Dissociation Phenomenon of Erythrocyte Agglutination and Its Application to Assay of Functional Activity of the Complement System in Clinical Laboratory.

OBJECTIVE: The objective of the study was to validate the dissociation phenomenon of erythrocyte agglutination which is based on erythrocyte fragments and to apply it in the functional activity assay of the complement system. METHODS: The dissociation-agglutination effect of erythrocyte fragments was validated by detecting the number of free erythrocytes after the action of erythrocyte fragments on agglutinated erythrocytes. The number of free erythrocytes produced after hemolysis of agglutinated erythrocytes caused by complements and complement activators(CAs) was detected by auto hematology analyzer and the results were indicated by mean hemoglobin concentration of erythrocytes (MCHC). We optimized the test conditions and validated the inter-batch stability, explored the resolution of the assay method, and assayed for the total complement activity (AC) and the CAs activated complement activity (ACA) in serum from patients and healthy individual groups. RESULTS: Erythrocyte fragments have a dissociative effect on agglutinated erythrocytes. The auto hematology analyzer was able to detect AC and ACA, where AC showed an inverse correlation with MCHC, and ACA demonstrated a positive correlation with MCHC. The inter-batch CV of AC, ACA, and ACA/AC was found to be 5%, 9%, and 11.7%, respectively, with good stability. The study found that serum samples from acute phase reaction patients showed significant differences in ACA compared with healthy individuals, with a p value of 0.018; serum samples from patients with nephrotic syndrome showed significant differences in AC, ACA, and ACA/AC compared with healthy individuals, with p values of 0.014, 0.002, and 0.041, respectively. CONCLUSION: Erythrocyte fragments have dissociation-agglutination effect. The complement system immunological functional detection method, based on this effect, has potential clinical application value due to its sensitivity and accuracy.

A multi-country comparative study of two treponemal tests for the serodiagnosis of syphilis amongst men who have sex with men (MSM): Chemo-luminescent assay vs Treponema pallidum particle agglutination assay.

INTRODUCTION: International guidelines recommend routine screening for syphilis (aetiological agent: Treponema pallidum subspecies pallidum) amongst key populations and vulnerable populations using tests detecting treponemal and non-treponemal antibodies. Whilst treponemal tests have high sensitivities and specificities, they differ regarding subjective or objective interpretation, throughput and workload. Chemiluminescence immunoassays (CLIAs) are cost- and time-effective automated methods for detecting treponemal antibodies. The Treponema pallidum particle agglutination assay (TPPA) has been considered the "gold standard" treponemal assay, however, this includes a highly manual procedure, low throughput and subjective interpretation. The present multi-country study evaluated the ADVIA Centaur® Syphilis CLIA (Siemens Healthcare) assay compared to the reference SERODIA-TP·PA® (Fujirebio Diagnostics) for the serodiagnosis of syphilis amongst men who have sex with men (MSM). METHOD: 1,485 MSM were enrolled in Brighton (UK), Malta, and Verona (Italy) as part of a larger WHO multi-country and multi-site ProSPeRo study. Ethical approval was obtained. Serum was tested with the ADVIA Centaur® Syphilis CLIA assay and SERODIA-TP·PA®, in accordance with the manufacturers' instructions, for a first round of validation. A second round of validation was carried out for discrepant results that were additionally tested with both Western Blot (Westernblot EUROIMMUN®) and an Immunoblot (INNO-LIA, Fujirebio Diagnostics). Sensitivity, specificity, positive and negative predictive value (PPV and NPV), likelihood ratios (positive/negative), and the Diagnostic Odds Ratio (DOR)/pre-post-test probability (Fagan's nomogram) were calculated. RESULTS: Out of 1,485 eligible samples analysed in the first phase, the SERODIA-TP·PA® identified 360 positive and 1,125 negative cases. The ADVIA Centaur® Syphilis CLIA assay (Siemens) identified 366 positives, missclassifying one TPPA-positive sample. In the second phase, the ADVIA Centaur® Syphilis CLIA resulted in 1 false negative and 4 false positive results. Considering the syphilis study prevalence of 24% (95% CI: 22-26.7), The sensitivity of the ADVIA Centaur® Syphilis CLIA assay was 99.7% (95% CI: 98.5-100), and the specificity was 99.4% (95% CI: 98.7-99.7). The ROC area values were 0.996 (95% CI: 0.992-0.999), and both the PPV and NPV values were above 98% (PPV 98.1%, 95% CI: 96.1-99.2; NPV 99.9%, 95% CI: 99.5-100). CONCLUSIONS: The ADVIA Centaur® Syphilis CLIA assay showed similar performance compared to the SERODIA-TP·PA®. Considering the study is based on QUADAS principles and with a homogeneous population, results are also likely to be generalisable to MSM population but potentially not applicable to lower prevalence populations routinely screened for syphilis. The automated CLIA treponemal assay confirmed to be accurate and appropriate for routine initial syphilis screening, i.e. when the reverse testing algorithm is applied.

