Automated point-of-care testing for ABO agglutination test: proof of concept and validation.

BACKGROUND AND OBJECTIVES: ABO-incompatible red blood cell transfusions still represent an important hazard in transfusion medicine. Therefore, some countries have introduced a systematic bedside ABO agglutination test checking that the right blood is given to the right patient. However, this strategy requires an extremely time-consuming learning programme and relies on a subjective interpretation of ABO test cards agglutination. We developed a prototype of a fully automated device performing the bedside agglutination test that could be completed by reading of a barcoded wristband. This POCT checks the ABO compatibility between the patient and the blood bag. MATERIALS AND METHODS: Proof of concept and analytical validation of the prototype has been completed on 451 blood samples: 238 donor packed red blood cells, 137 consecutive unselected patients for whom a blood group determination had been ordered and on 76 patient samples selected with pathology that could possibly interfere with or impair performances of the assay. RESULTS: We observed 100% concordance for ABO blood groups between the POCT and the laboratory instrument. CONCLUSION: These preliminary results demonstrate the feasibility of ABO determination with a simple POCT device eliminating manipulation and subjective interpretation responsible for transfusion errors. This device should be linked to the blood bank system allowing all cross-check of the results.

Comparative evaluation of gel column agglutination and erythrocyte magnetized technology for red blood cell alloantibody titration.

Antibody titration is traditionally performed using a conventional test tube (CTT) method, which is subjected to interlaboratory variations because of a lack of standardization and reproducibility. The aim of this study is to compare newer methods such as get column technology (GCT) and erythrocyte magnetized technology (EMT) for antibody titration in terms of accuracy and precision. Patient serum samples that contained immunoglobin G (IgG) red blood cell (RBC) alloantibodies of a single specificity for Rh or K anitgens were identified during routine transfusion service testing and stored. Titration and scoring were performed separately by and stored. Titration and scoring were performed separately by different laboratory personnel on CTT, GCT, and EMT. Testing was performed a total of three times on each sample. Results were analyzed for accuracy and precision. A total of 50 samples were tested. Only 20 percent of samples tested with GCT shoed titers identical to CTT, whereas 48 percent of samples tested with EMT showed titers identical to CTT. Overall, the mean of th titer difference from CTT was higher using GCT (+0.31) compared with that using EMT (+0.13). Precision shown by CTT was 30 percent, EMT was 76 percent, and GCT was 92 percent on repeat testing. GCT showed higher titer values in comparison with CTT but was found to be the most precise. EMT titers were comparable to CTT, and its precision was intermediate. Further studies to validate this method are required.

Development of a slide agglutination assay for detection of blastomycosis.

Blastomycosis, caused by the thermally dimorphic fungus Blastomyces dermatitides, which is endemic to eastern regions of the USA, is commonly misdiagnosed as a viral or bacterial infection and therefore treated improperly. Over the years, many immunodiagnostic assays to aid in the diagnosis of blastomycosis have been developed; however, a reliable assay for use in local clinics still remains elusive. Procedures for a slide agglutination assay for detection of antibody in serum from rabbits immunized with B. dermatitidis were evaluated with antigenic preparations from B. dermatitidis adsorbed to polystyrene microparticles. Yeast-phase lysates from five isolates of B. dermatitides: namely ER-593 (Eagle River, WI, USA), ER-598 (Eagle River, WI, USA), 48938 (India), B5896 (Mt. Iron, MN, USA), and T-58 (TN, USA) were evaluated for their sensitivity and specificity. Sensitivities of the lysates ranged from 29% to 83% whereas specificities ranged from 13% to 100%. Lysate ER-593 provided the most promising results with a sensitivity of 82% and specificity of 100%. This study provides suggests that a simple rapid slide agglutination assay for detecting blastomycosis may be used for screening patients with suspected B. dermatitidis infection.

Antenatal monitoring of anti-D and anti-c: could titre scores determined by column agglutination technology replace continuous flow analyser quantification?

