A Quantitative Human Red Blood Cell Agglutination Assay for Characterisation of Galectin Inhibitors.

Galectins are a family of beta-galactoside-binding proteins that are characterised by their carbohydrate recognition domain (CRD) and include galectin-1 and galectin-3. These galectins have been implicated in numerous diseases due to their pleiotropic nature, including cancer and fibrosis, with therapeutic inhibitors being clinically developed to block the CRD. One of the early methods developed to characterise these galectins was the hemagglutination of red blood cells. Although it is insightful, this approach has been hampered by a lack of sensitivity and accurate quantification of the agglutination observed. In this study, we aimed to validate a more precise and quantitative method to enable the further investigation of differences between galectins in respect to agglutination induction in different blood groups, as well as the characterisation of small molecule inhibitors. Quantification of hemagglutination was shown to be optimal using U-bottom plates imaged and analysed with FIJI ImageJ rather than flat-bottom plates read for absorbance on an optical density plate reader. Galectin-3-induced red blood cell agglutination efficacy increased significantly from blood group O to A to B. However, for both the galectin-1 monomer and concatemer, a more comparable effect was observed between blood group B and O, but with more potent effects than in blood group A. Inhibition assays for both galectin-3 and galectin-1 induced-hemagglutination were able to demonstrate clear concentration responses and expected selectivity profiles for a set of small-molecule glycomimetics, confirming the historical profiles obtained in biochemical binding and functional cellular assays.

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.

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.

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.

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.

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.

Bitiscetin-3, a Novel C-Type Lectin-like Protein Cloned from the Venom Gland of the Viper Bitis arietans, Induces Platelet Agglutination and Inhibits Binding of Von Willebrand Factor to Collagen.

Bitiscetin-1 (aka bitiscetin) and bitiscetin-2 are C-type lectin-like proteins purified from the venom of Bitis arietans (puff adder). They bind to von Willebrand factor (VWF) and-at least bitiscetin-1-induce platelet agglutination via enhancement of VWF binding to platelet glycoprotein Ib (GPIb). Bitiscetin-1 and -2 bind the VWF A1 and A3 domains, respectively. The A3 domain includes the major site of VWF for binding collagen, explaining why bitiscetin-2 blocks VWF-to-collagen binding. In the present study, sequences for a novel bitiscetin protein-bitiscetin-3-were identified in cDNA constructed from the B. arietans venom gland. The deduced amino acid sequences of bitiscetin-3 subunits α and ß share 79 and 80% identity with those of bitiscetin-1, respectively. Expression vectors for bitiscetin-3α and -3ß were co-transfected to 293T cells, producing the heterodimer protein recombinant bitiscetin-3 (rBit-3). Functionally, purified rBit-3 (1) induced platelet agglutination involving VWF and GPIb, (2) did not compete with bitiscetin-1 for binding to VWF, (3) blocked VWF-to-collagen binding, and (4) lost its platelet agglutination inducing ability in the presence of an anti-VWF monoclonal antibody that blocked VWF-to-collagen binding. These combined results suggest that bitiscetin-3 binds to the A3 domain, as does bitiscetin-2. Except for a small N-terminal fragment of a single subunit-which differs from that of both bitiscetin-3 subunits-the sequences of bitiscetin-2 have never been determined. Therefore, by identifying and analyzing bitiscetin-3, the present study is the first to present the full-length α- and ß-subunit sequences and recombinant expression of a bitiscetin-family toxin that blocks the binding of VWF to collagen.

Peptidoglycan recognition protein-S1 (PGRP-S1) from Diaphania pyloalis (Walker) is involved in the agglutination and prophenoloxidase activation pathway.

