Cell Agglutination Research Articles

A view of immunologic male infertility from the perspective of preserving seminal fluid homeostasis

The basis of the pathogenesis of immunologic male infertility is the production of antisperm antibodies against sperm antigens by plasmocytes. Extracellular nucleic acids, among other antigens, are perceived as DAMPs. The clearance system for biological fluids is complex. Endonucleases are responsible for the utilization of extracellular DNA. Among endonucleases, DNase 1 is the most studied. Its concentration in biological fluids is higher than others. For the first time, we determined the concentration of DNase 1 in the seminal fluid of men of reproductive age. DNase 1 concentration values were compared with the level of antisperm antibodies in the ejaculate, after which the relationship between the enzyme level and standard spermogram parameters was determined. The material for the study was the ejaculate of 44 relatively healthy men of reproductive age. During spermiological analysis, macroscopic and microscopic parameters of seminal fluid were determined, the concentration of deoxyribonuclease 1 (DNase 1) in the material was determined by enzyme immunoassay, and the presence of antisperm antibodies was assessed using a direct diagnostic method – the MAR test. Statistical processing of the obtained data was carried out using analytical software PAST v. 4.06. Reference intervals for DNase 1 concentrations were calculated using the MedCalc v. 17.4 program. The relationship between spermogram parameters and DNase 1 concentration was assessed by calculating Spearman’s linear rank correlation coefficients. As a result, a negative correlation was found between the DNase 1 ratio and such ejaculate parameters as liquefaction time (rS = -0.37; p = 0.013) and viscosity (rS = -0.37; p = 0.013), as well as the level of antisperm antibodies A (rS = -0.43; p = 0.003) and G (rS = -0.33; p = 0.027), the mucous component of the ejaculate (rS = -0.37; p = 0.012) and agglutination of male germ cells (rS = -0.33; p = 0.029). According to the data obtained, the optimal level of DNase 1 is directly related to the quality of the ejaculate. Determination of DNase 1 concentration can serve as an additional diagnostic criterion for male immunological infertility. These data will expand our understanding of a man’s fertile potential.

ABO-A2 red cell agglutination titres: Not the solution to improved ABO-A2-incompatible transplant eligibility assessment

ABO-A2 red cell agglutination titres: Not the solution to improved ABO-A2-incompatible transplant eligibility assessment

Antigen specificity affects analysis of natural antibodies.

Natural antibodies are used to compare immune systems across taxa, to study wildlife disease ecology, and as selection markers in livestock breeding. These immunoglobulins are present prior to immune stimulation. They are described as having low antigen specificity or polyreactive binding and are measured by binding to self-antigens or novel exogenous proteins. Most studies use only one or two antigens to measure natural antibodies and ignore potential effects of antigen specificity in analyses. It remains unclear how different antigen-specific natural antibodies are related or how diversity among natural antibodies may affect analyses of these immunoglobulins. Using genetically distinct lines of chickens as a model system, we tested the hypotheses that (1) antigen-specific natural antibodies are independent of each other and (2) antigen specificity affects the comparison of natural antibodies among animals. We used blood cell agglutination and enzyme-linked immunosorbent assays to measure levels of natural antibodies binding to four antigens: (i) rabbit erythrocytes, (ii) keyhole limpet hemocyanin, (iii) phytohemagglutinin, or (iv) ovalbumin. We observed that levels of antigen specific natural antibodies were not correlated. There were significant differences in levels of natural antibodies among lines of chickens, indicating genetic variation for natural antibody production. However, line distinctions were not consistent among antigen specific natural antibodies. These data show that natural antibodies are a pool of relatively distinct immunoglobulins, and that antigen specificity may affect interpretation of natural antibody function and comparative immunology.

Routine maternal ABO/Rhesus D blood typing can alert of massive foetomaternal haemorrhage.