OBJECTIVE: To determine if column agglutination technology (CAT) for titration of anti-D and anti-c concentrations produces comparable results to those obtained by continuous flow analyser (CFA). BACKGROUND: Anti-D and anti-c are the two commonest antibodies that contribute to serious haemolytic disease of the foetus and neonate (HDFN). Current practice in the UK is to monitor these antibodies by CFA quantification, which is considered more reproducible and less subjective than manual titration by tube IAT (indirect antiglobulin test). CAT is widely used in transfusion laboratory practice and provides a more objective endpoint than tube technique. MATERIALS AND METHODS: Antenatal samples were (i) quantified using CFA and (ii) titrated using CAT with the reaction strength recorded by a card reader and expressed as a titre score (TS). RESULTS: The TS rose in accordance with levels measured by quantification and was able to distinguish antibody levels above and below the threshold of clinical significance. CONCLUSION: CAT titre scores provided a simple and reproducible method to monitor anti-D and anti-c levels. The method was sensitive to a wide range of antibody levels as determined by quantification. This technique may have the potential to replace CFA quantification by identifying those cases that require closer monitoring for potential HDFN.

Modifications influencing Widal test reactivity in a novel microplate assay.

Reliability of the Widal tube agglutination test has been the subject of many controversies over the years. This study was performed to assess the effect of certain modifications on the performance of Widal test in a novel microplate assay. Sera from 37 patients (21 males; 16 females) (mean age 28 +/- 7 years) were tested in the Immunology Unit at King Khalid University Hospital, Riyadh. Among them were 26 patients with suspected typhoid fever and 11 had bacteriologically confirmed diagnosis of Salmonella infection. The modifications included either the use of 0.5% bovine serum albumin (BSA), absorption of sera with sheep red blood cells (SRBC) or heat inactivation of sera. Compared with Widal tube agglutination test, microplate assay with SRBC absorption of the sera from patients with suspected typhoid fever was not only associated with enhancement of detection titers for both H (p < or = 0.001) and O (p < or = 0.005) Salmonella agglutinins but also the percentage of reactivity. The presence of BSA augmented detection titers for Salmonella H agglutinins (p < or = 0.02) only. Heat inactivation of sera however was found to be associated with reduction in the detectable titers for both H (p < or = 0.03) and O (p < or = 0.01) agglutinins. Increased titers of Salmonella agglutinins were also evident in 11 patients with confirmed diagnosis of Salmonella infection. The novel microplate agglutination assay using the SRBC absorption was associated with enhancement in Widal test reactivity and appears to be a useful alternative for the diagnosis of Salmonella infection.

Quantitative determination of fibrinogen of patients with coronary heart diseases through piezoelectric agglutination sensor.

Fibrinogen can transform fibrin through an agglutination reaction, finally forming fibrin polymer with grid structure. The density and viscosity of the reaction system changes drastically during the course of agglutination. In this research, we apply an independently-developed piezoelectric agglutination sensor to detect the fibrinogen agglutination reaction in patients with coronary heart diseases. The terminal judgment method of determining plasma agglutination reaction through piezoelectric agglutination sensor was established. In addition, the standard curve between plasma agglutination time and fibrinogen concentration was established to determinate fibrinogen content quantitatively. The results indicate the close correlation between the STAGO paramagnetic particle method and the method of piezoelectric agglutination sensor for the detection of Fibrinogen. The correlation coefficient was 0.91 (γ = 0.91). The determination can be completed within 10 minutes. The fibrinogen concentration in the coronary heart disease group was significantly higher than that of the healthy control group (P < 0.05). The results reveal that high fibrinogen concentration is closely correlated to the incurrence, development and prognosis of coronary heart diseases. Compared with other traditional methods, the method of piezoelectric agglutination sensor has some merits such as operation convenience, small size, low cost, quick detecting, good precision and the common reacting agents with paramagnetic particle method.

