research paper

A rapid, inexpensive and disposable point-of-care blood test

for sickle cell disease using novel, highly specific monoclonal
antibodies

Charles T. Quinn,1 Mary C. Paniagua,1

Robert K. DiNello,2 Anand Panchal2
and Mark Geisberg2
1

Cincinnati Childrens Hospital Medical Center,

Cincinnati, OH, and 2Silver Lake Research

Corporation, Azusa, CA, USA
Received 7 April 2016; accepted for publication
24 June 2016
Correspondence: Mark Geisberg, Silver Lake
Research Corporation, PO Box 787, Azusa, CA
91702, USA.
E-mail: mgeisberg@silverlakeresearch.com

Sickle cell disease (SCD) is a significant healthcare burden worldwide, but

most affected individuals reside in low-resource areas where access to diagnostic testing may be limited. We developed and validated a rapid, inexpensive, disposable diagnostic test, the HemoTypeSCTM, based on novel
monoclonal antibodies (MAbs) that differentiate normal adult haemoglobin
(Hb A), sickle haemoglobin (Hb S) and haemoglobin C (Hb C). In competitive enzyme-linked immunosorbent assays, each MAb bound only its
target with <0
1% cross-reactivity. With the HemoTypeSCTM test procedure,
the sensitivity for each variant was <5
0 g/l. The accuracy of HemoTypeSCTM was evaluated on 100 whole blood samples from individuals with
common relevant haemoglobin phenotypes, including normal (Hb AA,
N = 20), carrier or trait (Hb AS, N = 22; Hb AC, N = 20), SCD (Hb SS,
N = 22; Hb SC, N = 13), and Hb C disease (Hb CC, N = 3). The correct
haemoglobin phenotype was identified in 100% of these samples. The accuracy of the test was not affected by Hb F (094
8% of total Hb) or Hb A2
(05
6% of total Hb). HemoTypeSCTM requires <1 ll of whole blood and
no instruments or power sources. The total time-to-result is <20 min.
HemoTypeSCTM may be a practical solution for point-of-care testing for
SCD and carrier status in low-resource settings.
Keywords: sickle cell disease, haemoglobinopathy, point of care, lateral flow
immunoassay, monoclonal antibody.

Sickle cell disease (SCD), a group of related genetic disorders

of haemoglobin, is most prevalent in sub-Saharan Africa and
in populations with African ancestry. In equatorial Africa,
the prevalence of sickle trait is 1040% (Piel et al, 2010;
Mulumba & Wilson, 2015). SCD also occurs elsewhere
throughout the world, especially in India, the Americas, Europe and the Middle East. More than 400 000 children are
born with SCD and >5 000 000 with sickle trait every year
(Piel et al, 2013). Taken together, the worldwide population
at risk for SCD or sickle trait is over 2 billion. It has been
estimated that 5090% of newborns with SCD in Africa die
in early childhood (Grosse et al, 2011), typically undiagnosed
until late in the course of the disease (Chakravorty & Williams, 2015). In many low-resource environments, haemoglobin electrophoresis and neonatal screening programmes for
haemoglobinopathies are available only as regional or pilot
programmes (Tshilolo et al, 2008; Makani & Roberts, 2016).
Therefore, it is likely that few individuals in these areas will
know their own carrier status, and most will be unaware of
2016 John Wiley & Sons Ltd, British Journal of Haematology

their risk of having children with SCD (World Health Organization, 2010).
An accurate diagnosis is the first step in improving the
outcomes of individuals with SCD. In high-resource environments, such as the United States, United Kingdom and
many European countries, universal newborn screening for
haemoglobinopathies has clearly decreased childhood
mortality from SCD (Telfer et al, 2007; Quinn et al, 2010).
Diagnostic testing for haemoglobinopathies, whether by
screening newborns or later in life, involves a number of
laboratory methods, including various electrophoretic techniques (gel- or capillary-based), high-pressure liquid chromatography (HPLC), mass spectrometry and genetic testing.
All of these methods require special equipment, oftentimes expensive, and highly trained personnel. However,
over 80% of the population at risk for SCD reside in lowand middle-income countries. The high cost and technological barriers of standard diagnostic methods prohibit their
implementation.