Spontaneous massive foetomaternal haemorrhage (SM-FMH) is a rare yet critical condition that poses substantial risk to foetal health and survival. Existing data indicate that many cases may be undiagnosed. The current study aimed to investigate and validate the utility of identifying mixed field red blood cell (RBC) agglutination during maternal blood typing as a diagnostic aid for SM-FMH. Retrospective analysis of medical records from neonates born at our tertiary, university-affiliated medical centre between 2016 and 2023 was performed. Diagnosis of SM-FMH was based on neonates born with severe anaemia (haematocrit [HCT] <15%) within the first 24 h post-delivery with positive maternal Kleihauer-Betke (KB) test. Maternal ABO/Rhesus D (RhD) blood typing results were scrutinized with the primary objective of assessing the ability to identify dual RBC populations in cases clinically diagnosed with SM-FMH. Among 29,192 neonates studied, a mere 0.02% (5 cases) exhibited severe SM-FMH. Notably, a mixed field RBC agglutination was discerned in 80% (4/5) of these cases. This study underscores the significance of detecting mixed field RBC agglutination during antepartum maternal ABO/RhD blood typing as a potential indicator for SM-FMH. Increased awareness among blood bank technology specialists and obstetricians regarding these laboratory findings could prove instrumental in saving foetal lives.

Quantitative and equipment-free paper-based agglutination assay of bacterial cells.

Background: point-of-care (POC) tests are useful for bedside/home applications, emergencies, frequent follow-ups, and resource-limited areas. Limited quantitative and equipment-free POC assays have been reported. This study aims to develop, validate, and apply a simple, quantitative, paper-based POC assay. Methods: wax-channeled paper treated with specific anti-Brucella and anti-Salmonella antibodies was used for distance-based chromatographic elution of stained bacterial cell agglutinations. Results: a qualitative paper-based agglutination POC test was developed using color intensity, tail appearance, and "+/-" signs that clearly distinguish the positive and negative results. The optimization of the test for paper type, microfluidic channel design, antibody and bacterial cell concentrations, and elution methods was carried out. Quantitative assay transformation was successfully developed using the color intensity of the original reaction zone, intensity of elution tail, and distance-based migration that correspond to bacterial agglutination size. The migration distance of eluted bacterial agglutination bands corresponds to the target concentration with good linearity and minimal variability. Reporting of colored band migration with numbers using microfluidic patterns was used to enhance non-technical end-user applications. A distance-based POC assay prototype was then successfully used for the accurate detection of known and unknown samples in comparison with standard assays. Conclusions: the migration distance of an eluted stained bacterial agglutination correlated with anti-bacterial antibody concentrations. A simple, cheap, quantitative, and equipment-free paper-based POC assay of bacterial cell agglutination was developed. This test can be used for simple "+/-" results, thermometer-like quantification, or text reporting with numbers corresponding to target concentrations. The assay has extended applications to different human disease biomarkers.

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.

The role of a novel secretory peptidoglycan recognition protein with antibacterial ability from the Chinese Oak Silkworm Antheraea pernyi in humoral immunity

Peptidoglycan recognition proteins (PGRPs) are a family of pattern recognition receptors that play a critical role in the immune response of invertebrates and vertebrates. Herein, the short ApPGRP-D gene was cloned from the model lepidopteran Antheraea pernyi. Quantitative PCR (qPCR) confirmed that ApPGRP-D is an immune-related protein and that the expression of ApPGRP-D can be induced by microorganisms. ApPGRP-D is a broad-spectrum pattern recognition protein that activates the prophenoloxidase cascade activation system and promotes the agglutination of microbial cells. Likely due to its amidase activity, ApPGRP-D can inhibit the growth of E. coli and S. aureus. In addition, we demonstrated for the first time that zinc ions, as important metal coenzymes, could promote multiple functions of ApPGRP-D but not its amidase activity.