Advances in risk predictive performance of pre-symptomatic type 1 diabetes via the multiplex Antibody-Detection-by-Agglutination-PCR assay.

Introduction: Achieving early diagnosis of pre-symptomatic type 1 diabetes is critical to reduce potentially life-threatening diabetic ketoacidosis (DKA) at symptom onset, link patients to FDA approved therapeutics that can delay disease progression and support novel interventional drugs development. The presence of two or more islet autoantibodies in pre-symptomatic type 1 diabetes patients indicates high-risk of progression to clinical manifestation. Method: Herein, we characterized the capability of multiplex ADAP assay to predict type 1 diabetes progression. We obtained retrospective coded sera from a cohort of 48 progressors and 44 non-progressors from the NIDDK DPT-1 study. Result: The multiplex ADAP assay and radiobinding assays had positive predictive value (PPV)/negative predictive value (NPV) of 68%/92% and 67%/66% respectively. The improved NPV stemmed from 12 progressors tested positive for multiple islet autoantibodies by multiplex ADAP assay but not by RBA. Furthermore, 6 out of these 12 patients tested positive for multiple islet autoantibodies by RBA in subsequent sampling events with a median delay of 2.8 years compared to multiplex ADAP assay. Discussion: In summary, multiplex ADAP assay could be an ideal tool for type 1 diabetes risk testing due to its sample-sparing nature (4µL), non-radioactiveness, compatibility with widely available real-time qPCR instruments and favorable risk prediction capability.

A 4-fold or greater decrease in TPPA titers may indicate effective BPG treatment in primary syphilis.

BACKGROUND: In the majority of clinical environments, the treponema pallidum particle agglutination (TPPA) test is known for its higher specificity compared to the rapid plasma reagin (RPR) test and is commonly employed for the diagnosis of syphilis, but their use for serological monitoring after syphilis therapy is controversial. OBJECTIVES: We aim to evaluate whether the TPPA titers is suitable for monitoring syphilis treatment efficacy. METHODS: At first, 232 patients with primary syphilis were recruited. Serological testing was performed at baseline (initial visit) and at 6 months (±1 month) after benzathine penicillin G (BPG) treatment. Second, New Zealand white male rabbits were infected with Treponema pallidum (T. pallidum) to evaluate the changes in TPPA titers after BPG therapy. Finally, we compared the TPPA titers in the culture supernatant of rabbit splenocytes stimulated with T. pallidum with or without BPG. RESULTS: After 6 months of treatment, 150 (64.7%) of 232 primary syphilis patients achieved serological cure, and 82 (35.3%) had adverse outcomes. Among 110 patients with TPPA titers decreased by more than fourfold, 109 of them were serological cure patients (≥4-fold decrease in RPR titers) (P ＜ 0.0001). In the rabbit model of syphilis, the TPPA titers was significantly decreased in the treatment subgroup (P = 0.016) and remained constant (±2-fold) or increased (≥4-fold) in the nontreatment subgroup. In addition, T. pallidum resulted in a positive TPPA titers in the culture supernatant of splenocytes (median titers was 1: 80), while BPG could directly reduce the TPPA titers in the culture supernatant (median titers was 1: 40) (P = 0.032). CONCLUSIONS: A 4-fold or greater decrease in TPPA titers may indicate effective treatment in primary syphilis. Combining TPPA titers with RPR titers results may potentially aid in the early diagnosis of syphilis.