doi: 10.1111/bjh.14298

C. T. Quinn et al
Recently, several rapid diagnostic methods have been
described, including assays based on differential erythrocyte
density (Kumar et al, 2014), differential wicking of Hb S and
Hb A through filter paper (Yang et al, 2013), and a polyclonal antibody-based capture immunoassay (Kanter et al,
2015). While these represent significant advances in diagnostic capabilities for SCD, each has deficiencies. Each method
requires an instrument as either an integral part of the procedure or to achieve maximum sensitivity and specificity
(McGann et al, 2016; Piety et al, 2016), which can be prohibitive in low-resource settings. In addition, none of these
methods achieve 100% accuracy with or without instrumentation. In the case of the immunoassay device (SickleSCAN,
BioMedomics, Inc., Research Triangle Park, NC, USA), an
independent evaluation identified limitations to the specificity and sensitivity of the device in clinical settings, which
could be due to the use of polyclonal antibodies (McGann
et al, 2016).
We set out to develop monoclonal antibodies (MAbs)
specific for each of Hb A, Hb S and Hb C to the exclusion
of the other variants. There have been reports of such MAbs
(Jensen et al, 1985; Rosenthal et al, 1995; Epstein et al,
1996), but, to our knowledge, none has resulted in fieldusable immunoassays. These major haemoglobin variants differ from Hb A by a single amino acid at position 6 of the
mature b-globin chain (glutamate to valine for Hb S, glutamate to lysine for Hb C), perhaps explaining the difficulties
encountered by previous attempts to make such MAbs. We
successfully produced MAbs with the desired specificities,
and then produced lateral flow immunoassay test strips in a
competitive format the HemoTypeSCTM test. The procedure
for HemoTypeSCTM requires <1 ll of blood, no instrumentation or power sources, and has a total time-to-result of
<20 min. We then investigated the performance and accuracy
of these simple devices using whole blood from individuals
with known haemoglobin phenotypes. The results of this
evaluation indicate that the HemoTypeSCTM test is a promising new method for rapid, point-of-care determination of
haemoglobin phenotype in low-resource settings.

Methods
Monoclonal antibodies
Female 6- to 10-week-old Balb/C or BDF-1 mice were immunised with immunogen constructs containing the N-terminal
sequences of human Hb A (VHLTPEEKSAVTAL),
human Hb S (VHLTPVEKSAVTAL) and human Hb C
(VHLTPKEKSAVTAL). Splenocyte fusions were performed as
described (Shirahata et al, 1998; Greenfield, 2013) and the
resulting clones were screened by direct and competitive
enzyme-linked immunosorbent assays (ELISAs) as described
below. Hybridoma clones were selected if they (i) bound to
the correct Hb variant, (ii) did not bind to the other two
variants, (iii) could be competed by diluted, lysed blood
2

samples from homozygous donors of the correct phenotype

but not of the other two phenotypes, and (iv) were of the
IgG isotype, as determined by a commercial kit (SBA Clonotyping System AP, Southern Biotechnology Associates,
Birmingham, AL, USA; Cat. # 5300-04). Clones were stabilised by multiple rounds of limiting dilution subcloning
and cryopreserved. The MAbs described here are commercially available from Silver Lake Research Corporation,
Azusa, CA, USA (http://www.silverlakeresearch.com). MAbs
for subsequent ELISAs and lateral flow test strip construction
were purified by Protein G chromatography (Sigma-Aldrich,
St. Louis, MO, USA; Cat. # P4691) or other methods.