Acute paroxysmal cold hemoglobinuria upon dengue fever: A case report

Rationale: Dengue fever is capable of inciting the formation of transient polyclonal antibodies directed at red blood cell antigens, resulting in complement-mediated hemolysis, leading to intravascular hemolysis and hemoglobinuria. Patient’s concern: A 12-year-old male patient who recovered from dengue fever a week ago had red blood cell agglutination, spherocytes, and engulfment of red blood cells (erythrophagocytosis) by monocytes and neutrophils on routine hematological peripheral blood smear. The unexpected blood smear results prompted the lab physicians to investigate autoimmune hemolytic anemia, which revealed a monospecific positive direct antiglobulin test for complement (C3d, C3b) and the presence of Donath-Landsteiner antibody. Diagnosis: Paroxysmal cold hemoglobinuria (PCH), secondary to dengue fever. Interventions: Oxygen supplements, antibiotics, intravenous immunoglobulins, steroid therapy, and packed cell transfusions were administered. Outcomes: The patient’s condition was improved following the therapy. Lessons: Post-dengue PCH is a rare complication that requires a thorough peripheral smear examination for erythrophagocytosis, as advanced hematology analyzers fail to detect such findings.

Reduction of hemagglutination induced by a SARS-CoV-2 spike protein fragment using an amyloid-binding benzothiazole amphiphile

COVID-19 infection is associated with a variety of vascular occlusive morbidities. However, a comprehensive understanding of how this virus can induce vascular complications remains lacking. Here, we show that a peptide fragment of SARS-CoV-2 spike protein, S192 (sequence 192-211), is capable of forming amyloid-like aggregates that can induce agglutination of red blood cells, which was not observed with low- and non-aggregated S192 peptide. We subsequently screened eight amyloid-binding molecules and identified BAM1-EG6, a benzothiazole amphiphile, as a promising candidate capable of binding to aggregated S192 and partially inhibiting its agglutination activity. These results provide new insight into a potential molecular mechanism for the capability of spike protein metabolites to contribute to COVID-19-related blood complications and suggest a new therapeutic approach for combating microvascular morbidities in COVID-19 patients.

C-type lectin 12B/4E of black rockfish (Sebastes schlegelii) macrophages as pattern recognition receptors in the antibacterial mechanism of exploration

As lower vertebrates, fish have both innate and adaptive immune systems, but the role of the adaptive immune system is limited, and the innate immune system plays an important role in the resistance to pathogen infection. C-type lectins (CLRs) are one of the major pattern recognition receptors (PRRs) of the innate immune system. CLRs can combine with pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs) to trigger NF-κB signaling pathway and exert immune efficacy. In this study, Ssclec12b and Ssclec4e of the C-type lectins, were found to be significantly up-regulated in the transcripts of Sebastes schlegelii macrophages stimulated by bacteria. The identification, expression and function of these lectins were studied. In addition, the recombinant proteins of the above two CLRs were obtained by prokaryotic expression. We found that rSsCLEC12B and rSsCLEC4E could bind to a variety of bacteria in a Ca2+-dependent manner, and promoted the agglutination of bacteria and blood cells. rSsCLEC12B and rSsCLEC4E assisted macrophages to recognize PAMPs and activate the NF-κB signaling pathway, thereby promoting the expression of inflammatory factors (TNF-α, IL-1β, IL-6, IL-8) and regulating the early immune inflammation of macrophages. These results suggested that SsCLEC12B and SsCLEC4E could serve as PRRs in S. schlegelii macrophages to recognize pathogens and participate in the host antimicrobial immune process, and provided a valuable reference for the study of CLRs involved in fish innate immunity.

Sample-to-answer lateral flow assay with integrated plasma separation and NT-proBNP detection

Through enabling whole blood detection in point-of-care testing (POCT), sedimentation-based plasma separation promises to enhance the functionality and extend the application range of lateral flow assays (LFAs). To streamline the entire process from the introduction of the blood sample to the generation of quantitative immune-fluorescence results, we combined a simple plasma separation technique, an immunoreaction, and a micropump-driven external suction control system in a polymer channel-based LFA. Our primary objective was to eliminate the reliance on sample-absorbing separation membranes, the use of active separation forces commonly found in POCT, and ultimately allowing finger prick testing. Combining the principle of agglutination of red blood cells with an on-device sedimentation-based separation, our device allows for the efficient and fast separation of plasma from a 25-µL blood volume within a mere 10 min and overcomes limitations such as clogging, analyte adsorption, and blood pre-dilution. To simplify this process, we stored the agglutination agent in a dried state on the test and incorporated a filter trench to initiate sedimentation-based separation. The separated plasma was then moved to the integrated mixing area, initiating the immunoreaction by rehydration of probe-specific fluorophore-conjugated antibodies. The biotinylated immune complex was subsequently trapped in the streptavidin-rich detection zone and quantitatively analyzed using a fluorescence microscope. Normalized to the centrifugation-based separation, our device demonstrated high separation efficiency of 96% and a yield of 7.23 µL (= 72%). Furthermore, we elaborate on its user-friendly nature and demonstrate its proof-of-concept through an all-dried ready-to-go NT-proBNP lateral flow immunoassay with clinical blood samples.Graphical

