Article
pubs.acs.org/ac
Validation of Paper-Based Assay for Rapid Blood Typing
Mohammad Al-Tamimi,*,† Wei Shen,*,† Rania Zeineddine,‡ Huy Tran,‡ and Gil Garnier*,†
†
Australian Pulp and Paper Institute, Department of Chemical Engineering, Monash University, Australia
Dorevitch Pathology, Australia
‡
ABSTRACT: We developed and validated a new paper-based assay for the detection of
human blood type. Our method involves spotting a 3 μL blood sample on a paper surface
where grouping antibodies have already been introduced. A thin film chromatograph tank was
used to chromatographically elute the blood spot with 0.9% NaCl buffer for 10 min by
capillary absorption. Agglutinated red blood cells (RBCs) were fixed on the paper substrate,
resulting in a high optical density of the spot, with no visual trace in the buffer wicking path.
Conversely, nonagglutinated RBCs could easily be eluted by the buffer and had low optical
density of the spot and clearly visible trace of RBCs in the buffer wicking path. Different paper
substrates had comparable ability to fix agglutinated blood, while a more porous substrate like
Kleenex paper had enhanced ability to elute nonagglutinated blood. Using optimized
conditions, a rapid assay for detection of blood groups was developed by spotting blood to
antibodies absorbed to paper and eluted with 200 μL of 0.9% NaCl buffer directly by pipetting. RBCs fixation on paper
accurately detected blood groups (ABO and RhD) using ascending buffer for 10 min or using a rapid elution step in 100/100
blood samples including 4 weak AB and 4 weak RhD samples. The assay has excellent reproducibility where the same blood
group was obtained in 26 samples assessed in 2 different days. Agglutinated blood fixation on porous paper substrate provides a
new, simple, and sensitive assay for rapid detection of blood group for point-of-care applications.
A
Few point-of-care assays that can be done without laboratory
equipment or blood collection have been developed.13−16 Most
of these tests require pretreatment of blood or reconstitution of
antibody, are adversely affected by prolonged storage,17 and
could have a high percentage of errors in interpreting the
results (18.2−39.8%) leading to erroneous transfusion.16,18−21
Development of simple, rapid, and reliable assays for blood
grouping would be of great value for bedside compatibility
checks and quick blood grouping in emergency scenarios and in
situations where there is no access to laboratory facilities such
as in rural areas, military facilities, and in developing countries.
Recently, multiple studies have highlighted the promising use
of paper for diagnostic and environmental applications,
especially in developing countries and for point-of-care
applications.22−26 A new paper-based assay was reported
recently for the rapid detection of blood grouping through
application of blood to a filter paper presoaked with antibodies
which leads to the formation of a plasma separation band with
the agglutinated blood.27 Detection of blood group using blood
separation was applied to only a few blood samples obtained
from normal volunteers. Despite the simplicity of this approach,
blood wicking through capillary absorption occurs within
seconds, which could limit the antibody−antigen interaction
necessary for agglutinated blood formation, should blood flow
not be perfectly controlled.2,8 Weak and/or slow agglutination
could occur without the formation of a clear separation band.
The development of any assay for medical diagnostic
ccurate assessment of human blood group is critical for
safe blood transfusion and transplantation medicine.1 The
blood group is determined based on the presence or absence of
certain antigens on red blood cells (RBCs).2,3 In the last
century, 328 different antigens have been identified on RBCs
and classified into 30 different blood groups, among them ABO
and RhD blood groups are still the most important.2,4 Every
year about 75 million units of blood are collected worldwide to
be used for treatment of multiple clinical conditions or for life
saving procedures.5 One third of unscreened blood transfusions
can lead to a hemolytic transfusion reaction that might be fatal.2
Accordingly, identification of ABO and RhD blood group for
both blood recipient and donor is mandatory to ensure
compatibility before commencement of blood transfusion.1,2
The identification of blood group is generally performed
using specific antibodies against RBC antigens that induce
RBCs agglutination. Agglutinated RBCs can be detected using
multiple diagnostic assays, including conventional tube test,
microplate and solid phase assays, gel column agglutination,
and affinity column technology.3,6−9 Recently, advanced but
highly technical assays for blood grouping have been reported
including gene sequencing of DNA10,11 and flow cytometrybased assays.12 The assays currently available are highly
sensitive and specific, objective, and reliable for detection of
blood groups.8 The major disadvantages of these assays are the
need for special laboratory instruments operated by trained
laboratory personnel, the long time required for the procedure
(10−30 min), and the high cost of these tests.6−8 Furthermore,
these assays routinely require 6 mL of blood collected by
syringe.
