Laser incubation for the rapid detection of red cell alloantibodies in human blood samples.

BACKGROUND AND OBJECTIVES: Pre-transfusion antibody screening requires the detection and identification of immunoglobulin G (IgG) antibodies against red blood cells (RBCs). Using the indirect antiglobulin test (IAT), plasma-RBC solutions are incubated at 37°C in gel cards, typically by heating block technology. Here, we apply the newly developed laser incubation method to detect RBC alloantibodies in the plasma from human donors. MATERIALS AND METHODS: Donated human plasma samples (N = 128) containing clinically significant IgG antibodies directed against Rh (D, C, c, Cw and E), Kell (K and Kpa ), Duffy (Fya and Fyb ), Kidd (Jka ) and MNS (S) blood group system antigens were tested by the indirect antiglobulin test (IAT). Samples were heated to 37°C by infrared laser (980 nm) for incubations of up to 5 min. Samples were also incubated in a heating block for comparison. RESULTS: When heating by laser, the presence of an alloantibody is detected after only a 1-min incubation for 96% of samples. No samples required longer than 3 min of laser incubation in order to detect the antibody. For all samples, incubation by laser gave the same or stronger result within 5 min. No samples required longer than 5 min to achieve an equivalent result to that of the 5-min heating block incubation. The laser was not found to damage cells or antibodies. CONCLUSION: Laser incubation provides comparable results in shorter time frames than the heating block. Laser incubation can rapidly detect even very weak antibodies.

Column Agglutination Assay Using Polystyrene Microbeads for Rapid Detection of Antibodies against SARS-CoV-2.

Rapid serology platforms are essential in disease pandemics for a variety of applications, including epidemiological surveillance, contact tracing, vaccination monitoring, and primary diagnosis in resource-limited areas. Laboratory-based enzyme-linked immunosorbent assay (ELISA) platforms are inherently multistep processes that require trained personnel and are of relatively limited throughput. As an alternative, agglutination-based systems have been developed; however, they rely on donor red blood cells and are not yet available for high-throughput screening. Column agglutination tests are a mainstay of pretransfusion blood typing and can be performed at a range of scales, ranging from manual through to fully automated testing. Here, we describe a column agglutination test using colored microbeads coated with recombinant SARS-CoV-2 spike protein that agglutinates when incubated with serum samples collected from patients recently infected with SARS-CoV-2. After confirming specific agglutination, we optimized centrifugal force and time to distinguish samples from uninfected vs SARS-CoV-2-infected individuals and then showed concordant results against ELISA for 22 clinical samples, and also a set of serial bleeds from one donor at days 6-10 postinfection. Our study demonstrates the use of a simple, scalable, and rapid diagnostic platform that can be tailored to detect antibodies raised against SARS-CoV-2 and can be easily integrated with established laboratory frameworks worldwide.

Wash-free paper diagnostics for the rapid detection of blood type antibodies.

Identification of specific antibodies in patient plasma is an essential part of many diagnostic procedures and is critical for safe blood transfusion. Current techniques require laboratory infrastructure and long turnaround times which limits access to those nearby tertiary healthcare providers. Addressing this challenge, a novel and rapid paper-based antibody test is reported. We validate antibody detection with reverse blood typing using IgM antibodies and then generalise the validity by adapting to detect SARS CoV-2 (COVID-19) antibodies in patient serum samples. Reagent red blood cells (RBC) are first combined with the patient plasma containing the screened antibody and a droplet of the mixture is then deposited onto paper. The light intensity profile is analyzed to identify test results, which can be detected by eye and/or with image processing to allow full automation. The efficacy of this test to perform reverse blood typing is demonstrated and the performance and sensitivity of this test using different paper types and RBC reagents was investigated using clinical samples. As an example of the flexibility of this approach, we labeled the RBC reagent with an antibody-peptide conjugate to detect SARS CoV-2 (COVID-19) antibodies in patient serum samples. This concept could be generalized to any agglutination-based antibody diagnostics with blood plasma.