Expression of haemagglutinin-neuraminidase of newcastle virus genotype VIII in insect cell Sf9

The hemagglutinin-neuraminidase (HN) protein is the surface glycoprotein of Newcastle disease virus (NDV) which is involved in the process of virus entry into host cells. HN exhibits the capability to induce agglutination of chicken red blood cells, thereby contributing to the viral infection of the host. As a consequence, HN is a potential antigen for the production of subunit vaccines for protecting poultry from NDV infections. In this study, the hn gene coding for the HN antigen of NDV genotype VIII was artificially synthesized, ligated into donor vector pFastBacHT A and integrated into the baculovirus genome for the purpose of HN expression in Spodoptera frugiperda 9 (Sf9) cells. The recombinant HN protein was successfully expressed in Sf9 cells in the SF900 III medium. The expressed recombinant HN protein, with a size of approximately 63 kDa, exhibited hemagglutinin activity (HA) of 4log2 when interacting with chicken erythrocyte fluid. The findings of this study lay the foundation for subsequent research endeavors aimed at the development of Newcastle disease virus (NDV) vaccines tailored for poultry in Vietnam.

Short Prewarming at 41°C to Correct the Interference in Samples with the Presence of Cold Agglutination.

Cold agglutinins (CAs) in blood samples can cause a reversible agglutination of red blood cell (RBC) which result in an incorrect complete blood count (CBC). So, it is important to explore new simple and feasible treatment conditions for clinical work. The CAs group included 32 samples with CAs. The parameters of CBC at room temperature or after prewarming at 37°C or 41°C for different time periods were compared. The consistency and correlation of those parameters were analyzed. The morphology of erythrocytes in the CAs group was observed manually. The control group included 45 samples without CAs and prewarmed at 37°C or 41°C for different time periods. The differences were also analyzed. CAs have a significant effect on CBC. After prewarming at 37°C or 41°C the interferences are all corrected. Consider prewarming at 37°C for 120 minutes as the standard procedure. The consistency and correlation analysis showed there was no statistical difference between the results of each subgroup and standard group, except the MCHC of group 41°C 10 minutes. The correlation of parameters between all subgroups and the standard group is satisfied. Microscopic examination showed no RBC aggregation or fragmentation after prewarming at 41°C or 37°C. According to the maximum bias requirements for expert performance in Validation, Verification, and Quality Assurance of Automated Hematology Analyzers, 2nd Edition (CLSI H26-A2), the differences in overall results in control group are negligible. The 41°C 2 minutes prewarming method is a rapid and effective way for treating samples with CAs. It is an efficient way to obtain more reliable CBC results, without specific instruments.

Successful Management of Total Plasma Exchange for Hemolytic Cold Agglutinin Disease.

Therapeutic plasma exchange (TPE) is a strategy for treating cold agglutinin disease (CAD) in order to manage hemolytic complications. However, there are no reports of hemolysis during TPE. A 41-year-old man with secondary CAD was unable to undergo initial TPE because of red blood cell agglutination and hemolysis in his extracorporeal circulation. To avoid low temperatures, the patient and extracorporeal circulation were kept warm by covering and heating them, and finally, he was able to successfully receive TPE three times. Although our approach still has room for improvement, our management protocol appears to be an effective treatment modality for such cases.

