBS for seven differ-
ent samples. These DBS samples included all relevant hemoglobin
patterns for the diagnosis of SCD, including homozygous SS and SC,
heterozygous hemoglobin S and C trait, and three samples had >80%
HbF. All seven DBS samples yielded clear positive bands and the cor-
rect hemoglobin patterns were accurately identified in all cases, with-
out loss of sensitivity or specificity.
Effect of storage conditions
The manufacturer recommendations suggest that the devices and
reagents be stored between 2 and 308C, but the conditions in sub-
Saharan Africa often reach temperatures above 308C. In order to
investigate the effects of high temperature, three devices (including
reagents) were stored at 378C for 1 month. After exposure to this
high temperature, these devices all gave identical results to those
stored at room temperature. Similarly, blood samples stored at 48C
for up to 1 month still gave accurate bands, compared to fresh sam-
ples (Supporting Information, Fig. 3).
Consistency of results over time
The Sickle SCAN assay is designed to be a POC assay with results
available immediately at the time of testing, but durability of the vis-
ual result interpretation over time was also assessed. In general, the
test result was clear within 1 min after application to the cartridge,
but scoring was performed after 5 min per manufacturer’s recom-
mendations to allow complete wicking of the sample and accurate
scoring. Over time, the result indicator bands demonstrated some
mild discoloration when left at room temperature, but the interpreta-
tion of the results remained easy to read and interpret for many days.
Used devices were stored at 2208C with repeat interpretation after 4
days for 60 samples, and after 90 days for 26 samples; the scoring
was identical (60/60) after 96 hr and nearly identical (25/26) after 90
days, indicating substantial stability of the results over time.
Interobserver agreement
The test demonstrated excellent interobserver agreement with a
kappa value of 0.93 (z 5 33.17, P < 0.0001) based upon the final
hemoglobin pattern. The kappa values for the detection of HbA, HbS,
and HbC were 0.96 (Z 5 19.69, P < 0.0001), 0.95 (Z 5 19.43,
P < 0.0001), and 1.00 (Z 5 20.42, P < 0.0001), respectively.
䊏 Discussion
This pilot study demonstrates the ease, sensitivity, specificity, and
reliability of the prototype Sickle SCAN POC device to diagnose both
sickle cell trait and disease. A recently published report using the
same device, performed in collaboration with the manufacturer, eval-
uated 137 samples and demonstrated 100% sensitivity and specificity
for the diagnosis of all HbA, HbS, and HbC disorders [22]. In that
report, however, the diagnosis of a positive or negative band was
determined by scanning the assay strip, analyzing the color intensity
using a custom-coded algorithm, and using preset intensity cutoffs
for each band. Testing also included an additional 71 patients at the
POC with known HbA, HbS, and HBC genotypes with near-perfect
sensitivity and specificity for these samples [22]. Our experiments
were performed simultaneous to, and without knowledge of, this
recently published work. We confirm that the POC device has great
promise, but our carefully designed experiments demonstrate some
important differences from this recent publication. Specifically, two
important differences in our report are (1) the sensitivity and speci-
ficity are very good but not perfect and (2) there is a substantial vari-
doi:10.1002/ajh.24232
Sickle cell point-of-care assay
ability in the intensity of the HbA band with the potential of missing
the presence of HbA. We identified several additional strengths of
this POC device, including a high degree of accuracy in the presence
of high HbF, using DBS samples, and in hot conditions, along with
excellent interobserver agreement in result interpretation. We also
identified limitations of this new assay including potential interfer-
ence by abnormal hemoglobins, variable intensity of the HbA band,
and a small but real learning curve for those interpreting the test.
Our data purposefully included all tested and scored samples with-
out any “training period,” in order to report on potential errors in
assay performance and interpretation of results that could be made by
truly na€ıve and untrained observers. The observers were instructed to
follow the manufacturer’s direction and score any potential bands visi-
ble at 5 min, even if faint. The scoring of individual HbA, HbS, and
HbC bands did not always correlate with the actual percentages, indi-
cating that this prototype is qualitatively accurate, but should not be
used for quantitative assessments. In general, HbS and HbC bands
were much stronger than the HbA band, regardless of the actual per-
centages present in the sample. Even a faint HbA band indicated that
HbA was likely present, compared to stronger bands when either HbS
or HbC was present. Importantly, however, HbS and HbC in both
homozygous (disease) and heterozygous (trait) samples were accurately
reported, although our findings suggest ways for improving the device
and identifying clinical settings where the device may have limitations.
The results of this study are encouraging, but several limitations of the
Sickle SCAN device were noted with potential areas for improvement fol-
lowing hands-on use. The most problematic issue was inconsistency in
the intensity of the HbA band. As per the package insert, the current pro-
totype uses polyclonal antibodies to detect each hemoglobin type. After
evaluation of the POC device through these experiments, a potential area
of improvement is to either improve the affinity or concentration of the
HbA antibody or to use monoclonal instead of polyclonal antibodies. Our
results were still encouraging and mostly accurate despite the faint HbA
band, but the quality of the test would be greatly improved with a stronger
and more consistent HbA signal. Since this test would be especially useful
for screening neonatal samples, where the presence of a small (<10%)
amount of HbA can make the difference between a disease and carrier
state, this improvement would greatly improve the utility of the Sickle
SCAN assay. However, even in this prototype, the presence of HbS and
HbC was always indicated by a strong band and was not missed in any of
our samples. Another limitation of this device was the potential cross-
reactivity of some important Hb variants with the HbA band, notably
HbD and HbE. The reason for this cross-reactivity is presumably reten-
tion of HbA epitopes around the Glu residue at position 6, but critically
no tested variant had cross-reactivity with HbS or HbC. However, the
possibility of a false-positive HbA band suggests that the appearance of
HbA and HbS on the test strip may not always mean sickle cell trait. Var-
iants such as Hb D-Los Angeles (beta 121 Glu > Gln, HBB:c.364G > C),
HbE (beta 26 Glu > Lys, HBB:c.79G > A), and Hb O-Arab (beta 121
Glu > Lys, HBB:c.364G > A) that are pathological in the compound het-
erozygous state with HbS, but retain the HbA epitope at position 6, may
be incorrectly identified as HbAS. A third potential limitation is that
HbSb1-thalassemia may be difficult or impossible to differentiate from
the HbAS carrier state, depending on the limits of detection for HbA. In
these experiments, the strength and sensitivity of the HbA band appeared
to vary with different lots of reagents and test cartridges. Finally, a fourth
potential limitation is that our results were interpreted in a laboratory set-
ting by pediatric hematologists. An important follow-up study will
include evaluating the accuracy of this device when used in actual
limited-resource settings by local laboratory technicians or healthcare
providers.