Recognition of invading foreign exogenous pathogen is the first step to initiate the innate immune response of insects, which accomplished by the pattern recognition receptors (PRRs). Peptidoglycan recognition proteins (PGRPs) serve as an important type of PRRs, which activate immune response by detecting peptidoglycan of microbial cell wall. In this study, we have cloned the full-length cDNA of PGRP gene called PGRP-S1 from the Diaphania pyloalis (Walker). The open reading frame (ORF) of D. pyloalis PGRP-S1 encodes 211 amino acids which containing a secretion signal peptide and a canonical PGRP domain. Multisequence alignment revealed that PGRP-S1 possess the amino acid residues responsible for zinc binding and amidase activity. D. pyloalis PGRP-S1 exhibited the highest transcript level in fat body and followed in head. The mRNA concentration dramatically increased after an injection of Escherichia coli or Micrococcus luteus. Purified recombinant PGRP-S1 exhibit binding ability to peptidoglycans from Staphylococcus aureus or Bacillus subtilis and cause intensive agglutination of E. coli, M. luteus or S. aureus in the presence of zinc ions. Furthermore, phenoloxidase activity significantly increased when the plasma from larvae was incubated with recombinant PGPR-S1 and peptidoglycans from B. subtilis or M. luteus simultaneously. These results implied that PGRP-S1 was a member involving the prophenoloxidase activation pathway. Overall, our results indicated that D. pyloalis PGRP-S1 serve as a PRR to participate in the recognition of foreign pathogen and prophenoloxidase pathway stimulation.

An l-rhamnose-binding lectin from Nile tilapia (Oreochromis niloticus) possesses agglutination activity and regulates inflammation, phagocytosis and respiratory burst of monocytes/macrophages.

Rhamnose-binding lectins (RBLs), a Ca2+-independent lectin family, are widely present in vertebrates and invertebrates, which involve in the innate immune response. However, the functional characterization and related regulation mechanisms of RBLs remain unclear in teleost fish. In this study, an l-rhamnose-binding lectin-like (OnRBL-L) was identified and functionally characterized from Nile tilapia (Oreochromis niloticus). The open reading frame of OnRBL-L is 678 bp encoding 225 aa. The sequence of OnRBL-L has relatively conservative characteristic peptide motifs, including YGR, DPC, and KYL-motif. Expression analysis showed that OnRBL-L was abundantly distributed in intestine tissue, and widely existed in all detected tissues. Meanwhile, the expression of OnRBL-L increased significantly in vivo (liver, spleen, head kidney, intestine, gills and peripheral blood) and in vitro (monocytes/macrophages) following challenges with two important tilapia pathogenic bacteria Streptococcus agalactiae and Aeromonas hydrophila. In addition, the recombinant OnRBL-L was found to bind and agglutinate S. agalactiae and A. hydrophila. Furthermore, OnRBL-L could participate in non-specific cellular immune defense, including reducing the expression of pro-inflammatory factors (IL-6、IL-8 and TNF-α), and enhancement of the phagocytosis and respiratory burst of MO/MФ. Overall, our results provide new insights into the understanding of RBL as an important pattern recognition molecule and regulator in non-specific cell immunity in an early vertebrate.

Protein Diversity and Immune Specificity of Hemocyanin From Shrimp Litopenaeus vannamei.

Hemocyanin is an important non-specific innate immune defense molecule with phenoloxidase, antiviral, antibacterial, hemolytic, and antitumor activities. To better understand the mechanism of functional diversity, proteomics approach was applied to characterize hemocyanin (HMC) expression profiles from Litopenaeus vannamei. At first, hemocyanin was purified by Sephadex G-100 and DEAE-cellulose (DE-52) columns from shrimp serum, and 34 protein spots were identified as HMC on the 2-DE gels. Furthermore, we found that 9 HMC spots about 75 or 77 kDa were regulated by Streptococcus agalactiae and Vibrio parahaemolyticus infection at 6, 12, and 24 h. In addition, 6 different pathogen-binding HMC fractions, viz., HMC-Mix, HMC-Vp, HMC-Va, HMC-Vf, HMC-Ec, and HMC-Sa, showed different agglutinative and antibacterial activities. Moreover, lectin-blotting analysis showed significant differences in glycosylation level among HMC isomers and bacteria-binding HMC fractions. Particularly, the agglutinative activities of the HMC fractions were almost completely abolished when HMC was deglycosylated by O-glycosidase, which suggest that O-linked sugar chains of HMC played important roles in the innate immune recognition. Our findings demonstrated for the first time that L. vannamei HMC had molecular diversity in protein level, which is closely associated with its ability to recognize diverse pathogens, whereas glycan modification probably contributed to HMC's diversity and multiple immune activities.