A comparison of three column agglutination tests for red blood cell alloantibody identification.

OBJECTIVE: Commercial kits of column tests for pre-transfusion testing have progressively replaced conventional tube tests in most laboratories. Aim of this study was to compare three commercial test cell panels for the identification of irregular red blood cell (RBC) alloantibodies. Overall, 44 samples with a positive indirect antiglobulin test (IAT) by routine testing were used for comparison of following panels: Ortho RESOLVE® panelC (Ortho Clinical Diagnostics (OCD), Milan, Italy), ID-DiaPanel(-P) (Bio-Rad Laboratories, CA, USA) and Identisera Diana(P) (Grifols, Barcelona, Spain). Column agglutination techniques were used, with microtubes containing either microgel (Bio-Rad/Grifols) or glass bead microparticles (Ortho). RESULTS: Alloantibody identification was possible in 38 samples, of which identical identification was shown in 33 samples by all methods. The remaining samples showed differences between certain methods, with the gel card system being superior to the glass card system for analyzing stored samples Considering that not all samples were evaluated in all three methods, the concordance rate reached 100% between Bio-Rad and Grifols, 90.5% between Bio-Rad and OCD, 86.5% between OCD and Grifols and 90.5% between all methods. Although differences in sensitivities were seen for specific antibodies, the three methods showed comparable performance for the identification of RBC alloantibodies.

Chamfer-Type Capillary Stop Valve and Its Microfluidic Application to Blood Typing Tests.

This paper presents a novel design of a capillary stop valve with a chamfered side that can be used as a flow regulator to hold an injected microfluid in the valve position in a capillary force-driven microfluidic device. Biochemical analysis can be conducted if the chamfer-type valves are placed at strategic positions according to the test protocol. Hence, the stored reagent can be dragged out of the valve for further reaction when the specimen passes through. However, countercurrent phenomena were observed in the commonly used T-type capillary stop valve (without the chamfered side). In blood typing tests, the countercurrent led to incomplete dragging and the fluid stopped flowing at the complicated mixing channel; thus, the blood typing reaction was attenuated. On the contrary, the chamfer-type valve reduced the countercurrent phenomena and ameliorated the blood typing reaction. Consequently, agglutination results can be easily discriminated from nonagglutination cases.

Measuring electrical and mechanical properties of red blood cells with double optical tweezers.

Red blood cell (RBC) aggregation in the blood stream is prevented by the zeta potential created by its negatively charged membrane. There are techniques, however, to decrease the zeta potential and allow cell agglutination, which are the basis of most of antigen-antibody tests used in immunohematology. We propose the use of optical tweezers to measure membrane viscosity, adhesion, zeta potential, and the double layer thickness of charges (DLT) formed around the cell in an electrolytic solution. For the membrane viscosity experiment, we trap a bead attached to RBCs and measure the force to slide one RBC over the other as a function of the velocity. Adhesion is quantified by displacing two RBCs apart until disagglutination. The DLT is measured using the force on the bead attached to a single RBC in response to an applied voltage. The zeta potential is obtained by measuring the terminal velocity after releasing the RBC from the trap at the last applied voltage. We believe that the methodology proposed here can provide information about agglutination, help to improve the tests usually performed in transfusion services, and be applied for zeta potential measurements in other samples.

A novel piezoelectric immunoagglutination assay technique with antibody-modified liposome.