A simple agglutination system for rapid antigen detection from large sample volumes with enhanced sensitivity.

Lateral flow assays (LFAs) provide a simple and quick option for diagnosis and are widely adopted for point-of-care or at-home tests. However, their sensitivity is often limited. Most LFAs only allow 50 µL samples while various sample types such as saliva could be collected in much larger volumes. Adapting LFAs to accommodate larger sample volumes can improve assay sensitivity by increasing the number of target analytes available for detection. Here, a simple agglutination system comprising biotinylated antibody (Ab) and streptavidin (SA) is presented. The Ab and SA agglutinate into large aggregates due to multiple biotins per Ab and multiple biotin binding sites per SA. Dynamic light scattering (DLS) measurements showed that the agglutinated aggregate could reach a diameter of over 0.5 µm and over 1.5 µm using poly-SA. Through both experiments and Monte Carlo modeling, we found that high valency and equivalent concentrations of the two aggregating components were critical for successful agglutination. The simple agglutination system enables antigen capture from large sample volumes with biotinylated Ab and a swift transition into aggregates that can be collected via filtration. Combining the agglutination system with conventional immunoassays, an agglutination assay is proposed that enables antigen detection from large sample volumes using an in-house 3D-printed device. As a proof-of-concept, we developed an agglutination assay targeting SARS-CoV-2 nucleocapsid antigen for COVID-19 diagnosis from saliva. The assay showed a 10-fold sensitivity enhancement when increasing sample volume from 50 µL to 2 mL, with a final limit of detection (LoD) of 10 pg mL-1 (∼250 fM). The assay was further validated in negative saliva spiked with gamma-irradiated SARS-CoV-2 and showed an LoD of 250 genome copies per µL. The proposed agglutination assay can be easily developed from existing LFAs to facilitate the processing of large sample volumes for improved sensitivity.

Flagellar-based motility accelerates IgA-mediated agglutination of Salmonella Typhimurium at high bacterial cell densities.

Introduction: Secretory IgA (SIgA) protects the intestinal epithelium from enteric pathogens such as Salmonella enterica serovar Typhimurium (STm) through a process known as immune exclusion, where invading bacteria are aggregated via antibody cross-linking, encased in mucus, and then cleared from the intestinal tract via peristalsis. At high cell densities, the STm aggregates form a tightly packed network that is reminiscent of early bacterial biofilms. However, the underlying mechanism of how SIgA mediates this transition from a motile and invasive state to an avirulent sessile state in STm is currently unknown. Methods: In this report, we developed and validated a methodology known as the "snow globe" assay to enable real-time imaging and quantification of STm agglutination by the mouse monoclonal IgA Sal4. Results: We observed that agglutination in the snow globe assay was dose-dependent, antigen-specific, and influenced by antibody isotype. We determined that flagellar-based motility was a prerequisite for rapid onset of agglutination, even at high cell densities where cell-cell contacts are expected to be frequent. We also investigated the roles of individual cyclic-di-GMP metabolizing enzymes previously implicated in motility and biofilm formation in Sal4 IgA-mediated agglutination. Discussion: Taken together, our results demonstrate that IgA-mediated agglutination is a dynamic process influenced by bacterial motility and cell-cell collisions. We conclude that the snow globe assay is a viable platform to further decipher the molecular and genetic determinants that drive this interaction.