Droplet-based blood group antibody screening with laser incubation.

Detection of blood group antibodies is a crucial step for blood transfusion recipients and pregnant women to prevent potentially fatal haemolytic reactions. Due to the short, non-bridging structure of such antibodies (IgG), the indirect antiglobulin test (IAT) is required, complete with a thermal incubation phase. This incubation step, where the sample must be heated to 37 °C for several minutes, has hitherto prevented chip- and paper-diagnostics from performing a complete IAT and instead required the IAT to be performed away from the patient beside in a laboratory setting with specialist equipment - significantly delaying blood transfusions. With recent laser technology for immunohaematology, a single blood droplet can be heated. This study presents a simple diagnostic where a single 15 µL droplet sits on hydrophobic PTFE film and is heated by laser. The result of the test is then determined via placement of a paper strip where passive wicking and filtration of the sample separates positive from negative results. We demonstrate that this diagnostic can accurately and sensitively detect blood group antibodies, with results quickly read by eye without further specialist equipment or training, with potential to lead to a point-of-care antibody screen.

A rapid paper-based blood typing method from droplet wicking.

Paper-based diagnostics are leading the field of low-cost, point of care analytical techniques. However, large scale testing facilities such as hospitals are still primarily using the gel column agglutination technique. This is because paper-based systems are single use tests that are generally more time consuming and less automatable than traditional methods. Here, we present a novel, rapid and scalable, paper-based blood typing method that can produce test results in under ten seconds. We believe this is the fastest blood typing test that is appropriate for large scale automation. The test consists of placing a drop of antibody solution on paper, followed by a drop of blood on the same locus, and measuring the evolution of blood stain area as a function of time. Positive reactions for both forward and reverse tests have significantly slower growth rates and smaller final stain sizes when compared to negatives. We analyse the effect paper type, red blood cell concentration, antibody specificity (A, B and D) and antibody dilution have on the sensitivity and reproducibility of the technique. A high sensitivity is found in papers with a low density and thickness. The optimum red blood cell concentration is determined from a balance between wicking rate, strength of reaction and optical contrast. A and B antibodies give more sensitive results than D; however, the D antigen can still be successfully identified. This technique has the potential to significantly cut down the time and cost of blood typing tests and enable design of a new high throughput and fully automatable system.

Paper Diagnostic for Direct Measurement of Fibrinogen Concentration in Whole Blood.

The ability to diagnose and treat critically bleeding patients can save more than 2 million lives a year. Diagnosing hypofibrinogenemia is essential in these patients. Recently, with the development of new handheld diagnostics, fibrinogen concentration can be measured rapidly at the point of care. However, these diagnostics can only work with plasma and hence need blood cells to be separated before use. In this study, we demonstrate a handheld fibrinogen diagnostic that works with whole blood. The test works by (1) forming a premixed droplet of a whole blood sample and thrombin solution on a solid surface, (2) allowing it to clot, and (3) dropping a paper strip on top. The further that blood moves down the strip, the lower the fibrinogen concentration. The diagnostic can easily measure plasma fibrinogen concentrations below 1.6 g/L for blood samples with hematocrits between 40 and 50%. Furthermore, diluting blood samples not only increases the test's sensitivity but also eliminates the effect of hematocrit and thrombin inhibitors. The test can be completed in 3-4 min, making it suitable for diagnosing early hypofibrinogenemia and allowing for fibrinogen replacement therapy in critically bleeding patients.

Rapid Gel Card Agglutination Assays for Serological Analysis Following SARS-CoV-2 Infection in Humans.