A simple rapid piezoelectric immunoagglutination assay (PEIA) technique with antibody-modified liposome has been developed for direct quantitative detection of human immunoglobulin G (hIgG). This technique is based on specific agglutination of antibody-coated liposome particles in the presence of the corresponding antigen, which can be monitored by the frequency shift of a piezoelectric device. Compared with conventional piezoelectric assays, this liposome-based PEIA does not require the immobilization of antigen or antibody on the quartz crystal surface, making the developed technique especially useful for rapid and renewable immunochemical determination. To alleviate non-specific adsorption of serum proteins, modification of the quartz crystal surface by different protocols and the composition of the assay medium have been investigated. The results indicate that the background interference can be substantially minimized through modifying the quartz crystal surface with a bovine serum albumin (BSA) layer and introducing an appropriate amount of BSA in the assay medium. The effects of the liposome composition, the liposome concentration and the concentration of poly(ethylene glycol) (PEG) in the assay medium, have also been investigated. The frequency responses of the liposome-based PEIA are linearly correlated to hIgG concentration in the range of 0.05-6 microg mL(-1) with a detection limit of 50 ng mL(-1).

In situ, dual-mode monitoring of organ-on-a-chip with smartphone-based fluorescence microscope.

The use of organ-on-a-chip (OOC) platforms enables improved simulation of the human kidney's response to nephrotoxic drugs. The standard method of analyzing nephrotoxicity from existing OOC has majorly consisted of invasively collecting samples (cells, lysates, media, etc.) from an OOC. Such disruptive analyses potentiate contamination, disrupt the replicated in vivo environment, and require expertize to execute. Moreover, traditional analyses, including immunofluorescence microscopy, immunoblot, and microplate immunoassay are essentially not in situ and require substantial time, resources, and costs. In the present work, the incorporation of fluorescence nanoparticle immunocapture/immunoagglutination assay into an OOC enabled dual-mode monitoring of drug-induced nephrotoxicity in situ. A smartphone-based fluorescence microscope was fabricated as a handheld in situ monitoring device attached to an OOC. Both the presence of γ-glutamyl transpeptidase (GGT) on the apical brush-border membrane of 786-O proximal tubule cells within the OOC surface, and the release of GGT to the outflow of the OOC were evaluated with the fluorescence scatter detection of captured and immunoagglutinated anti-GGT conjugated nanoparticles. This dual-mode assay method provides a novel groundbreaking tool to enable the internal and external in situ monitoring of the OOC, which may be integrated into any existing OOCs to facilitate their subsequent analyses.

Immediate HbA1c results. Performance of new HbA1c system in pediatric outpatient population.

OBJECTIVE: This study compared the performance of a new device that uses an IA to measure HbA1c in 9 min with a 1-microliter capillary blood sample with AC and CE methods in both nondiabetic and diabetic pediatric patients. RESEARCH DESIGN AND METHODS: Two hundred seven pediatric subjects (103 nondiabetic, 104 with insulin-dependent diabetes mellitus) had HbA1c measured with the IA method and compared with total GHb values determined by AC and HbA1 by the CE method with the same whole-blood capillary aliquot. Glucose values were also obtained from the same blood samples. RESULTS: Correlations and regression analyses show excellent correspondence between the three assays. The correlation between the AC and CE methods is 0.98 (P less than 0.001) with a slope of 1.615 +/- 0.0125 and intercept of 4.00 +/- 0.20. The correlation between the IA and AC methods is 0.99 (P less than 0.001) with a slope of 0.608 +/- 0.007 and intercept of 1.326 +/- 0.066. The correlation between the IA and CE methods is 0.97 (P less than 0.001), with a slope of 0.983 +/- 0.018 and intercept of 1.122 +/- 0.153. The average difference and average percentage difference between methods were also significant (P less than 0.001), reflecting the differences in GHb components measured. There was a significant correlation (P less than 0.001) between each method and glucose values (IA r = 0.72, AC r = 0.70, CE r = 0.73). Within-run precision for IA ranged from 1.7 to 3.5% and between-run precision 2.7 to 4.1%. CONCLUSIONS: Study results suggest that the IA method gives extremely accurate and reliable values over the clinical range of interest. The instrument is small, portable, easy to use, and provides information within 9 min for both physicians and patients.

Simultaneous detection of pathogenic bacteria using agglutination test based on colored silica nanoparticles.