A patient with the highly suspected B cell lymphoma accompanied by the erythrocytes cold agglutination: Case report.

RATIONALE: Cold agglutinins are related with B cell lymphoproliferative disorder and lymphoma, and can agglutinate red blood cells (RBCs) at an optimum temperature of 3-4°C, which is the undergoing cause of RBCs cold agglutination. RBC cold agglutination may lead to an extreme abnormality of RBC parameters of complete blood count (CBC). PATIENT CONCERNS: The present study reports a case of an old patient with severe infectious fever and anemia presenting extremely abnormal levels of RBC parameters in CBC and a sand-like appearance of blood on tube wall. The validating tests indicated the presence of the RBCs cold agglutination and the highly suspected B cell lymphoma. DIAGNOSES: The 37°C-incubation corrected the CBC results of the patient, and the microscopic observation and flow cytometry analysis of blood and marrow indicated many abnormal B lymphocytes. Subsequently, the patient was diagnosed with a highly suspected B-cell lymphoma. INTERVENTIONS: The blood with a sand-like appearance was reanalyzed to validate the cold agglutination by 37°C-water incubation. The smears of peripheral blood and marrow were made for morphological observation by using optical microscopy. Moreover, the clusters of differentiation of the white blood cells were analyzed to confirm the type of abnormal white blood cells with a flow cytometer. OUTCOMES: The RBCs cold agglutination was validated, and the highly suspected B cell lymphoma was proved as the undergoing cause. LESSONS: This case focuses on the discovery and solutions of RBCs cold agglutination, and emphasizes the importance of microscopic observation in the exploration of undergoing causes of cold agglutination.

In situ characterization of the agglutination of lectins via cross-linking of carbohydrates by time-resolved measurement of forward static light scattering.

We present real-time observations of a structurally variable process for cross-linking agglutination between multivalent lectins and glycoclusters using a small-angle forward static light scattering (F-SLS) technique. In this study, a cross-linking agglutination reaction was carried out using a tetravalent Neu5Acα2,6LacNAc-glycocluster and Sambucus sieboldiana agglutinin (SSA). The scattering intensity of time-resolved F-SLS increased with formation of the Neu5Acα2,6LacNAc-glycocluster-SSA cross-linked complex. Using this approach, fine sequential cross-linking agglutination between glycoclusters and lectins was observed in real-time. The rate of increase in the intensity of time-resolved F-SLS increased with the concentration of sialo-glycoclusters and SSA. Structural analysis based on the fractal dimension using time-resolved F-SLS patterns revealed that the density of the aggregates changed with progression of the cross-linking reaction until equilibrium was reached. This is the first report to evaluate the cross-linking agglutination reaction between glycoclusters and lectins and analysis of the subsequent structure of the obtained aggregates using time-resolved measurements of F-SLS.

Antibody detection by agglutination-PCR (ADAP) assays for the analysis of tissue transglutaminase autoantibodies in celiac disease.