ELISA
The methodology of indirect competitive ELISA has
been described previously (Kemeny & Challacombe, 1988).
Briefly, ferrous stabilised human Hb A (Sigma-Aldrich; Cat.
# H0267) and Hb S (Sigma-Aldrich; Cat. # H0392), were
used as capture antigens. As Hb C is not commercially available, an analogue, BSA-C, was produced by conjugating
bovine serum albumin (Sigma-Aldrich; Cat. # A2934), to a
synthetic peptide containing the N-terminal 14 amino acids
of Hb C (VHLTPKEKSAVTAL) through the heterobifunctional linker e-maleimidocaproic acid N-hydroxysuccinimide
ester (EMCS; Prochem, Rockford, IL, USA, Cat. # CL-222)
(Chen et al, 2003). Ninety-six-well enzyme immunoassay
plates were coated with 50 ll of 1 lg/ml of each capture
antigen for 1 h and washed three times with PBS containing
0
05% Tween 20 (PBST). For direct ELISA, antibodies were
added to the wells at 50 ll/well. For competitive ELISAs,
25 ll of lysed blood samples, diluted in H2O, were added as
competitors to the wells, followed by addition of 25 ll of
anti-haemoglobin antibodies (1029-31, 1029-13, and 1029-8)
to a final concentration of 1 lg/ml and incubated for
45 min. After three additional washes, 50 ll of alkaline phosphatase (AP)-conjugated goat anti-mouse immunoglobulin G
(American Qualex, San Clemente, CA, USA) was added at
1 lg/ml in PBST and incubated for 45 min. The plates were
again washed three times with PBST and 50 ll of 1 mg/ml
p-nitrophenyl phosphate substrate (Thermo Fisher Scientific,
Waltham, MA, USA) was added to the wells. The plates were
read at 405 nm after allowing the substrate to develop for
30 min. Data points were performed in triplicate. Data are
presented as % of maximal reading (in the absence of competitor) after subtraction of background optical density (OD)
(in the absence of primary antibody), mean of triplicates.

HemoTypeSCTM lateral flow test strips

The methods for construction of lateral flow immunochromatographic test strips have been well described (Wong &
Tse, 2008). Briefly, for the HemoTypeSCTM test, Hb A, Hb S
and BSA-C (described above), as well as an irrelevant mouse
immunoglobulin, were conjugated to colloidal gold
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Rapid Test for SCD

nanoparticles and dried onto the surface of polypropylene
test vials. Capture-purified MAbs 1029-31, 1029-13 and
1029-8, as well as the control capture antibody (goat antimouse-Ig polyclonal serum) were deposited in discrete lines
onto the surface of plastic-backed nitrocellulose membranes.
Test strips were comprised of laminated fiberglass sample
pads, antibody-impregnated nitrocellulose and a cellulosic
wick, so that a liquid sample can flow sequentially through
the three materials in order. An assay buffer, containing
detergents and non-specific blocking reagents, was used in
the procedure to rehydrate the dried gold conjugate and
dilute the lysed blood sample. The test procedure for the
HemoTypeSCTM test entailed: (i) lysis of the blood sample by
adding 1 ll whole blood to 1 ml of distilled H2O, (ii) rehydration of the dried gold conjugate in the test vial with
150 ll of assay buffer, (iii) adding 15 ll of lysed blood to
the fiberglass portion of the test strip, (iv) placing the test
strip into the test vial, (v) allowing the liquid to migrate
through each portion of the test strip for 10 min, and (vi)
taking the test strip out of the vial and visually determining
the presence or absence of a red-coloured line at each of four
locations (representing, in order, Hb C, Hb S, Hb A and
Control). Following the competitive principle, the absence
of a line indicates the presence of the specific haemoglobin
variant.

Blood samples for clinical validation

Blood samples for clinical validation of the HemoTypeSCTM
were obtained from the Erythrocyte Diagnostic Laboratory at
Cincinnati Childrens Hospital Medical Center (CCHMC).
Standard clinical diagnostic methods were followed for identification and quantitation of haemoglobins. Briefly, all samples were first tested by capillary zone electrophoresis [CZE;
CAPILLARYSTM 2 FLEX Piercing (Sebia, Inc., Norcross, GA,
USA)] or HPLC [Alliance Separation Module 2690/2695,
dual wavelength absorbance detector 2487 (Waters, Inc., Milford, MA, USA)]. Detected Hb variants that were consistent
with HbS or HbC in the heterozygous state by CZE or HPLC
were confirmed by acid agarose gel electrophoresis. Other
variants and HbS and HbC in apparent homozygous or compound heterozygous states were confirmed by both acid agarose gel electrophoresis and isoelectric focusing. Total
haemoglobin concentration of blood samples and spiked
solutions of Hb A and Hb S were determined by the pyridine-haemochromagen method (Barr & Guo, 2015) and confirmed by direct spectroscopy (Prahl, 2012).
A convenience sample set of approximately 100 blood
samples was obtained to represent normal individuals, the
trait state for Hb S and Hb C, the two major forms of SCD
(Hb SS and Hb SC), and haemoglobin C disease (Hb CC).
All blood samples were left-over, de-identified specimens that
were originally obtained for clinically-indicated diagnostic
testing (haemoglobin analysis) that would otherwise have
been discarded. Therefore, the requirement for informed
2016 John Wiley & Sons Ltd, British Journal of Haematology