© 2011 American Chemical Society
Received: November 7, 2011
Accepted: December 12, 2011
Published: December 12, 2011
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resuspended by gentle shaking, and agglutination was recorded
according to the degree of agglutination as 4+ to 1+ against a
well-lit background.3,8
Agglutinated RBCs Fixation on Paper. Fresh 10 μL droplets
of Anti-A, Anti-B, and Anti-D antibody solutions were spotted
at 2 cm from the lower edge of blotting, filter, or Kleenex paper
and allowed to be absorbed completely for 30 s. A volume of 3
μL of undiluted blood was added to the center of each antibody
spot and allowed to interact for 30 s. The paper was then
suspended in 0.9% NaCl buffer in a thin film chromatography
tank about 1 cm from the lower edge to ensure the blood spot
remained above the buffer level and the buffer was allowed to
elute the paper by capillary absorption for 10 min (chromatographic elution). The paper was left to dry at room temperature
on a blotting paper for another 10 min and elution of RBCs
spotted on specific and nonspecific antibodies was observed.
The elution distance of RBCs was measured from the upper
edge of the original blood spot. The intensity of the blood spot
after elution was quantified by measuring the mean optical
density of the red color on a digital picture captured by an
Olympus camera (μ-9010) and analyzed using ImageJ software
(National Institute of Health). Each red-green-blue (RGB)
digital image was corrected by subtracting the background
using a rolling ball radius of 300 pixels. The entire image was
corrected for brightness to give a background optical density of
about 0 which minimizes variability due to lighting conditions.
The red spots were accurately outlined and the mean optical
density was measured.
Optimization of Agglutinated Blood Fixation on Paper.
To optimize agglutinated blood fixation on a paper substrate,
the elution distance and the mean optical density of the blood
spot after elution were compared using different paper
substrates (blotting, filter, and Kleenex paper) treated with
the same antibody and chromatographically eluted with the
same buffer. Serially diluted antibodies in 0.9% NaCl buffer
(total volume of 100 μL) from different clones were compared
using the same paper substrate and eluted with the same
elution buffer. Similarly, blood from the same donors collected
into different anticoagulants (citrate or EDTA) and eluted with
different buffers for different durations were compared. Using
the optimized conditions, the blood group was identified with
rapid elution by applying 3 μL of blood on grouping antibodies
absorbed to paper and allowed to interact for 30 s then eluted
with 200 μL of 0.9% NaCl buffer droplet applied dropwise by
pipet.
Statistical Analysis. The elution distance and the optical
density of the blood spot after elution were reported as mean ±
standard deviation (SD). The mean elution distance and the
mean optical density of blood collected from different donors
spotted over specific versus nonspecific antibodies were
compared using an unpaired two-tailed t test. To compare
the elution distance and the mean optical density in more than
two groups, one way analysis of variance (ANOVA) was
applied. Statistical analysis was performed using GraphPad
Prism (version 5) software with P < 0.05 considered significant.
applications requires thorough characterization and validation
as the assay should have a high rate of sensitivity and specificity
and needs to be reproducible with low interassay and intraassay variability. This is even more critical for developing assays
to detect human blood groups as the consequences of
misidentification of the blood group could be fatal should
incompatible blood be transfused. The application of the new
assay to a reasonable number of healthy and nonhealthy
individuals compared to standard assays is essential.
In this study, we report the development and validation of a
simple and rapid paper-based assay for human blood grouping.
Agglutinated RBCs are fixed onto paper interfiber spaces while
nonagglutinated RBCs can be eluted easily with 0.9% NaCl
buffer. This approach using an eluent was selected to remove
the effect of blood−antibody contact time from the study,
therefore simplifying the procedure to better characterize the
robustness of the paper based assay for blood typing. The
different factors that affect the performance of the assay are
characterized and the robustness of the assay is tested by
detecting blood groups (ABO and RhD) successfully for 100
blood samples within 1 min, including 8 samples with weak AB
and RhD antigen.