High-throughput and rapid serology assays to detect the antibody response specific to severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) in human blood samples are urgently required to improve our understanding of the effects of COVID-19 across the world. Short-term applications include rapid case identification and contact tracing to limit viral spread, while population screening to determine the extent of viral infection across communities is a longer-term need. Assays developed to address these needs should match the ASSURED criteria. We have identified agglutination tests based on the commonly employed blood typing methods as a viable option. These blood typing tests are employed in hospitals worldwide, are high-throughput, fast (10-30 min), and automated in most cases. Herein, we describe the application of agglutination assays to SARS-CoV-2 serology testing by combining column agglutination testing with peptide-antibody bioconjugates, which facilitate red cell cross-linking only in the presence of plasma containing antibodies against SARS-CoV-2. This simple, rapid, and easily scalable approach has immediate application in SARS-CoV-2 serological testing and is a useful platform for assay development beyond the COVID-19 pandemic.

Rapid, hand-held paper diagnostic for measuring Fibrinogen Concentration in blood.

Critical bleeding causes over 2 million deaths a year. Early hypofibrinogenemia is a strong predictor of mortality in critically bleeding patients. The early replenishment of fibrinogen can significantly improve outcomes. However, over replenishment can also be dangerous. Furthermore, there is no rapid, cheap, hand-held diagnostic that can aid critically bleeding patients in fibrinogen replacement therapy. In this study, we have developed a hand-held paper diagnostic that measures plasma fibrinogen concentrations. The diagnostic has the potential to be used as a point of care device both inside and outside of hospital settings. It can vastly reduce the time to treatment for fibrinogen replacement therapy. The diagnostic is a two-step process. First, thrombin and plasma are added onto horizontially-orientated paper strips where the fibrinogen is converted into fibrin, drastically increasing the plasma's hydrophobicity. Second, an aqueous blue dye is pipetted onto the strips and allowed to wick through the fibrin. The distance the blue dye wicks through the strip correlates precisely to the fibrinogen concentration. The diagnostic can provide results within a minute. It can distinguish low fibrinogen concentrations (ie. <2 g/L) from normal fibrinogen concentrations. It shows remarkable reproducibility between healthy individuals. It is unaffected by common blood conditions such as acidosis, blood alcohol, severe hypertriglyceridemia, severe haemolysis and warfarin administration. Finally, it is unaffected by humidity and can withstand cold temperatures.

Photothermal incubation of red blood cells by laser for rapid pre-transfusion blood group typing.

Safe blood transfusion requires compatibility testing of donor and recipient to prevent potentially fatal transfusion reactions. Detection of immunoglobulin G (IgG) antibodies requires incubation at 37 °C, often for up to 15 minutes. Current incubation technology predominantly relies on slow thermal-gradient dependent conduction. Here, we present rapid optical heating via laser, where targeted illumination of a blood-antibody sample in a diagnostic gel card is converted into heat, via photothermal absorption. Our laser-incubator heats the 75 µL blood-antibody sample to 37 °C in under 30 seconds. We show that red blood cells act as photothermal agents under near-infrared laser incubation, triggering rapid antigen-antibody binding. We detect no significant damage to the cells or antibodies for laser incubations of up to fifteen minutes. We demonstrate laser-incubated immunohaematological testing to be both faster and more sensitive than current best practice - with clearly positive results seen from laser incubations of just 40 seconds.

Rapid paper diagnostic for plasma fibrinogen concentration.

Fibrinogen is a blood protein that is essential for clotting. It is converted into the polymer fibrin by the blood enzymes thrombin and factor XIIIa. Fibrinogen is one of the first proteins to be depleted in heavily bleeding patients. Patients with early hypofibrinogenemia need urgent fibrinogen replenishment to prevent the onset of haemorrhage and death. However, currently there is no rapid, sensitive, cheap and easy-to-use fibrinogen assay that can detect fibrinogen concentrations. In this study, we have developed a new paper-based diagnostic to quantify the fibrinogen concentration in blood at room temperature. This diagnostic is a 2-step process: first, plasma is added onto thrombin-treated paper strips where fibrinogen is converted to fibrin; then the strips are placed into an aqueous dye bath where elution occurs. The test operates by measuring the change in hydrophobicity, which increases with fibrinogen concentration under otherwise constant conditions. The diagnostic can precisely measure fibrinogen concentration within the range of 0-2 g L-1, which is ideal for the clinical diagnosis of hypofibrinogenemia. Furthermore, testing needs only 12 µL of plasma, 60 mU of thrombin and 7.5 minutes of testing. This diagnostic has the potential to revolutionise point of care testing and save many lives.