consent was waived, and the study was exempt from fullboard review by the local Institutional Review Board. Each
sample was phenotyped using standard clinical methods (see
above) and used in the HemoTypeSCTM procedure as
described. Results were read visually by two operators and
photographed; a third individual confirmed the interpretation of results from electronic images. All three individuals
interpretations of each HemoTypeSCTM result were concordant in all samples.

Results
Monoclonal antibodies specific for Hb A, Hb S, and Hb C
Monoclonal antibodies were produced by screening hybridoma clones for exclusive binding to purified Hb A and Hb
S, and to BSA conjugated to synthetic peptides with the
N-terminal sequence of Hb C (purified Hb C is not commercially available). MAbs were further selected by competing
the binding to the specific antigen by lysed blood from Hb
AA, Hb SS and Hb CC donors. In this format, specificity of
antibodies is determined by testing for binding of free antibody with free antigen in solution, which decreases the binding of the antibody to a capture antigen immobilised on a
surface. In the indirect competitive ELISA format, the
selected MAbs (1029-31 for Hb A, 1029-13 for Hb S and
1029-8 for Hb C) each bound exclusively to their respective
haemoglobin variant and that binding was competed by lysed
blood samples containing the respective haemoglobin variant
(Fig 1). This set of three competitive ELISAs correctly identified each relevant homozygous and heterozygous phenotype,
including normal (Hb AA), carrier (Hb AS and Hb AC),
sickle cell disease (Hb SS and Hb SC) and Hb C disease (Hb
CC). No competition by fetal haemoglobin (Hb F, a2c2) or
by Hb A2 (a2d2) was seen with any of these three MAbs
(data not shown). Taken together, these results are consistent
with the binding of each MAb being dependent on the presence of a variant-specific residue in position 6 of the Hb b
chain. For at least the anti-Hb A MAb 1029-31, the binding
is also dependent on the presence of b-chain-specific residues
in positions 9 and 12 of the Hb b chain, as these positions
are different in the d chain of Hb A2.
In dilution experiments in the same competitive ELISAs,
whole blood of the targeted phenotype competed the binding of each antibody at dilutions up to 1:1051:106, while little or no competition was seen with blood of the nontargeted phenotypes even at dilutions of 1:100 (Fig 2). The
IC50 (half maximal inhibitory concentration) of each targeted Hb variant was at least 1000-fold lower than the IC50
of the other two variantsin fact, the non-targeted variants
did not reach 50% inhibition of binding at any measured
concentration. These results indicate that these MAbs are
capable of specifically binding each haemoglobin variant in
solution, with <0
1% cross-reactivity with non-targeted
variants.
3

C. T. Quinn et al

Fig 1. Binding of anti-haemoglobin MAbs to SCD-associated haemoglobin variants. ELISA plates were coated with Hb A (Panel A),
Hb S (Panel B) and BSA conjugated to the N-terminal peptide of Hb
C (Panel C). Monoclonal antibodies (MAbs) 1029-31, 1029-13 and
1029-8 were applied in the presence of competitor haemoglobins whole blood of known Hb phenotype, lysed and diluted 1:400 in
H2O or no competitor (H2O only). After washing, binding of
MAb to the plate surface was determined by addition of secondary
antibody (goat anti-mouse-Ig conjugated to alkaline phosphatase),
followed by addition of the colourimetric phosphatase substrate
p-nitrophenyl phosphate. Results were determined by optical density
at 405 nm (OD405). For each antigen/MAb/competitor combination,
data are presented as percentage of maximum MAb binding to
that antigen (defined as OD405 of highest-binding MAb with no
competitor).