■
EXPERIMENTAL SECTION
Materials, Blood, and Antibodies. Antibodies against
RBC antigens approved for human blood grouping including
Anti-A IgM antibodies (clones 10090, 51000), Anti-B IgM
antibodies (clones 10091, BX 48000), and Anti-D IgM
antibodies (clones 20093, MS 201, 11270) were obtained
from Lateral Grifols, Australia. Epiclone Anti-A, Anti-B, and
Anti-D IgM antibodies were purchased from CSL, Australia.
The standard Drink Coster blotting paper 280 g−2 was from
Fibrosystem AB, Sweden. Whatman filter paper (no. 4) was
purchased from Whatman International Ltd., England. Kleenex
towel paper manufactured by Kimberly-Clark, Australia, was
also purchased. Analytical grades of NaCl, KCl, Na2HPO4, and
KH2PO4 were purchased from Sigma-Aldrich, USA. Anticoagulated blood (heparin, citrate, and EDTA) was collected
from adult volunteers with a known blood group or using
unidentified discarded blood samples from Dorevitch Pathology, Melbourne. The blood group was identified by a diagnostic
laboratory using the standard gel card assay (Lateral Grifols,
Australia). In this assay, agglutinated RBCs are trapped in the
gel while nonagglutinated RBCs travel through the gel to the
bottom of the tube by centrifugation. According to the traveling
distance of agglutinated RBCs, the results can be graded from
4+ to 1+.2,7,8 Weak AB and weak RhD were identified as
samples with weak agglutination of grade 1+ or 2+ by the gel
card assay. Blood samples were stored at 4 °C and analyzed
within 7 days of collection.
Methods. Confirmation of Donor’s Blood Type. The
recorded blood group of study participants was determined
using the standard gel assay test performed by a diagnostic
laboratory. The samples were retested independently using the
conventional slide test to confirm blood typing; briefly, 20 μL
of a 20% suspension of RBCs (20 parts of RBCs to 80 parts of
0.9% NaCl buffer) was mixed with 20 μL of Anti-A, Anti-B, and
Anti-D antibodies on a labeled glass slide for 2 min at room
temperature and blood agglutination was observed. For samples
with weak agglutination, a conventional tube test was
performed by mixing one drop of 3% RBCs suspension with
one drop of different antibodies at at 37 °C for 1 h and
centrifuged at 1000 rpm for 1 min, the bottom of the tube was
■
RESULTS
Fixation of Agglutinated Blood on Paper Substrates.
The effect of chromatographic elution on agglutinated and
nonagglutinated blood spotted on a porous structure was
investigated. Blood was spotted on grouping antibodies
absorbed to paper and then chromatographically eluted with
0.9% NaCl buffer for 10 min. Agglutinated blood consisted of
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Figure 1. Agglutinated blood fixation on blotting, filter, and Kleenex paper. Blood from donors with different blood groups (A+, B+, O+, and O−)
spotted on Anti-A, Anti-B, and Anti-D antibodies absorbed to blotting paper (a), filter paper (b), and Kleenex paper (c) and chromatographically
eluted with 0.9% NaCl buffer for 10 min. Agglutinated blood was fixed on the paper substrate, resulting in a high optical density of the spot, with no
visual trace in the buffer wicking path, while nonagglutinated blood has low optical density of the spot and a clearly visible trace in the buffer wicking
path.
distance of nonagglutinated blood was significantly higher
with Kleenex paper compared to blotting or filter paper (Table
1).
The mean optical density of the blood spotted on specific
antibodies after elution was significantly higher compared to
blood spotted on nonspecific antibodies on all paper substrates
(blotting paper 125 ± 13 versus 96 ± 14; P < 0.0001, filter
paper 115 ± 16 versus 74 ± 20; P < 0.0001, and Kleenex paper
100 ± 24 versus 7.9 ± 8; P < 0.0001) (Figure 2b).
Furthermore, the mean optical density of blood spotted to
nonspecific antibodies after elution was significantly lower with
Kleenex paper compared to blotting or filter paper, respectively
(8 ± 8 versus 96 ± 14 and 74 ± 20; P < 0.0001). There was no
detectable overlap between optical densities for agglutinated
and nonagglutinated blood with Kleenex paper. A cutoff mean
optical density point can be determined to achieve optimal
sensitivity (all agglutinated blood samples have optical density
above the cutoff point) and optimal specificity (all nonagglutinated blood samples have optical density below the
cutoff point) with Kleenex paper only (Figure 2b).