BACKGROUND & AIMS: Tissue transglutaminase autoantibodies (tTGA) are used as diagnostic markers of celiac disease. Different methods have been developed for the detection of tTGA of which enzyme-linked immunosorbent assays (ELISA), radiobinding assays (RBA) and electrochemiluminescence (ECL) assays are the most commonly used. Here we aimed to evaluate a novel antibody detection by agglutination-PCR (ADAP) assay for the detection of tTGA. METHODS: Included were 126 children with untreated celiac disease (UCD), 64 disease controls (DC), 21 children with potential celiac disease (PCD), and 1501 children from the general population. Tissue TGA were determined using an automated ADAP assay platform and compared with two RBAs for the detection of IgA-tTG and IgG-tTG, respectively. RESULTS: ADAP detected tTGA in 123/126 (97.6%) UCD children compared with 122/126 (96.8%) using RBA-IgA-tTG and RBA-IgG-tTG (p > 0.9999), respectively. Among DC, ADAP detected 5/64 (7.8%) children with tTGA compared with 4/64 (6.3%) with RBA-IgA-tTG (p > 0.9999) and 8/64 (12.5%) with RBA-IgG-tTG (p = 0.5600), respectively. Tissue TGAs were equally detected in children with PCD in both assays. In the general population, 4/1501 (0.3%) were tTGA positive using ADAP compared with 3/1501 (0.2%) for RBA-IgA-tTG and RBA-IgG-tTG (p > 0.9999), respectively. The area under the curves (AUCs) were 0.998 for ADAP, 0.994 for RBA-IgA-tTG, and 0.999 for RBA-IgG-tTG, respectively. CONCLUSIONS: No difference in specificity and sensitivity of tTGA for the diagnosis of celiac disease was reported between ADAP and RBA. ADAP could be recommended as the first-line screening method of larger populations for celiac disease.

AIE-Active Glycomimetics Triggered Bacterial Agglutination and Membrane-Intercalating toward Efficient Photodynamic Antiseptic.

Opportunistic infections caused by Pseudomonas aeruginosa (P. aeruginosa) are particularly difficult to treat due to the altered membrane permeability and inherent resistance to conventional antibiotics. Here, a cationic glycomimetics is designed and synthesized with aggregation-induced emission (AIE) characteristics namely TPyGal, which self-assembles into the spherical aggregates with galactosylated surface. TPyGal aggregates can effectively cluster P. aeruginosa through multivalent carbohydrate-lectin interactions and auxiliary electrostatic interactions and subsequently trigger membrane-intercalating, which results in efficient photodynamic eradication of P. aeruginosa under white light irradiation by in situ singlet oxygen (1 O2 ) burst to disrupt bacterial membrane. Furthermore, the results demonstrate that TPyGal aggregates promote the healing of infected wounds, indicating the potential for clinical treatment of P. aeruginosa infections.

Agglutination and hemolytic crossmatching to determine transfusion reaction differences between large and small breed goats.

BACKGROUND: Blood transfusions are performed frequently in goats, but crossmatches are rarely performed. HYPOTHESIS/OBJECTIVES: Determine differences in the frequency of agglutination and hemolytic crossmatch reactions between large and small breed goats. ANIMALS: Healthy adult goats, 10 large and 10 small breed. METHODS: Two hundred eighty major and minor agglutination and hemolytic crossmatches: 90 large breed donor to large breed recipient (L-L), 90 small breed donor to small breed recipient (S-S), 100 large breed donor to small breed recipient (L-S). A linear mixed model with treatment group (L-L, S-S, L-S) as a fixed effect and individual crossmatch as a random effect was used to identify variations in reaction frequency among groups and individuals. RESULTS: Frequency of major agglutination reactions for L-L, S-S, and L-S were 3/90 (3.3%), 7/90 (7.8%), and 10/100 (10.0%), respectively. Frequency of major hemolytic reactions for L-L, S-S, and L-S were 27/84 (32.1%), 7/72 (9.7%), and 31/71 (43.7%). Individual pairings and groupings had no effect on agglutination reactions. Individual pairings had no effect on the frequency of hemolytic reactions. For major hemolytic crossmatches, pairwise comparisons identified higher frequencies of reactions when comparing L-L to S-S (P = .007) and L-S to S-S (P < .001). CONCLUSION AND CLINICAL IMPORTANCE: Goats experience increased frequencies of hemolytic reactions compared to agglutination. Significant increases in hemolysis were seen between large breed donors and small breed recipients, compared to small breed pairings. Additional studies are required to determine correlations between crossmatches and transfusion reactions.

Reduction-Responsive Supramolecular Sheets for Selective Regulation of Facultative Anaerobe Agglutination.