Fig 2. Cross-reactivity of anti-haemoglobin MAbs with Hb A, Hb S

and Hb C. ELISA plates were coated with Hb A for monoclonal antibody (MAb) 1029-31 (Panel A), with Hb S for MAb 1029-13 (Panel B)
and with BSA-C for MAb 1029-8 (Panel C). Competitive ELISAs were
performed as in Fig 1, using lysed whole blood from phenotyped
homozygous donors (Hb AA, Hb SS, and Hb CC) as competitors.
Blood samples were serially diluted in H2O. Data are presented as percentage of maximum MAb binding (with no competitor).

HemoTypeSCTM test strip

The HemoTypeSCTM test was designed as a competitive lateral flow immunochromatographic assay (LFA) (Ngom et al,
2010) in order to translate the performance of the three

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Rapid Test for SCD

competitive ELISAs into a rapid, inexpensive test kit that
requires no instrumentation and few procedural steps. The
HemoTypeSCTM test strip incorporates each of three independent competitive immunoassays for Hb A, Hb S and Hb C.
Each immunoassay comprises red-coloured colloidal gold
particles conjugated to variant-specific antigens Hb A, Hb S
and BSA-C, and the corresponding capture MAbs (1029-31
for Hb A, 1029-13 for Hb S and 1029-8 for Hb C) immobilised at a specific location on the LFA test strip (Fig 3A).
The flow of the sample through the strip carries the particles
through capture MAb-impregnated membrane lines. In the
absence of the specific haemoglobin variant, the three MAbconjugated colour particles bind to their specific immobilised
antigens, producing red-coloured lines on the test strip. The
presence of each haemoglobin variant in a sample is indicated
by the absence of a colour signal at a specific line location on
the test strip (Fig 3B, C). The test strip also has a control
line to indicate that sample flow through the strip proceeded
correctly.
It was found that the optimal visual result was obtained
using a 15-ll sample of 1:1000 diluted whole blood. One ll
of whole blood was diluted in 1 ml H2O to lyse erythrocytes
immediately. The testing procedure entails adding the 15-ll
sample to the strip, then placing the strip into a sample vial
containing rehydrated colloidal gold particles. The presence

of each line on the test strip is determined visually. The

entire procedure is completed in about 20 min.
In the competitive LFA format, the limit of detection
(LOD) of the assay is determined by the minimal concentration of analyte that completely eliminates the colour signal at
the capture MAb location. The sensitivity of the HemoTypeSCTM test for each Hb variant was determined using samples of decreasing concentrations of Hb A, Hb S and Hb C,
created by spiking blood from Hb SS donors with Hb A, Hb
AA blood with Hb S, and Hb AA blood with Hb CC blood,
respectively (Fig 4). Using the measured total haemoglobin
concentration and the fraction of total Hb represented by the
variant Hb, it was determined that the LOD of HemoTypeSCTM was <4
0 g/l for Hb A, <5
0 g/l for Hb S, and <2
0 g/l
for Hb C. In a typical heterozygous sample with a total haemoglobin concentration of 150 g/l, these sensitivities correspond to <2
7% for Hb A, <3
3% for Hb S and <1
3% for
Hb C.

Clinical validation of HemoTypeSCTM using whole blood

samples
We tested whole blood samples from 100 unique individuals
with established haemoglobin phenotypes (Table I). The test
procedure was performed at CCHMC by personnel with no

Fig 3. HemoTypeSCTM test strip. (A) Design of

competitive lateral flow immunoassay. The top
panel shows a schematic of the lateral flow test
strip with a sample containing Hb S () and
colloidal gold particles conjugated to Hb A
(), Hb S (), BSA-C(), and an irrelevant
mouse immunoglobulin (*). The test strip has
monoclonal antibodies (MAbs) 1029-8 (antiHb C), 1029-13 (anti-Hb S), 1029-31 (anti-Hb
A) and goat anti-mouse-immunoglobulin
deposited at lines C, S, A, and Control, respectively. Flow of this sample through the test
strip (bottom panel) results in the capture of
red gold particles at the C, A, and Control
lines of the strip, but not at the S line, where
the Hb S from the sample competes for the
MAb binding sites. (B) Appearance of test strip
results for Hb SS phenotype; (C) Schematic of
appearance of test results for each relevant
phenotype.