Optimization of Agglutinated Blood Fixation on
Paper. The different variables that affect agglutinated blood
fixation on paper were analyzed to understand the factors that
influence the assay performance and to improve the assay
sensitivity and specificity. The different paper substrates had
comparable ability to fix agglutinated blood. However, Kleenex
paper had enhanced ability to elute nonagglutinated RBCs as
shown by the higher elution distance and the decreased mean
optical density of the blood spot after elution (Figure 2 and
Table 1). Multiple brands of commercial towel paper were
blood spotted over specific antibodies including blood group A
with Anti-A, blood group B with Anti-B, and blood group RhD
positive (+) with Anti-D. Agglutinated blood resists elution and
remains fixed on the same spot. However, nonagglutinated
blood, which is the blood spotted on nonspecific antibodies,
can be eluted easily by ascending buffer which leads to the
formation of a faint blood spot (Figure 1).
While agglutinated blood could be fixed on the three
different paper substrates investigated (blotting, filter, and
Kleenex paper, Figure 1), the elution distance was greater with
Kleenex (Figure 1c), compared to blotting paper (Figure 1a) or
filter paper (Figure 1b). The nonagglutinated blood spot after
elution was almost invisible on Kleenex paper (Figure 1c) but
faintly visible on blotting or filter paper (Figure 1a,b). To
confirm this observation, blood collected from 31 donors
having different blood groups (A, B, AB, O, Rh+, and Rh−)
was spotted on Anti-A, Anti-B, and Anti-D antibodies absorbed
to blotting, filter, and Kleenex paper and subjected to
chromatographic elution with 0.9% NaCl buffer for 10 min
followed by measurement of the elution distance and the mean
optical density of the blood spot. Blood spotted on specific
antibodies absorbed to papers (blotting, filter, and Kleenex
paper) always resisted elution with minimal variability among
different donors, while blood spotted on nonspecific antibodies
could be eluted easily. There was no detectable overlap in the
elution distance with blood spotted on specific antibodies
compared to nonspecific antibodies on all paper substrates
(Figure 2a and Table 1). The elution distance was significantly
higher with nonagglutinated blood compared to agglutinated
blood with all paper substrates. Additionally, the elution
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both agglutinated and nonagglutinated blood (Table 1). The
RBCs elution distance increased significantly with increased
exposure time to elution buffer and was affected significantly by
lowering the pH of the buffer to 2.5 (Table 1). NaCl buffer
with low pH could elute both agglutinated and nonagglutinated
blood mostly because the antigen−antibody binding was
affected by pH of the media and the interaction was optimal
at neutral pH.2,8 In summary, agglutinated blood fixation in
paper was significantly affected by the paper substrate, antibody
concentration and clone, and the elution buffer, but there was
no significant effect for antibody or blood storage at 4 °C for up
to 7 days.
Detection of Blood Groups Using Agglutinated Blood
Fixation on Paper. Using the optimized conditions for
agglutinated blood fixation on paper, a rapid assay for the
detection of blood groups was developed (rapid elution). A 3
μL droplet of undiluted blood was deposited on Anti-A, Anti-B,
and Anti-D antibodies freshly absorbed to Kleenex or filter
paper. Blood was allowed to interact with the antibody for 30 s
before elution with a 200 μL droplet of 0.9% NaCl buffer
deposited slowly. Visual observation of fixation (clearly visible
blood spot with no visible trace at buffer wicking path) versus
elution (faint or invisible blood spot and clearly visible RBCs
trace at buffer wicking path) was used to identify the samples
blood group using chromatographic elution for 10 min or rapid
elution with 200 μL applied directly by pipetting (Figures 1 and
4). Blood obtained from 100 donors including 4 weak AB and 4
weak RhD with known blood grouping was determined by the
standard assay performed by a diagnostic laboratory and
confirmed by a conventional slide or tube test. Agglutinated
blood fixation detected the blood group accurately in all blood
samples including the difficult samples with weak RBC
antigens. This indicated a high sensitivity rate for the assay
(Table 2 and Figure 5). A clearer distinction between fixed and
eluted RBCs was obtained with Kleenex paper compared to
filter paper (Figures 1 and 4). Two of the four samples with
weak RhD antigen had incomplete fixation of blood deposited
on the Anti-D antibody. However, these weak RhD samples
could easily be distinguished from the negative RhD for which
most RBCs were eluted (Figure 5). Day to day reproducibility
of the assay was tested by performing the assay on 26 donors
having different blood groups on 2 different days. The same
blood group was detected for all the blood samples on the 2
different days, showing excellent reproducibility.