Stimuli-responsive supramolecular materials have promising biological applications because of their ability to rapidly undergo significant structural changes in response to diverse stimuli. Herein, supramolecular sheets assembled via charge-transfer interactions between the pyrene moiety of a d-mannose-containing amphiphile and 7,7,8,8-tetracyanoquinodimethane (TCNQ) are reported. The supramolecular sheets show reduction-responsive behavior, in which their disassembly is triggered by the reduction of TCNQ by sodium sulfide. In an anaerobic environment, the sheet structure remains intact and the exposed d-mannose moieties induce the agglutination of facultative anaerobes, thereby inhibiting bacterial growth. In contrast, in an aerobic environment, the reduction of TCNQ by the hydrogen sulfide generated by facultative anaerobes causes sheet disassembly. This enables continuous bacterial growth, because the collapsed sheets cannot induce agglutination. Thus, this study presents a novel supramolecular material for the selective regulation of facultative anaerobe growth according to the external environment.

Collection and processing of hematopoietic progenitor cell products at risk of presenting with cold agglutination.

BACKGROUND AIMS: Cold agglutinins are commonly identified in transfusion laboratories and are defined by their ability to agglutinate erythrocytes at 3-4°C, with most demonstrating a titer >64. Similarly, cryoglobulins can precipitate from plasma when temperatures drop below central body temperature, resulting in erythrocyte agglutination. Thankfully, disease associated from these autoantibodies is rare, but unfortunately, such temperature ranges are routinely encountered outside of the body's circulation, as in an extracorporeal circuit during hematopoietic progenitor cell (HPC) collection or human cell therapy laboratory processing. When agglutination occurs ex vivo, complications with the collection and product may be encountered, resulting in adverse events or product loss. Here, we endeavor to share our experience in preventing and responding to known cases at risk of or spontaneous HPC agglutination in our human cell therapy laboratory. CASE REPORTS: Four cases of HPC products at risk for, or spontaneously, agglutinating were seen at our institution from 2018 to 2020. Planned modifications occurred, including ambient room temperature increases, tandem draw and return blood warmers, warm product transport and extended post-thaw warming occurred. In addition, unplanned modifications were undertaken, including warm HPC product processing and plasma replacement of the product when spontaneous agglutination of the product was identified. All recipients successfully engrafted after infusion. CONCLUSIONS: While uncommon, cold agglutination of HPC products can disrupt standard processes of collection and processing. Protocol modifications can circumvent adverse events for the donor and minimize product loss. Such process modifications should be considered in individuals with known risks for agglutination going to HPC donation/collection.

Pre-transfusion Testing Using Crossmatching Agglutination Reaction Grades Combined With Rh Subgroup Phenotyping in Patients With Autoantibodies: A Three-year Experience at a Tertiary Hospital.

Background: The currently recommended pre-transfusion testing techniques for patients with autoantibodies are complex, time-consuming, and labor-intensive. Therefore, although the red blood cell (RBC) selection method using crossmatched RBC agglutination reaction grades (i.e., the "least incompatible" transfusion) is discouraged, many institutions still use it. We aimed to evaluate the effectiveness of this method combined with Rh subgroup phenotyping. Methods: We retrospectively investigated RBC transfusions from January 2019 to December 2021 in patients presenting as auto-control-positive via antibody identification (auto-control (+) group), where Rh subgroup phenotype-matched RBCs were selected based on the agglutination reaction grades of crossmatched units. For each study patient, an auto-control-negative patient was matched based on age, sex, department, and pre-transfusion Hb levels (auto-control (-) group). The mean Hb change per unit, transfusion-associated symptom/sign reports, and agglutination reaction grades upon crossmatching were analyzed. Results: In the auto-control (+) group, the Hb change per unit among different agglutination reaction grades of transfused RBCs and among different relative grades of transfused RBCs and crossmatching auto-controls was not significantly different (P=0.392 and P= 0.132, respectively). No significant difference was observed in Hb changes and transfusion-associated symptom/sign occurrence between the auto-control (+) and auto-control (-) groups (P=0.121 and P=0.822, respectively). In addition, no definite evidence of hemolysis in the auto-control (+) group was observed in the medical record review. Conclusions: Together with Rh subgroup phenotyping, selecting the RBC unit with the lowest agglutination reaction grade upon crossmatching does not adversely affect transfusion efficiency.