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C. T. Quinn et al
F and Hb A2 because they are not recognized by the anti-Hb
MAbs in HemoTypeSCTM, and the presence or absence of Hb
F (094
8% of total Hb in this sample set) or Hb A2 (0
5
7% of total Hb in this sample set) does not affect the
results of haemoglobin phenotyping. Similarly, the test is
blind to Hb Barts.
We tested whole blood samples from 11 additional unique
individuals with established haemoglobin phenotypes that the
HemoTypeSCTM test strip was not designed to identify and
differentiate (Table II). All non-Hb S and non-Hb C structural variants were not identified by the test strip. Heterozygotes for these variants appeared to have only Hb A. One
compound heterozygote for Hb S and Hb D (Hb SD disease)
appeared to have Hb S and Hb A, indicating that anti-Hb A
MAb cross-reacts with Hb D. An individual with sickle-b0thalassaemia was correctly identified to have only Hb S present. An individual with sickle-b+-thalassaemia was correctly
identified to have both HbS and HbA present, but this qualitative determination does not differentiate it from HbS trait.

Discussion

Fig 4. Limits of detection of HemoTypeSCTM for Hb A, Hb S, and

Hb C. For determination of the limit of detection of HemoTypeSCTM
for each haemoglobin variant, tests were performed on samples of
Hb SS blood spiked with Hb A to known concentrations (06
0 g/l,
Panel A); samples of Hb AA blood spiked with Hb S (Panel B); and
samples of Hb AA blood spiked with Hb CC blood of known total
Hb and known percentage of Hb C (Panel C). The result windows
of HemoTypeSCTM test strips are shown for each sample. The limit
of detection of the HemoTypeSCTM test for each Hb variant is the
lowest concentration of that variant that results in a total absence of
the respective result line in the test results.

prior training, and the results were interpreted by two observers (who reported no discrepant results). The correct haemoglobin phenotype was identified in 100% of these samples
by the HemoTypeSCTM test strip, including normal haemoglobin type (Hb AA), sickle trait (Hb AS), Hb C trait (Hb
AC), homozygous sickle cell anaemia (Hb SS), sickle-haemoglobin C disease (Hb SC), and haemoglobin C disease (Hb
CC). In this sample set, sensitivity and specificity were 100%
for each Hb variant that the test was designed to identify
(Hb A, Hb S and Hb C). The test had 100% accuracy across
a broad range of clinically relevant concentrations of these
Hb types (Hb A: 698
2% of total Hb; Hb S: 4
386
9%; Hb
C: 4
599
4%). As expected, the test was blind to both Hb
6

The HemoTypeSCTM test is a promising new method for

rapid, point-of-care determination of haemoglobin phenotypes, specifically for the major subtypes of SCD and related
carrier states, in low-resource settings. HemoTypeSCTM
requires <1 ll of blood and no instrumentation or power
sources. The interpretation of test results is visual and qualitative, so extensive training is not required. The total timeto-result is <20 min. The test is designed for use at the site
of blood collection, without the need to prepare a dried
blood spot or transport it elsewhere for testing. This timeframe gives healthcare providers the opportunity for parent
education and immediate intervention, without the disadvantage of locating the affected individuals after a long delay of
obtaining test results. The simple test-strip design minimises
the costs of production and packaging for low-resource settings. The estimated cost of materials for this version of
HemoTypeSCTM is less than U.S. $0
25. Similar tests using
the same format (competitive LFA), but for other analytes,
have a shelf life of >2 years at ambient temperatures of up to
40C, which is a key advantage of this method.
One advantage of the competitive format is the absence of
a prozone effect. In sandwich-type assays, high concentrations of analyte can give a low signal or a false negative
result. (Gillet et al, 2009) This is caused by an excess of antigen relative to the binding sites on the sandwich antibodies,
so that all sites are saturated by antigen without a sandwich
being formed. Assay developers must use a variety of procedural adjustments to counteract potential prozone effects.
This is a particular problem in detecting haemoglobin variants, which can be present at >150 mg/ml in whole blood
samples. In the competitive format used in HemoTypeSCTM,
any concentration of Hb above the LOD results in a total
elimination of signal, thus the prozone effect does not exist.
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Rapid Test for SCD

Table I. Results of HemoTypeSCTM for the diagnosis of individuals
with normal haemoglobin type, sickle cell trait, Hb C trait, sickle cell
anaemia, sickle-Hb C disease, and Hb C disease.