Autoimmune hemolytic anemia in a preoperative patient: a case report and literature review

Background: In patients undergoing elective surgery, it is known that as many as one-third have preoperative anemia. Preoperative anemia is associated with an almost five-fold increase in the likelihood of postoperative mortality. So, it is essential to know the cause of anemia so that the management is appropriate so that surgery can be performed safely and prevent patients from dying on the operating table or postoperatively. This case report aims to describe the pathogenesis of the laboratory findings in a patient with autoimmune hemolytic anemia (AIHA) who was accidentally diagnosed during preoperative screening and the management performed. Case: A 65-year-old man with an enlarged prostate was scheduled for elective surgery. Preoperative examination revealed anemic right and left conjunctiva, decreased erythrocytes, hemoglobin, hematocrit, and increased MCV, MCH, and MCHC, peripheral blood smear dominated by red cell agglutination and positive direct Coomb's test. The patient was given methylprednisolone 62.5 mg intravenously before transfusion and given a transfusion of 2 units of slow drip packed red cells (PRC) under close supervision. There were no transfusion reactions during and after transfusion. There was an increase in Hb after transfusion so that the patient could be operated on. Conclusion: The patient in this case was diagnosed with AIHA based on preoperative laboratory findings. Laboratory results showed evidence of autoimmune hemolytic anemia with decreased erythrocytes, hemoglobin, hematocrits, spherocytes, and agglutination on peripheral blood smear and positive Coomb's test. Rapid identification and treatment of anemia help improve preoperative conditions.

Targeting Complement Inhibition in Cold Hemolysis; A Multifaceted Approach Using Sutimlimab and Washed Red Cell Transfusions

Introduction: Cold agglutinin syndrome (CAS) is a transient disorder mediated by autoantibodies that cause red cell agglutination at cold temperatures. CAS is often associated with infections rather than malignant or autoimmune conditions. Sutimlimab, a monoclonal antibody directed against the complement component C1s, was previously shown to improve anemia and fatigue in cold agglutinin disease (CAD) in single-arm studies. However, its use in CAS has not yet been elucidated. Here, we describe a case where complement inhibition using the novel C1s inhibitor, as well as washed and warmed red cell transfusions, were used to treat CAS in the setting of a rare autoantibody directed toward the Pr antigen Case report: A healthy 46-year-old woman presented with severe acute hemolytic anemia: with nadir hemoglobin of 4.7, hematocrit of 13.4%, 0.2% of reticulocytes, total bilirubin of 9.8 mg/dL, and unconjugated bilirubin of 8.4 mg/dL. A peripheral blood smear review revealed severe anemia with agglutination of red blood cells. An extensive infectious workup was negative including COVID-19, Parvovirus B19, CMV, EBV, CMV, and HIV. Over a period of two weeks, she received three doses of IVIG, prednisone, and 17 transfusions units of warmed red blood cells in the setting of persistent hemolytic anemia. Despite the evidence of severe hemolysis, her reticulocyte count remained very low (&amp;lt;0.5%). A bone marrow biopsy showed trilineage hematopoiesis with erythroid hypoplasia and maturation arrest. A direct Coombs test was intermittently positive but probably confused by IgG infusions, with anti-I and anti-i titers of 1:64, and positive for C3. Thermal amplitude studies showed agglutination at 30°C, 37°C, and room temperature. Red blood cell antibodies were not identified in the eluate. The indirect antiglobulin test was negative for ficin-treated RBCs showing no agglutination. This led the reference laboratory to report that the cold agglutination was directed against the anti-pr antigen and that this patient had clinically significant anti-Pr antibodies. The patient was kept warm and started on Danazol and Sutimlimab. However, the patient continued to have hemolysis after transfusions. After receiving her second dose of Sutimlimab and in the context of persistent hemolytic anemia, she underwent another bone marrow biopsy on day 34 after presentation. The biopsy showed hypercellular bone marrow with erythroid hyperplasia and complete maturation. However, this was not reflected in reticulocytosis until day 44. The patient received her third dose of Sutimlimab, and red blood cell transfusions were washed in addition to being warmed, which resolved hemolysis after transfusion reactions. She received a total of 30 red blood cell transfusions since presentation. The patient's anemia improved, with a hemoglobin level of 8.8 mg/dL and a reticulocyte count of 13%, and she was discharged on Day 51 after presentation (figure 1). She received one more dose of Sutimlimab (total of four doses) with complete resolution of her anemia, a normal reticulocyte count, normal LDH, negative Coombs study, and negative cold agglutinin titers. Conclusion: Here, we describe a challenging case of CAS caused by a rare Pr autoantibody, with a failure of first-line treatments for CAS. This report is the first to describe the use of Sutimlimab as a treatment for CAS. After initiating Sutimlimab, the patient continued to experience significant hemolysis shortly after transfusions, exhibiting hemoglobinuria and an inadequate increase in hematocrit. Only when the red blood cell transfusions were washed to remove complement, the hemolytic reactions ceased and significant increases in hematocrit occurred. This finding may be a consideration in other cases of cold agglutinin anemia. As CAS is a transient disorder, Sutimlimab could ultimately be discontinued once the hemolysis resolved. This case illustrates the role of complement inhibition in CAS and the potential need to warm and wash red blood cells in severe cases of CAS.