The DUF1943 and VWD domains endow Vitellogenin from Crassostrea gigas with the agglutination and inhibition ability to microorganism.

Vitellogenin (Vg) is the major precursor of the egg-yolk proteins, which mainly acts as an energy reserve molecule for providing nutrients during embryonic development. Vg also plays an immune function in vertebrates such as fish, but there are few studies on the immune function of Vg in invertebrates. In the present study, a Vg homologue (CgVg) was identified and characterized in oyster Crassostrea gigas. There are three domains in the CgVg protein, including a Vitellogenin_N domain, a domain of unknown function 1943 (DUF1943) and a von Willebrand factor type D domain (VWD). The mRNA transcripts of CgVg were detected in all tested tissues with high expression in the gonad, hepatopancreas and haemocytes, which was 466.29-, 117.15- and 57.49-fold (p < 0.01) of that in adductor muscle, respectively. After Vibrio splendidus stimulation, the mRNA expression level of CgVg in haemocytes increased significantly at 6, 12 and 24 h, which was 1.97-, 3.58- and 1.3-fold (p < 0.01) of that in the seawater group, respectively. The immunofluorescence assay showed that positive signals of CgVg protein were mainly located at the cytoplasm of haemocytes. The recombinant protein of DUF1943 domain (rDUF1943) and VWD domain (rVWD) was able to bind lipopolysaccharide (LPS), mannose (MAN), peptidoglycan (PGN) and poly (I:C), as well as Gram-positive bacteria (Staphylococcus aureus and Micrococcus luteus), Gram-negative bacteria (Escherichia coli and V. splendidus) and fungi (Pichia pastoris). rDUF1943 exhibited stronger agglutination activity towards S. aureus, M. luteus, E. coli, V. splendidus and P. pastoris, while agglutination was only observed in the rVWD group towards P. pastoris. The rVWD inhibited the growth of E. coli, S. aureus and V. splendidus, while no antibacterial activity was detected in rDUF1943 group. Collectively, CgVg not only functioned as a pattern recognition receptor (PRR) to bind various microorganisms and PAMPs, but also as an immune effector participating in the clearance of invaders, in which DUF1943 and VWD domain were mainly responsible for agglutinating and inhibiting microorganism respectively.

Validation of a cage-side agglutination card for Dal blood typing in dogs.

BACKGROUND: Although 98% of the canine population is Dal-positive, Dal-negative dogs are more common in some breeds such as Doberman Pinschers (42.4%) and Dalmatians (11.7%), and finding compatible blood for these breeds may be challenging, given limited access to Dal blood typing. OBJECTIVES: To validate a cage-side agglutination card for Dal blood typing and determine the lowest packed cell volume (PCV threshold) at which interpretation remains accurate. ANIMALS: One-hundred fifty dogs, including 38 blood donors, 52 Doberman Pinschers, 23 Dalmatians and 37 anemic dogs. Three additional Dal-positive canine blood donors were included to establish the PCV threshold. METHODS: Dal blood typing was performed on blood samples preserved in ethylenediaminetetraacetic acid (EDTA) <48 hours using the cage-side agglutination card and a gel column technique (gold standard). The PCV threshold was determined using plasma-diluted blood samples. All results were read by 2 observers, blinded to each other's interpretation and to the sample's origin. RESULTS: Interobserver agreement was 98% and 100% using the card and gel column assays, respectively. Overall, the sensitivity and specificity of the cards were 86%-87.6% and 96.6%-100%, respectively, depending on the observer. However, 18 samples were mistyped using the agglutination cards (15/18 by both observers): 1 false-positive (Doberman Pinscher), and 17 false-negative samples including 13 anemic dogs (PCV range, 5%-24%; median, 13%). The PCV threshold allowing reliable interpretation was determined to be >20%. CONCLUSIONS AND CLINICAL IMPORTANCE: Dal agglutination cards are reliable as a cage-side test, but results should be interpreted cautiously in severely anemic patients.

