Validation of Paper-Based Assay for Rapid Blood Typing - American

Dec 12, 2011 - ABSTRACT: We developed and validated a new paper-based assay for the detection of human blood type. Our method involves spotting a 3 μ...
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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 1661

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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.

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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

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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 1662

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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 1663

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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

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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. 1664

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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 1665

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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 1666

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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.

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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

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (G.G.); wei.shen@monash. edu (W.S.); [email protected] (M.A.-T.).

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ACKNOWLEDGMENTS This work was funded by an Australian Research Council linkage grant (Grant LP110200973).

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REFERENCES

(1) Daniels, G.; Reid, M. E. Transfusion 2010, 50, 281−289. (2) Daniels, G.; Bromilow, I. Essential Guide to Blood Groups, 2nd ed.; Wiley-Blackwell: Chichester, West Sussex, U.K., 2010. (3) Malomgre, W.; Neumeister, B. Anal. Bioanal. Chem. 2009, 393, 1443−1451. (4) Daniels, G.; Castilho, L.; Flegel, W. A.; Fletcher, A.; Garratty, G.; Levene, C.; Lomas-Francis, C.; Moulds, J. M.; Moulds, J. J.; Olsson, M. L.; Overbeeke, M.; Poole, J.; Reid, M. E.; Rouger, P.; van der Schoot, E.; Scott, M.; Sistonen, P.; Smart, E.; Storry, J. R.; Tani, Y.; Yu, L. C.;

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Analytical Chemistry

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dx.doi.org/10.1021/ac202948t | Anal. Chem. 2012, 84, 1661−1668