The utility of a rapid and accurate POC diagnostic device for SCD
as described here is twofold: (1) to accurately identify persons (primar-
ily infants and young children) affected by SCD and (2) to screen large
American Journal of Hematology, Vol. 91, No. 2, February 2016 209
McGann et al. RESEARCH ARTICLE

populations to identify carriers for abnormal and disease-causing
hemoglobin variants such as HbS and HbC. The primary benefit of
such an assay for the screening of infants and young children is that
the result is accurate and available immediately after sample collection
and testing, even in the newborn setting, and does not require finding
persons with positive results. As a screening test for older populations,
such as adolescents or young adults, this POC assay would be able to
identify carriers of HbS or HbC accurately, and allow education
regarding the genetic basis of SCD and the risks associated with the
carrier state. These two primary potential uses of this POC assay could
directly reduce the morbidity and mortality of SCD, and also could
potentially decrease the number of SCD births due to improved aware-
ness by those who carry these hemoglobin variants.
In addition to Sickle SCAN [22], there are several recently pub-
lished reports of other POC assays that also may have clinical utility
for identifying SCD [18–21]. One paper-based assay identifies the
presence of HbS by measuring the separation of HbS from non-HbS
using differential wicking in a paper matrix [18,19]. This test has the
benefit of providing semiquantitative %HbS, but does not specifically
identify the presence or absence of HbA, HbC, or other hemoglobin
types. In addition, the time to complete this assay is reportedly 35
min, which is significantly longer than both the Sickle SCAN device
and even more advanced laboratory techniques such as HPLC and
CZE. A second recently described POC assay separates erythrocytes
by density using aqueous multiphase systems in order to diagnose
SCD [20,21]. This test is simple and rapid (12 min), but requires at
least a basic centrifuge and some training. This test is able to identify
HbSS accurately, but cannot differentiate HbAS from HbAA, and
thus cannot identify the HbS or HbC carrier state or homozygous
HbCC [20,21]. Also, because dense cells are not present in newborns
with SCD due to high percentages of HbF, the density gradient device
is likely not suitable for neonatal screening [20,21]. However, this vis-
ual test was recently evaluated in a clinical setting in Zambia, and
demonstrated sensitivity of 86% and specificity of 60% for the diag-
nosis of homozygous HbSS disease [21]. In contrast to these two pub-
lished POC assays, the Sickle SCAN device has the benefits of quickly
identifying the presence or absence of HbA, HbS, and HbC, and
yields rapid and accurate results even in high-HbF samples. Its reli-
ability after prolonged exposure to high temperatures further suggests
that it may be suitable for screening newborns, young children, or
adults in hot climates for sickle cell disease and carrier states, includ-
ing the compound heterozygous HbSC disease commonly found in
West Africa.
Together, our analyses indicate that the Sickle SCAN POC device
is simple, robust, and highly sensitive for detecting HbA, HbS, and
HbC, even in very low percentages. The device has been designed as
a low-cost test to be used in resource poor settings such as sub-
Saharan Africa and India. The device easily and rapidly detected
common hemoglobins, but was not quantitative. Specificity was excel-
lent even in the presence of high HbF and unusual hemoglobin var-
iants, with the possible exception of hemoglobin variants that share
some cross-reactivity with HbA. The ability to obtain rapid and accu-
rate results for both sickle cell disease and trait, using either liquid
blood or dried blood spots, including those with newborn high-HbF
phenotypes, suggests that this device has great potential for use in
large-scale sickle cell screening programs in limited-resource settings.
It remains to be seen whether this test, with the potential for occa-
sional inaccurate results due to rare and abnormal hemoglobin patterns,
should be offered primarily as a screening tool to identify patients who
need further testing by standard techniques like IEF or CZE, or can be
considered a diagnostic tool that establishes an accurate Hb result at the
time of testing. Since our data demonstrate that the assay reliably and
easily detects the presence of pathological HbS and HbC, even in very
low percentages, we believe this test could be used as a standalone diag-
nostic test in settings where confirmatory or advanced testing is not
available. Although two-phased testing and subsequent confirmation is
routine in the US and other developed countries, it may be time for a
paradigm shift to think more practically at the cost-effectiveness of a
single rapid and accurate diagnostic test such as Sickle SCAN, even
admitting a small chance of missing rare conditions such as HbSD or
HbSE disease. Future studies with POC devices should focus on such
“real-world” applications of these assays, with evaluations and direct
feedback from the end-users and policy-makers from limited-resource
settings where the need for such testing is the greatest.
䊏 Acknowledgments
The authors thank Biomedomics for providing the POC test car-
tridges free of charge for testing, and allowing all results to be pub-
lished without any prior review of the data or the written manuscript.

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210 American Journal of Hematology, Vol. 91, No. 2, February 2016 doi:10.1002/ajh.24232