Figure 2. Quantitation of agglutinated blood fixation on blotting, filter,
and Kleenex paper. The elution distance (a) and the mean optical
density (b) of blood spotted on blotting, filter, and Kleenex paper
previously treated with specific and nonspecific antibodies and
chromatographically eluted with 0.9% NaCl for 10 min.
tested and it was found that the single sheet thick Kleenex
towel (Kimberly-Clark, Australia) provided the best fixation
and separation. As blood agglutination is induced by specific
antibody binding to targeted antigens on RBCs, blood spotted
on serially diluted Anti-A (10090), Anti-B (10091), and Anti-D
(20093) absorbed to Kleenex paper and eluted with 0.9% NaCl
buffer was investigated. Agglutinated blood fixation correlated
positively with the antibody concentration as the elution
distance increased significantly with the dilution of antibody
concentration (Figure 3a and Table 1). Tests using serially
diluted antibodies against the same RBCs antigen purified from
different clones were also investigated. Anti-A clone 51000,
Anti-B clone 10091, and Anti-D clone MS 201 had the best
ability to fix agglutinated RBCs (Figure 3b−d). There was no
effect on the elution distance of blood spotted on serially
diluted nonspecific antibodies purified from the different clones
(Figure 3, Table 1). Blood from the same donors added to fresh
antibodies or antibodies absorbed to paper and then stored at 4
°C or at ambient conditions for up to 7 days showed no
significant effect on agglutinated RBCs fixation (Table 1).
The ability of blood or buffer to diffuse into the dry antibody
spot absorbed to paper and stored at 4 °C or at ambient
conditions was slower compared to wet antibody added freshly
to paper. The elution distance of the remaining blood spot was
similar for blood collected into citrate or EDTA anticoagulant
tubes (Table 1), storing anticoagulated blood at 4 °C for up to
7 days also had no significant effect on the level of agglutinated
blood fixation on paper (Table 1). Different elution buffered
including 0.9% NaCl buffer (0.154 M NaCl, pH 7) and
phosphate buffer saline (80 mM NaH2PO4, 20 mM Na2HPO4,
100 mM NaCl, pH 7.4) had comparable ability to elute
nonagglutinated blood compared to distilled water that eluted
■
DISCUSSION
The fixation of RBCs on paper is affected by the paper
structure and chemical properties which governs the capillary
flow and chromatographic interaction of a complex fluid.
Different paper substrates including blotting, filter, and Kleenex
paper have comparable ability to fix agglutinated blood, while a
more porous substrate like Kleenex paper has an enhanced
ability to elute nonagglutinated blood. Nonagglutinated RBCs
can be chromatographically eluted with a buffer by capillary
action that can transport isolated RBCs from their original
blood spot. Blood elution in paper using capillary action is
affected by the blood viscosity and surface tension, nonspecific
interaction between RBCs and cellulose fibers, as well as
mechanical entrenchment of RBCs in interfiber spaces.24,27,28
Our results show that mechanical entrenchment leads to partial
elution of nonagglutinated RBCs in the thick and dense paper
substrates with small pores like blotting paper, while substrates
with larger pores such as Kleenex show enhanced elution.
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Table 1. Factors Affecting the Chromatographic Elution Distance of Blood Spotted on Specific and Nonspecific Antibodies
Including Paper Substrates, Antibody Concentration and Storage, Blood Collection, and Storage and Elution Buffera
a
P < 0.05 considered significant (unpaired two-tailed t test or one way ANOVA).