Nanocellulose Hydrogel for Blood Typing Tests.

The gel test is the most prevalent method for the forward and reverse blood typing tests. It relies on the controlled centrifugation of red blood cells (RBCs) and antibodies through a gel column. This noncontinuous matrix is currently based on microbeads that often lack sensitivity. For the first time, nanocellulose hydrogel is demonstrated as a sustainable and reliable medium for gel-based blood typing diagnostics. Gels with a minimum of 0.3 wt % TEMPO-oxidized cellulose nanofibers (0.92 mmol/g of carboxyl content) separate agglutinated and individual RBCs in the forward test. The addition of glycine is able to balance the osmotic pressure and reduce hemolysis to 5%, while retaining the electrostatic repulsion responsible for the gel network structure and its rheological properties. For the reverse typing, cellulose nanofibers are chemically cross-linked with hexamethylenediamine (HMDA), increasing the gel yield point 8-fold. Sodium chloride is added to achieve the osmolality found in the human plasma and limit cell lysis to 15%, without affecting the gel colloidal stability. Nanocellulose hydrogel constitutes a performant, low cost, and green soft material, providing clear and well-defined results for both blood grouping tests.

Activity and Longevity of Antibody in Paper-Based Blood Typing Diagnostics.

Paper-based diagnostics provide a low-cost, reliable and easy to use mode of blood typing. The shelf-life of such products, however, can be limited due to the reduced activity of reagent antibodies sorbed on the paper cellulose fibers. This study explores the effects of aging on antibody activity for periods up to 12 months on paper and in solution under different aging and drying conditions-air-dried, lyophilized, and kept as a liquid. Paper kept wet with undiluted antibody is shown to have the longest shelf-life and the clearest negatives. Antibody diluted with bovine serum albumin (BSA) protects against the lyophilization process, however, beyond 9 months aging, false positives are seen. Paper with air-dried antibodies is not suitable for use after 1 month aging. These results inform preparation and storage conditions for the development of long shelf-life blood grouping paper-based diagnostics.

Surface Engineering of Transparent Cellulose Nanocrystal Coatings for Biomedical Applications.

The concept of blood typing diagnostics using blood drops dried onto transparent cellulose nanocrystal thin film (∼35 nm) coatings has been demonstrated. The substrate onto which the blood drops are dried plays an important role in such tests, depending on surface composition, roughness, and wettability. The drying profile of three different fluid dispersions: model latex particles, reagent blood cells, and whole human blood was studied on a range of different surfaces, including cellulose nanocrystals (CNCs), regenerated cellulose, and several hydrophobic polymers, in order to understand the role of surface chemistry, roughness, and fluid dispersion properties. The morphology of these surfaces was investigated using atomic force microscopy, roughness was calculated, and wettability was explored via contact angle measurement. The morphology of dried drops of human blood on different cellulosic surfaces was compared in order to understand the importance of cellulose crystallinity. Well-defined dried blood drops were observed on random and aligned CNC surfaces, facilitating visualization of individual cells. A simple antibody-antigen test was used to demonstrate the effectiveness of the CNC substrate for blood testing, showing high and reproducible selectivity.

Duffy blood group (Fya & Fyb) analysis using surface plasmon resonance.