Aimed to explore an agglutination test which can simultaneously detect two pathogenic bacteria, an agglutination test based on colored silica nanoparticles (colored-SiNps) was established in this work. Monodisperse colored-SiNps were used as agglutination test carriers; red-SiNps and blue-SiNps were prepared by reverse microemulsion with C.I. Reactive red 136 and C.I. Reactive Blue 14. Then the red-SiNps were sensitized with antibodies against E. sakazaki and denoted as IgG-red-SiNps; The blue-SiNps were coated with antibodies against S. pullorum and S. Gallinarum and denoted as IgGblue- SiNps. The mixture solution of IgG-red-SiNps and IgG-blue-SiNps could simultaneously agglutinate with E. sakazakii and S. pullorum and S. gallinarum on glass slide. The E. sakazakii and S. pullorum and S. gallinarum could be simultaneously detected by agglutination test with obvious agglutination phenomena. The E. sakazakii and S. pullorum and S. gallinarum could both be detected in a range from 4×10(3) to 4×10(9) CFU/mL. The pullorum and S. gallinarum and E. sakazakii in the infected food sample were detected by mixture solution of IgG-red-SiNps and IgG-blue-SiNps too. This agglutination test was easy and rapid, it might be useful for in situ rapid detection method for simultaneously screening different pathogenic microorganisms of foods and feeds in the field.

Automated reading and processing of quantitative IgG, IgM, IgA, and IgE isotypic agglutination results in microplates. Development and application in parasitology-mycology.

Microplate agglutination techniques represent a simple and commonly used approach for the quantitative or qualitative isotypic analysis of specific antibodies. However, they require optical reading by the investigator and are thus prone to an important degree of variability. In order to solve some of the problems associated with the variability of optical readings, we have used an automatic reader scanning each of the 96 wells of a standard microplate in 32 different locations. The inherent advantages of the automatic reader were further maximized by coupling it to a dedicated computer running customized software designed to process data coming on-line from the spectrophotometer. This approach has been applied to the diagnosis of human toxoplasmosis and candidosis. Suspensions of Toxoplasma gondii tachyzoites or of sensitised erythrocytes were used for the determination of IgG antibodies or the quantification of IgM, IgA, or IgE specific isotypes. This procedure allows the simple and reproducible collection of objective results. Moreover, it permits a reduction in cut-off values and direct interpretation of results with automatic conversion of scores into titer, units, index, or into any other scale appropriate for standardization purposes.

Feasibility of Helicobacter pylori identification by a slide agglutination test.

Culture isolation and identification of Helicobacter pylori represents a considerable work load in clinical microbiology. The aim of this study was to test if antibody-mediated bacterial agglutination could be used for rapid identification of H. pylori. Rabbit antiserum against H. pylori strain I and against another strain, H. pylori 330, which was very weakly agglutinated (1+) by anti-H. pylori I serum, were mixed and used in a slide agglutination test. Of 107 consecutive clinical isolates tested, 101 (94%) strains showed 2+ or 3+ reaction using the antiserum mixture, whereas 6 (6%) strains could not be evaluated owing to autoagglutinability. Bacteria of a variety of other species, including Campylobacter spp., showed no agglutination with the antiserum mixture. The results support the notion that reliable identification of the majority of cultured H. pylori strains should be possible in less than 3 min by agglutination testing.

Identification of oxacillin-susceptible and oxacillin-resistant Staphylococcus aureus using commercial latex agglutination tests.

Four hundred thirteen Staphylococcus sp. were identified by Staphaurex, Staphaurex Plus, and BACTiStaph kits using tube coagulase as reference. Among 222 coagulase-positive isolates, 56 were oxacillin-resistant Staphylococcus aureus. All tests were accurate in distinguishing between coagulase-positive and -negative staphylococci with sensitivities and specificities > or = 97% and only nine discrepancies.

A piezoelectric immunoagglutination assay for Toxoplasma gondii antibodies using gold nanoparticles.