Patient
Category

Normal
(HbA A)*

20

Sickle cell
trait (Hb AS)

22

Hb C trait
(Hb AC)

20

Sickle cell
anaemia
(Hb SS)
Sicklehaemoglobin
C disease
(Hb SC)
Hb C disease
(Hb CC)

22

13

Range of
Hb levels in
clinical Hb
analysis (%)
A: 93
398
2
F: 02
A2: 1
84
7
A: 666
5
S: 4
340
8
F: 089
7
A2: 03
3
A: 8
166
C: 4
535
4
F: 087
4
A2: 04
1
S: 11
586
9
F: 088
5
A2: 05
7
S: 19
551
C: 16
745
F: 162
A2: 1
84
7
C: 5
299
4
F: 0
294
8
A2: 0

Results of
clinical
Hb analysis

Results of
HemoTypeSCTM

A (19)
A F (1)

A S (10)
A S F (4)
F A S (8)

AS

A C (5)
A C F (10)
F A C (5)

AC

S
S
F
S
S
F

S (6)
F (14)
S (2)
C (6)
C F (6)
S C (1)

C C (2)
F C (1)

Table II. Results of HemoTypeSCTM for other structural haemoglobin variants and thalassaemia.

Patient Category

Type

Hb I trait

a-globin
variant

Hb
G-Philadelphia
trait

a-globin
variant

a-thalassaemia
trait

a-globin
gene deletion

Hb D-Punjab
trait

b-globin
variant

Hb E trait

b-globin
variant

Hb O-Arab trait

b-globin
variant

Hb Hope trait

b-globin
variant

Hb Lepore trait

db fusion
product

Sicklehaemoglobin
D disease
Sickleb0-thalassaemia

Compound
heterozygous
SCD
Compound
heterozygous
SCD
Compound
heterozygous
SCD

SC

*Includes 2 individuals with increased Hb A2 (>3
3%).

All haemoglobin types detected are listed in order of decreasing
abundance (e.g., AF indicates Hb A > Hb F). The numbers in
parentheses indicate the number of individuals with the specified
haemoglobin pattern.
All haemoglobin types detected by the HemoTypeSCTM test strip.
The order in which the haemoglobins are listed is arbitrary.

HemoTypeSCTM was designed with highly-specific MAbs

to Hb A, Hb S and Hb C to differentiate the following conditions: normal Hb type (Hb AA), sickle cell trait (Hb AS),
Hb C trait (Hb AC), homozygous sickle cell anaemia (Hb
SS), sickle-Hb C disease (Hb SC) and Hb C disease (Hb
CC). The sensitivity and specificity of HemoTypeSCTM was
100% when tested against a panel of whole blood specimens
from 100 unique individuals with these combinations of haemoglobins. By comparison, no other rapid, non-instrument
test for haemoglobinopathies has reported specificities over
98
5% (Yang et al, 2013; Kumar et al, 2014; Kanter et al,
2015; McGann et al, 2016; Piety et al, 2016). This difference
may be significant because of the low prevalence of SCD,
even in areas of relatively high penetration of the genetic
trait. For example, in Uganda, with one of the worlds highest reported frequencies of Hb S trait carriers, the percentage
of children born with SCD is about 2% (Okwi et al, 2010).
A newborn screening test with a specificity of 98
5% could
potentially identify 1
5% of non-SCD infants as SCDpositive, in addition to correctly identifying the vast majority
2016 John Wiley & Sons Ltd, British Journal of Haematology