Use of the particle agglutination/particle agglutination inhibition test for antigenic analysis of SARS-CoV-2.

Background: The antigenicity of SARS-CoV-2 is a critical issue for the effectiveness of the vaccine, and thus, it should be phenotypically evaluated by serological assays as new field isolates emerge. The hemagglutination/hemagglutination inhibition (HA/HI) tests are well known as a representative method for antigenic analysis of influenza viruses, but SARS-CoV-2 does not agglutinate human or guinea pig red blood cells. Therefore, the antigenic analysis requires complicated cell-based assays using special equipment such as plate reader or ELISPOT analyzer. Methods: Based on the HA/HI tests for influenza viruses, we developed the particle agglutination/particle agglutination inhibition (PA/PAI) test to easily and rapidly quantify the virus and antibody using human angiotensin-converting enzyme 2 (hACE2)-bound latex beads. The virus titers were determined by mixing the beads and the virus from culture supernatant, settling it overnight, and then observing the sedimentation/agglutination pattern (PA test). The neutralization antibody titers were determined by mixing virus-infected hamster antisera in addition to the beads and virus (PAI test). Results: The PA titer was positively correlated with the plaque-forming units. The PAI titer using the hamster antisera clearly revealed the antigenic difference between the omicron and previous variants. The antigenic differences were supported by the results shown in other methods. Conclusions: The PAI test is an easy and rapid method to analyze the antigenicity of SARS-CoV-2.

Outcomes After Lysis of Adhesions and Dilator Placement for Treatment of Vulvovaginal Agglutination Due to Lichen Planus.

OBJECTIVE: The aim of the study is to determine intraoperative and postoperative surgical outcomes for the treatment of vulvovaginal agglutination secondary to lichen planus (LP) following a standard protocol using intraoperative dilator placement and postoperative intravaginal steroid use. MATERIALS AND METHODS: This was a retrospective chart review of patients who underwent surgical management of vulvovaginal agglutination due to LP following a protocol that included surgical lysis of vulvovaginal adhesions, intraoperative dilator placement and removal 48 hours later, and high-potency intravaginal corticosteroid and regular dilator use thereafter. Demographic and clinical data were abstracted from the medical record and analyzed using descriptive statistics. RESULTS: Thirty-four patients, with mean age 51.2 ± 11 years and body mass index 32.8 ± 8.5 kg/m 2 , underwent lysis of vulvovaginal adhesions between 1999 and 2021 with 8 different surgeons at a single institution. The mean preoperative, immediate postoperative, and 6-week postoperative vaginal lengths were 2.8 ± 1.8 cm ( n = 18), 8.0 ± 1.9 cm ( n = 21), and 7.9 ± 2.2 cm ( n = 16), respectively. The mean estimated blood loss intraoperatively was 16 ± 15 mL. No patients had a documented surgical site infection or reoperation within 30 days after surgery. Of patients who had it documented ( n = 26), 70% (18/26) reported postoperative sexual activity. Where documented, 100% (18/18) reported preoperative dyspareunia, while 17% (3/18) did postoperatively. Six percent (2/34) had recurrent severe agglutination and 3% (1/34) underwent reoperation. CONCLUSIONS: Lysis of vulvovaginal adhesions, intraoperative dilator placement, and postoperative intravaginal corticosteroids with dilator use is a safe and effective treatment option to restore vaginal length for those with vulvovaginal LP.