The serologic detection of anti-Toxoplasma gondii immunoglobulins plays a key role in the clinical diagnosis of Toxoplasmosis. In this paper, a simple, rapid and highly sensitive agglutination-based piezoelectric immunoassay has been firstly developed for directly detecting anti-T. gondii immunoglobulins in infected rabbit serum (IRS) and infected rabbit blood (IRB). The proposed technique is based on that the specific agglutination of antigen-coated gold nanoparticles, averaging 10nm in diameter, in the presence of the corresponding antibody causes a frequency change that is monitored by a piezoelectric device. In contrast to the commonly used piezoelectric assays, it possesses an attractive advantage in that the immobilization of antibody or antigen on the crystal is unnecessary. Use of a newly prepared sensing probe which was modified by a plasma-polymerized film (PPF) of n-butyl amine and further by a heparin layer resulted in a response-enhanced immunoagglutination and a high compatibility of the probe with biological samples. An appropriate reagent consisting of 1% normal rabbit serum (NRS) and 0.1% bovine serum albumin (BSA) for diluting the analytes were verified in counteracting the background interference of assay. Moreover, an optimization of assay medium composition with the addition of poly(ethylene glycol) (PEG) serving as immunoagglutination rate and sensitivity enhancer was investigated in detail. It is found that the developed immunoagglutination assay system is sensitive to dilution ratio of anti-T. gondii antibody as low as 1:5500. Analytical results of several specimens obtained using the developed technique are in satisfactory agreement with those given by the ELISA method, implying a promising alternative approach for detecting anti-T. gondii antibodies in the clinical diagnosis.

Standardisation of subjectively scored HIV immunoassays: developing a quality assurance program to assist in reproducible interpretation of results using an anti-HIV particle agglutination assay as a model.

Immunoassays such as particle agglutination assays, rapid tests and western or line blots are scored or read subjectively. These readings display intra- and inter-reader variability, as well as intra- and inter-laboratory variability. In the present study the consistency of scoring was assessed between readers both within and between two groups of scientists using the Serodia anti-HIV particle agglutination assay as an example of an assay scored subjectively. An anti-HIV positive sample in eight serial dilutions made to yield a full range of results expected for the assay was presented 12 times (96 test wells). Each dilution was placed randomly in a plate and tested with the Serodia anti-HIV particle agglutination assay then photographed. Participants in the two groups each scored the photographed plate independently and twice, 2 h apart. Each well was assigned a status (the consensus result of the four most experienced Australian readers) and each participant's results were compared with this status. The average percentage of wells assessed as 'correct' for the Group A participants was 86% (range 56-98%) and for the Group B participants was 67% 'correct' (range 46-88%). In general, strongly positive and negative wells were scored 'correctly'. The highest variations between scores were seen in the borderline positive dilutions +/- region. A quality assessment program based on the method used to obtain these results will be instituted in order to improve the consistency of scoring assays read subjectively.

Assessment of the AmnioStat-FLM immunoagglutination test for phosphatidylglycerol in amniotic fluid.

One hundred and eight amniotic fluids were assayed by the AmnioStat-FLM (A-FLM) immunological agglutination test for phosphatidylglycerol (PG) and simultaneously measured enzymatically for PG content. Of 52 amniotic fluids found to be PG negative by the A-FLM method, all had enzymatic PG concentrations less than or equal to 1.5 mumol/l. Conversely, of 56 amniotic fluids judged to be either PG positive or weak positive, all but five had enzymatic PG concentrations greater than 1.5 mumol/l. The sensitivity of the A-FLM assay employed clinically for predicting foetal lung maturity was 89% and the specificity was 100%. The overall predictive accuracy of the test could be improved by providing controls at lower, more appropriate PG concentrations. Ninety-one fluids analysed by the A-FLM kit were subsequently tested for the presence of PG by two-dimensional thin-layer chromatography (2D TLC). A 94%-concordance between the methods was found.