Sickleb+-thalassaemia

Results
of clinical
Hb analysis*

Results
of
HemoTypeSCTM

A: 70
8%
I: 27
2%
A2: 2%
A: 69
6%
F: 5
1%
G-Phil:
22
2%
A2: 1
4%
G2-Phil:
1
7%
A: 69
6%
F: 28
8%
A2: 1
3%
Barts: 0
3%
A: 57
4%
D: 39
7%
A2: 2
9%
A: 71
1%
E: 25
8%
A2: 3
1%
A: 58
5%
O-Arab +
A2: 41
55
A: 51
9%
Hope:
43
7%
A2: 3
8%
A: 81
1%
Lepore:
16
7%
A2: 2
2%
S+D: 72
6%
F: 24
8%
A2: 2
6%
S: 80
7%
F: 13
4%
A2: 5
9%
S: 80
7%
A: 4
4%
F: 12
1%
A2: 2
8%

AS

AS

*When Hb fractions could not be resolved by the electrophoretic

method, the sum of the two is indicated.
All haemoglobin types detected by the HemoTypeSCTM test strip.
The order in which the haemoglobins are listed is arbitrary.

of the 2% of true SCD infants as SCD-positive. In this case,

the predictive value of the positive result would be only
57%, which may result in unnecessary treatment for a large
number of individuals. In areas with <0
5% incidence of
SCD births, such as regions of Brazil (Lervolino et al, 2011),
the predictive value of the positive result of a test with
98
5% specificity would decrease to <25%. The specificity,
7

C. T. Quinn et al
sensitivity, and the predictive value of both positive and negative results of the HemoTypeSCTM test in this study were
100%; further studies are needed to ascertain whether this
accuracy can be approached when the test method is used in
low-resource settings.
When blood was tested from individuals with haemoglobinopathies for which HemoTypeSCTM was not designed to differentiate (i.e., not Hb S or Hb C), these structural variants
gave results consistent with Hb A. For sickle-b0-thalassaemia,
HemoTypeSCTM gave the correct Hb phenotype (presence of
Hb S only). Homozygous sickle cell anaemia (Hb SS) and
sickle-b0-thalassaemia have the same Hb phenotype, and
their clinical management is identical, so the conflation of
these genotypes with HemoTypeSCTM would have no direct
clinical consequences. For sickle-b+-thalassaemia, HemoTypeSCTM again gave the correct Hb phenotype (presence of Hb
A and Hb S). However, this non-quantitative result does not
differentiate it from sickle cell trait, where Hb A > Hb S,
while in sickle-b+-thalassaemia Hb S > Hb A. Sickle-haemoglobin D disease was misidentified as having Hb A and Hb
Sas expected and explained by the sequence identity in the
N-terminal region between b chains of Hb A and Hb D. The
SickleSCAN assay was shown to have the same limitations,
and any antibody-based test specific for N-terminal differences between the b chains of Hb A, S, and C can be
expected to perform in the same manner. The inability to
discriminate sickle-b+-thalassaemia and sickle-haemoglobin
D disease from sickle cell trait does have direct clinical consequences.
For all forms of protein-based testing, the differentiation
of sickle cell trait from sickle-b+-thalassaemia requires a
quantitative analysis. In both conditions, Hb A and Hb S will
be present but differ in proportion (Hb S > Hb A for sickleb+-thalassaemia). If applied to newborn screening, the
HemoTypeSCTM would properly identify three of the most
common forms of SCD without additional testing: homozygous sickle cell anaemia, sickle-haemoglobin C disease, and
sickle-b0-thalassaemia. In general, these are the most severe
forms of SCD. However, sickle-b+-thalassaemia, the third
most common form of SCD, and other rare compound
heterozygous disease forms, would not be properly identified
with one-stage testing.

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Author contributions
CTQ, MP, RKD, AP and MG performed the research. CTQ,
RKD, AP and MG analysed the data. CTQ and MG wrote
the paper.

Conflict of interest
RKD, AP and MG are employees of Silver Lake Research
Corporation. CTQ is a consultant for Silver Lake Research
Corporation. CTQ and MP receive research funding
from Silver Lake Research Corporation through a grant
from NIH-NHLBI to Silver Lake Research Corporation
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