Research advances and prospects of legume lectins.

Lectins are widely distributed proteins having ability of binding selectively and reversibly with carbohydrates moieties and glycoconjugates. Although lectins have been reported from different biological sources, the legume lectins are the best-characterized family of plant lectins. Legume lectins are a large family of homologous proteins with considerable similarity in amino acid sequence and their tertiary structures. Despite having strong sequence conservation, these lectins show remarkable variability in carbohydrate specificity and quaternary structures. The ability of legume lectins in recognizing glycans and glycoconjugates on cells and other intracellular structures make them a valuable research tool in glycomic research. Due to variability in binding with glycans, glycoconjugates and multiple biological functions, legume lectins are the subject of intense research for their diverse application in different fields such as glycobiology, biomedical research and crop improvement. The present review specially focuses on structural and functional characteristics of legume lectins along with their potential areas of application.

Magnetic field-enhanced agglutination as a readout for rapid serologic assays with human plasma.

Recent virus outbreaks have revealed a critical need for large scale serological assays. However, many available tests either require a cumbersome, costly apparatus or lack the availability of full automation. In order to address these limitations, we describe a homogeneous assay for antibody detection via measurement of superparamagnetic particles agglutination. Application of a magnetic field permits to overcome the limitations governed by Brownian translational diffusion in conventional assays and results in an important acceleration of the aggregation process as well as an improvement of the limit of detection. Furthermore, the use of protein-concentrated fluid such as 5 times-diluted human plasma does not impair the performances of the method. Screening of human plasma samples shows a strict discrimination between seropositive and seronegative samples in an assay duration as short as 14 s. The sensitivity of this method, combined with its quickness and simplicity, makes it a promising diagnostic tool.

Lymphocyte Aggregation in Low-Grade B-Cell Lymphoma.

Platelet and erythrocyte agglutination is known to happen in vitro due to EDTA or temperature-induced cold antibodies. Leukocyte agglutination is far less common, and its etiology is not always known. The 2 cases presented herein are of low-grade B-cell lymphomas consistent with splenic marginal-zone lymphoma that presented with lymphocyte agglutination. In Case A, the lymphocyte aggregates were not resolved by warming the sample or by non-EDTA anticoagulation. In Case B, the lymphocyte aggregates were largely resolved by warming the specimen at 37°C for 15 minutes. The 2 cases presented herein further show that the etiology of lymphocyte aggregation can have multiple causes, even within the same disease process.

A C-type lectin (OmCTL) in Onychostoma macrolepis: Binding ability to LPS, PGN and agglutinating activity against bacteria.

C-type lectins (CTLs) are calcium-dependent carbohydrate-binding proteins that mainly bind to carbohydrate-based or other ligands to mediate cell adhesion, recognize pathogens, and play important roles in the immune system. In the present study, a novel C-type lectin (OmCTL) isolated from Onychostoma macrolepis was investigated. The open reading frame of OmCTL comprises 468 bp, encoding a 155 amino acid polypeptide with an 18 amino acid putative signaling peptide. The predicted primary OmCTL structure contains a signal peptide, a single carbohydrate recognition domain (CRD) and an EPN/WND motif required for carbohydrate-binding specificity. Using tissue expression pattern analysis, OmCTL has been shownto be highly expressed in the liver, and is also detected in other tissues. OmCTL was significantly upregulated in the liver and spleen following infection with Aeromonas hydrophila, suggesting its involvement in immune response. The recombinant OmCTL protein (rOmCTL) agglutinated two gram-negative bacteria, Escherichia coli and A. hydrophila, in vitro in the presence of Ca2+, showing that it is a typical Ca2+-dependent carbohydrate-binding protein.Furthermore, rOmCTL purified from E. coli BL21 (DE3) strongly bound to LPS and PGN, as well as all tested bacteria in a Ca2+-independent manner. These results indicate that OmCTL plays a central role in the innate immune response and as a pattern recognition receptor that recognizes diverse pathogens among O. macrolepis.