biologically compatible, recyclable, and suitable for colorimetric
assays.22−24,26−28 Many recent studies have established simple
and useful ways of handling paper for biological and
environmental purposes including inkjet printing of reagents,
enzymes, and microfluidics patterns.25,26,30−32 Paper provides a
unique substrate for the detection of blood grouping due to its
porous structure which facilitates the fixation of agglutinated
blood compared to nonagglutinated blood. Additionally, the
driving force provided by the paper capillary action facilitates
RBCs elution by simple deposition of a buffer with no need for
pumping, centrifugation, or other washing techniques. Agglutinated blood fixation on Kleenex paper is simple and objective,
and the assay results are easy to interpretate by giving a simple
yes/no answer (fixed/eluted) rather than direct interpretation
of agglutination which can be subjective and requires basic
laboratory training.18,20 There was no detectable overlap at any
individual point in the wicking distance or mean optical density
of blood spotted over specific versus nonspecific antibodies
absorbed to Kleenex paper, indicating optimal sensitivity and
specificity rate. The assay requires only a small amount of
undiluted whole blood and the reaction is stable and recordable
Increasing the porous spaces in paper facilitates RBCs elution
as the RBCs leave the original spot. Agglutinated blood which
forms larger particles is fixed completely in the interfiber spaces
of the paper. Immobilization of antibodies to the paper surface
would further enhance agglutinated RBCs fixation through
specific antigen−antibody interaction, independent of RBCs
agglutinate formation. The wet strength resin typically added to
towel paper and filter paper was previously shown to enhance
antibody adsorption without affecting the antibody activity.28,29
The correlation between the degree of RBCs agglutination and
blood fixation on paper was confirmed by the critical role of
antibody concentration and clone, by the ionic and pH effect of
the elution buffer, and by incomplete fixation in two of the four
weak RhD samples. Antibody concentration and epitope
recognition, RBCs antigen density, ionic concentration and
pH of the medium, and antigen−antibody interaction time are
variables known to play a critical role in RBCs agglutination.2,3,8
There are many advantages of using paper as a substrate for
diagnostic applications. Paper is widely available, flexible,
disposable, and inexpensive; it wicks fluid through capillary
action; and cellulose, the main component of paper, is
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Figure 3. Effect of antibody dilution and antibody clone on the blood elution distance for specific and nonspecific blood interactions on Kleenex (n
= 8). Elution distance of blood spotted over (a) serially diluted Anti-A, Anti-B, and Anti-D antibodies, (b) serially diluted Anti-A from different
clones, (c) serially diluted Anti-B from different clones, and (d) serially diluted Anti-D from different clones.
Table 2. Detection of ABO (a) and RhD (b) Blood Groups
in 100 Donors Using Blood Fixation on Kleenex and Filter
Paper by Chromatographic Elution for 10 min or by Rapid
Elution with 200 μL of 0.9% NaCl Buffer
(a)
Figure 4. Schematic representation of the rapid blood group detection
method by fixation of agglutinated blood on filter and Kleenex paper.
Agglutinated blood resisted elution and formed a clearly visible red
spot with Kleenex and filter paper while nonagglutinated blood could
be eluted resulting in almost an invisible blood spot with Kleenex
paper and a faint blood spot with filter paper.
chromatographic elution for
10 min
rapid elution with 200 μL
applied directly by pipetting
blood group
(ABO)
blood
fixation with
Anti-A
blood
fixation with
Anti-B
blood
fixation with
Anti-A
blood
fixation with
Anti-B
A (n = 38)
B (n = 15)
AB (n = 6)
O (n = 37)
weak AB
(n = 4)
38/38
0/15
6/6
0/37
4/4
0/38
15/15
6/6
0/37
4/4
38/38
0/15
6/6
0/37
4/4
0/38
15/15
6/6
0/37
4/4
(b)
blood group
(RhD)
positive
(n = 72)
negative
(n = 24)
weak RhD
(n = 4)a
for future reference. The main drawback of the current rapid
point-of-care assays is the high error rate in interpreting the
results often due to inexperienced operator, inaccurate
detection of weak RBCs antigens, and false positive results in
certain groups of patients.16−21,33
We have recently reported a new paper-based assay for the
rapid detection of blood grouping through application of blood
to a filter paper presoaked with antibodies. The ABO blood
group can be detected by observing a plasma separation band
from the initial blood droplet that appears with agglutinated
blood.27 Detection of a separation band depends on the
formation of large viscous agglutinates that wicked at a slower
rate compared to plasma, leading to the formation of a
separation band. Antibody-induced RBCs agglutination necessitates sensitization and bridging of targeted antigens on RBCs,
which requires a proper interaction time. Blood wicking
a
chromatographic
elution for 10 min
rapid elution with 200 μL applied
directly by pipetting
blood fixation with
Anti-D
blood fixation with Anti-D
72/72
72/72
0/24
0/24
4/4
4/4
2/4 weak RhD samples have ∼70% fixation with Anti-D antibody.
through capillary absorption occurs within seconds which can
limit the efficacy and the extent of antibody-induced RBCs
agglutination and therefore limits plasma separation band
formation, should blood flow and blood−antibody contact time
not be perfectly controlled.2,8 In weak/slow agglutination,
which commonly occurs in individuals with low RBCs antigen
density, blood wicking proceeds without the formation of a
clear separation band which compromises the assay sensitivity.