The most widely known blood groups, ABO and RhD, have been extensively observed as having strong antibody-antigen interactions during blood typing. However, not all interactions show the same binding affinity. The Duffy blood group system, where Fya and Fyb antigens are the most clinically significant, are only available with an IgG antibody structure, and display weak binding interactions. While current blood typing techniques are well established, methods for quantifying the binding strength are more limited. Surface Plasmon Resonance (SPR) provides avenues for developing more robust detection methods, and serve as a sensitive quantification technique by itself. This study tested SPR for the detection of weaker antibody-antigen interactions using the Duffy blood groups, Fya and Fyb, as a model. This study shows a minimum threshold of antibody concentration is required for successful detection. Some instances of detection were successful using concentrated commercial anti-Fya and anti-Fyb solution during the incubation stage. However, these results were not fully reproducible. We found that a significant dissociation of the Duffy antigen-antibody complex occurs over time. A combination of factors affects the detection of the Duffy antigens using SPR; these include antibody concentration, antigen expression, and antigen structure. This results in weak, unstable and reversible antibody-antigen interactions which are currently limiting accurate and reproducible detection by SPR. Despite these issues, detection of Duffy antigens Fya and Fyb was demonstrated using SPR; however, further development is required for SPR to become a robust clinical blood typing technique.

Paper-based assay for red blood cell antigen typing by the indirect antiglobulin test.

A rapid and simple paper-based elution assay for red blood cell antigen typing by the indirect antiglobulin test (IAT) was established. This allows to type blood using IgG antibodies for the important blood groups in which IgM antibodies do not exist. Red blood cells incubated with IgG anti-D were washed with saline and spotted onto the paper assay pre-treated with anti-IgG. The blood spot was eluted with an elution buffer solution in a chromatography tank. Positive samples were identified by the agglutinated and fixed red blood cells on the original spotting area, while red blood cells from negative samples completely eluted away from the spot of origin. Optimum concentrations for both anti-IgG and anti-D were identified to eliminate the washing step after the incubation phase. Based on the no-washing procedure, the critical variables were investigated to establish the optimal conditions for the paper-based assay. Two hundred ten donor blood samples were tested in optimal conditions for the paper test with anti-D and anti-Kell. Positive and negative samples were clearly distinguished. This assay opens up new applications of the IAT on paper including antibody detection and blood donor-recipient crossmatching and extends its uses into non-blood typing applications with IgG antibody-based diagnostics. Graphical abstract A rapid and simple paper-based assay for red blood cell antigen typing by the indirect antiglobulin test.

Effect of cationic polyelectrolytes on the performance of paper diagnostics for blood typing.

We investigated the effect that two common types of cationic polyelectrolytes used in papermaking might have on the performance of paper diagnostics using blood typing as an example. The results were analyzed in terms of red blood cells (RBC) retention and antibody-antigen specificity. Two questions were addressed: (1) can poly(amido-amine) epichlorohydrin (PAE) typically used for paper wet strength affect the diagnostic performance? (2) can high molecular weight cationic polyacrylamide (CPAM) employed as retention aid enhance or affect the selectivity and sensitivity of paper diagnostics? A series of paper varying in type of fibers and drying process were constructed with PAE and tested for blood typing performance. Residual PAE has no significant effect on blood typing paper diagnostics under normal conditions. Positives are unaffected with PAE, while negatives lose slight sharpness as some RBCs are unselectively retained. CPAM, the most common retention aid, can also be used to retain cells and biomolecules on paper. Paper towel was treated with CPAM solutions varying in polymer concentration and charge density and tested for blood typing. We found that CPAM dried on paper can retain RBC. CPAM affects the negative tests by retaining non-specifically individual RBC on fibers. RBC retention increases non-linearly with the CPAM charge density and concentration. As expected, wet CPAM retain RBCs at concentrations higher than 0.1wt%. As paper diagnostics are becoming a reality, more realistic papers than the Whatman filter paper will be engineered. This study provides guidance on how best use the required polymeric wet-strength and retention agents.