Musa acuminata lectin exerts anti-cancer effects on HeLa and EAC cells via activation of caspase and inhibitions of Akt, Erk, and Jnk pathway expression and suppresses the neoangiogenesis in in-vivo models.

In the present study aimed to purify the lectin from the sap of Musa acuminata pseudostem and elucidate the apoptotic and angiogenic molecular mechanism in both in-vitro and in-vivo model. Mannose specific lectin was purified by using mannose affinity column chromatography and analyzed by RP-HPLC, SDS-PAGE, and PAS staining method. Furthermore, the protein was identified by MALDI-MS/MS. MAL effectively agglutinates trypsinized RBCs and showed effective cytotoxicity against various human cancer cell lines. MAL mitigates the cell proliferation, colony formation, cell migration, arrest the cell cycle in the G2/M phase, and induce apoptosis by altering the expression of apoptotic proteins/mRNA level (Bax and Bcl-2) via caspase 8/9, 3 dependent pathway in both in-vitro and in-vivo. Supporting this, in-vivo EAC tumor mice models prove the efficacy of MAL by inducing cell death and inhibiting the neovessel formation by targeting the MVD, inhibition of VEGF secretion, suppressing the expression of MMPs, HIF-1α, Flt-1, Akt, Jnk, and Erk1/2. More importantly, the MAL treatment leads to effective inhibition of tumor growth and an increase in the survivability of EAC mice. Our study summarizes that the MAL having a significant anticancer potential expressively degenerates the tumor development by inducing apoptosis and suppressing neoangiogenesis.

3D Reconstruction of HvRNASET2 Molecule to Understand Its Antibacterial Role.

Recent studies performed on the invertebrate model Hirudo verbana (medicinal leech) suggest that the T2 ribonucleic enzyme HvRNASET2 modulates the leech's innate immune response, promoting microbial agglutination and supporting phagocytic cells recruitment in challenged tissues. Indeed, following injection of both lipoteichoic acid (LTA) and Staphylococcus aureus in the leech body wall, HvRNASET2 is expressed by leech type I granulocytes and induces bacterial aggregation to aid macrophage phagocytosis. Here, we investigate the HvRNASET2 antimicrobial role, in particular assessing the effects on the Gram-negative bacteria Escherichia coli. For this purpose, starting from the three-dimensional molecule reconstruction and in silico analyses, the antibacterial activity was evaluated both in vitro and in vivo. The changes induced in treated bacteria, such as agglutination and alteration in wall integrity, were observed by means of light, transmission and scanning electron microscopy. Moreover, immunogold, AMPs (antimicrobial peptides) and lipopolysaccharide (LPS) binding assays were carried out to evaluate HvRNASET2 interaction with the microbial envelopes and the ensuing ability to affect microbial viability. Finally, in vivo experiments confirmed that HvRNASET2 promotes a more rapid phagocytosis of bacterial aggregates by macrophages, representing a novel molecule for counteracting pathogen infections and developing alternative solutions to improve human health.

Subgrupos A1, A2 y AINT en donantes efectivos del banco de sangre materno infantil CNS La Paz ­ Bolivia / Subgroups A1, A2 and AINT in effective donors of the maternal and child blood bank CNS La Paz - Bolivia