In this study, a rapid assay for detection of blood groups was
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countries. The variability in absolute optical density measurement of blood and the proper optical density cutoff value that
achieve the best sensitivity and specificity need to be
determined. Alternatively, using relative intensity of blood
spotted over specific antibodies compared to nonspecific
antibodies may be applied or samples compared to a positive
and negative control included as a reference standard. Analysis
of a larger number of samples obtained from normal volunteers
or hospitalized patients is warranted to assess the assay
sensitivity, specificity, and applicability.
■
CONCLUSIONS
In this study, a paper-based assay for rapid blood typing was
validated with 100 samples and the sensitivity of the test was
quantified. The variables investigated include paper structure,
antibody concentration and clone, elution solution, and blood
collection and storage conditions. RBCs agglutinated by specific
antigen−antibody interaction are fixed onto the paper
substrate, while nonagglutinated blood can be chromatographically eluted with NaCl buffer solution through capillary
action. Agglutinated RBCs fixation on paper depends on the
paper structure and characteristics, while different paper
substrates including blotting, filter, and Kleenex paper have
comparable ability to fix agglutinated blood; a more porous
substrate, like Kleenex paper, has an enhanced ability to elute
nonagglutinated blood. Antibody concentration and purification clone also play a critical role in the agglutinated RBCs
fixation, while antibodies absorbed to paper and stored at 4 °C
or at ambient conditions for up to 7 days have no significant
effect. A 0.9% NaCl elution buffer (pH ∼7) was superior to
distilled water in RBCs elution. RBCs fixation on paper
accurately detects blood groups (ABO and RhD) by chromatographic elution for 10 min or rapid elution with a 200 μL of
0.9% NaCl buffer applied directly by pipetting for all the 100
donors, including 8 donors with weak AB and RhD antigen.
The development and validation of quick, cheap, simple, and
instantaneous assays for blood typing can be of great value for
bedside compatibility checking prior to blood transfusion or the
quick identification of blood grouping in emergency scenarios
and any situations where no access to laboratory facilities is
available, such as remote rural areas, military facilities, and
developing countries.
Figure 5. Agglutinated RBCs fixation of weak RhD and AB samples
compared to normal samples. (a) Blood from weak RhD, normal RhD
+, and RhD − spotted over Anti-D antibody. Blood from weak AB,
normal AB, and O blood group spotted over Anti-A antibody (b) or
Anti-B antibody (c). All samples were chromatographically eluted with
0.9% NaCl for 10 min.
also developed by spotting blood to antibodies absorbed to
Kleenex or filter paper and eluted with 200 μL of 0.9% NaCl
buffer applied directly by pipetting. Blood was allowed to
interact with the antibody for 30 s to enhance antigen−
antibody interaction. This approach successfully identified
blood group in 100/100 blood samples including the weak
AB and RhD samples. Furthermore, RBCs fixation requires a
smaller volume of blood compared to RBCs wicking (3 μL
versus 20 μL), is much faster (1 min versus 4−10 min), and has
an elution distance which is measurable in centimeters
compared to the wicking distance measurable in millimeters.
Finally, RBCs fixation can be applied to dry antibodies
absorbed to paper.
Agglutinated blood fixation on porous paper substrate can be
used for visual determination of blood grouping for point-ofcare applications including the pretransfusion safety check
routinely performed before each blood transfusion in many
countries, for the quick identification of blood grouping in
emergency situations and in situations where no access to
laboratory facilities is available.16,18−20 Alternatively, by
measuring the optical density of blood spotted over specific
and nonspecific antibodies absorbed onto paper, the assay can
be applied for automated analysis using devices that measure
optical density, which may be invaluable in developing
■
AUTHOR INFORMATION
Corresponding Author
*E-mail: [email protected] (G.G.); wei.shen@monash.
edu (W.S.); [email protected] (M.A.-T.).
■
ACKNOWLEDGMENTS
This work was funded by an Australian Research Council
linkage grant (Grant LP110200973).
■
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