Además del sistema ABO, los subgrupos del mismo revisten gran importancia en inmunohematología, Los subgrupos A difieren tanto en el número de sitios antigénicos como en la configuración del antígeno eritrocitario. Los principales, A1 y A2 se diferencian en que los eritrocitos A1 son aglutinados por el anticuerpo Anti-A1 humano o por la Lectina Anti-A1 (Dolichos biflorus), y los eritrocitos A2 son aglutinados por la Lectina Anti-H (Ulex europaeus). MATERIALES Y MÉTODOS: se realizó un estudio descriptivo, de Corte Transversal, Se analizó los registros tanto físico y electrónico del Banco de Sangre, se incluyeron todos los donadores efectivos, mismos que fueron tipificados por el laboratorio de inmunohematología en el periodo de mayo a julio del 2018. Método empleado, aglutinación en tubo y en micro placa. RESULTADOS: en un total de 1599 donantes, se determinó que el grupo O tiene mayor frecuencia con un 84% y el menos frecuente fue el AB con un 0,66%. Según el grupo sanguíneo A y AB tenemos las siguientes frecuencias: A1 que representa el (73.3%), A2 el (15.9%), Aint el (5.65%), A1 B el (3.60%) y A2 B el (1.55%). La importancia clínica se basa en que algunas personas del grupo A2 transfundidas con A1 , pueden producir Anti-A1 que es un anticuerpo natural irregular activo a 22 ºC, pero en ocasiones está activo a 37ºC causando una reacción transfusional extravascular, por lo que, si no se cuenta con eritrocitos A2 , se recomienda transfundir eritrocitos grupo O.

In addition to the ABO system, its subgroups review great importance in Immunohematology. Subgroups A differ both in the number of antigenic sites and in the configuration of the erythrocyte antigen. The main ones, A1 and A2 differ in that A1 erythrocytes are agglutinated by human Anti-A1 antibody or by Anti-A1 Lectin (Dolichos biflorus), and A2 erythrocytes are agglutinated by Anti-H Lectin (Ulex europaeus). MATERIALS AND METHODS: a descriptive, cross-sectional study was conducted. The physical and electronic records of the Blood Bank were analyzed, all effective donors were included, which were typified by the Immunohematology Laboratory in the period of May. to July 2018. Method used, agglutination in tube and in microplate. RESULTS: in a total of 1599 protocols, it was determined that group O has the highest frequency with 84% and the least frequent was the AB with 0.66%. According to blood group A and AB we have the following frequencies: A1 representing (73.3%), A2 (15.9%), Aint (5.65%), A1B (3.60%) and A2B (1.55%). The clinical importance is based on the fact that some people in group A2 transfused with A1, can produce Anti-A1 which is an irregular natural antibody active at 22 ° C but sometimes it is active at 37 °C causing an extravascular transfusion reaction, so if A2 erythrocytes are not available, it is recommended to transfuse group O erythrocytes.

Sensitive detection of multiple islet autoantibodies in type 1 diabetes using small sample volumes by agglutination-PCR.

Islet autoantibodies are predominantly measured by radioassay to facilitate risk assessment and diagnosis of type 1 diabetes. However, the reliance on radioactive components, large sample volumes and limited throughput renders radioassay testing costly and challenging. We developed a multiplex analysis platform based on antibody detection by agglutination-PCR (ADAP) for the sample-sparing measurement of GAD, IA-2 and insulin autoantibodies/antibodies in 1 µL serum. The assay was developed and validated in 7 distinct cohorts (n = 858) with the majority of the cohorts blinded prior to analysis. Measurements from the ADAP assay were compared to radioassay to determine correlation, concordance, agreement, clinical sensitivity and specificity. The average overall agreement between ADAP and radioassay was above 91%. The average clinical sensitivity and specificity were 96% and 97%. In the IASP 2018 workshop, ADAP achieved the highest sensitivity of all assays tested at 95% specificity (AS95) rating for GAD and IA-2 autoantibodies and top-tier performance for insulin autoantibodies. Furthermore, ADAP correctly identified 95% high-risk individuals with two or more autoantibodies by radioassay amongst 39 relatives of T1D patients tested. In conclusion, the new ADAP assay can reliably detect the three cardinal islet autoantibodies/antibodies in 1µL serum with high sensitivity. This novel assay may improve pediatric testing compliance and facilitate easier community-wide screening for islet autoantibodies.