Prevalence of “weak B” phenotypes and their evaluation and differentiation in healthy blood donor population in Eastern India

Abstract BACKGROUND: Examples of group B red cells that react weakly or not at all with anti-B have been described. Subgroups of B such as B3, Bx, Bm, and Bel are rare and are less frequently reported. We studied the frequency of subgroups of B in our healthy blood donor population and serologically characterized and differentiated these subgroups. MATERIALS AND METHODS: The 9-year prospective study included 84,534 healthy blood donors. Initial blood grouping and antibody screening of all donor samples were performed using automated solid-phase assay. Any sample showing blood group discrepancy or weaker agglutination was subjected to further immunohematological investigations. RESULTS: Among 84,534 healthy donors, “B” blood group was found in 29,190 (34.53%). Weak B phenotypes were demonstrated in 9 (0.031%) B donors. Among the 9 weak B phenotypes, B3 was the most common followed by Bm. The frequency of B3, Bm, Bx, and Bel in our blood donor population was found to be 1 in 21,133, 1 in 28,178, 1 in 84,534, and 1 in 84,534, respectively. Red cell agglutination with anti-B and anti-AB varied from Wk+ to 2+ with or without mixed-field agglutination in the B3 and Bx phenotypes. Naturally occurring anti-B of immunoglobulin M type was detected in the Bx donor. Two (22.2%) of the 9 donors were found to be nonsecretor. Adsorption-elution demonstrated “B” antigen specificity in different strengths in Bm, Bx, and Bel phenotypes. CONCLUSION: We conclude that differentiating weak subgroups of “B” by serological assays is possible to a great extent with technical expertise. Mistyping weak subgroups of B as “O” group may lead to reporting errors and wrong blood transfusion. Therefore, blood centers in developing countries including India should establish simple techniques to detect and differentiate weak subgroups and develop procedures to ensure safe blood transfusion and transplantation.

The Cs a and Cs b Red Cell Antigens of the Cost Blood Group Collection Correspond to the HNA-3a and HNA-3b Neutrophil Antigens: Unexpected Twins with Implications for Sickle Cell Anemia

Recent genome-wide association and murine studies identified the human neutrophil antigen 3a/b polymorphism (HNA-3a/b) in SLC44A2 (rs2288904-G/A) as a risk factor in venous thromboembolism (VTE). We tested whether this polymorphism associates with clinical features in SCA patients in whose VTE and stroke are frequently encountered complications. Incidentally, we discovered that rare subjects with the homozygous HNA-3b/b genotype (genotype frequency &amp;lt; 2%) also carry the uncommon Cs(a-) red cell phenotype. Despite its discovery more than 50 years ago, the molecular basis of this red cell phenotype and the presumptive protein carrier for the antigens of the COST blood group collection (Cs a and Cs b) has remained unknown. Inspired by this fortuitous discovery, we hypothesized that rs2288904 and COST antigens are closely associated. Thus, we genotyped this SNP in a cohort of 35 Cs(a-) subjects and found that all of them showed a HNA-3b/b genotype, indicating that the high-frequency allele of rs2288904 (HNA-3a; 461G) encoding Arg154 might encode the Cs a antigen (COST1). Interestingly, anti-Cs a does not react with SLC44A2 null RBCs (exceptional VER- blood type) and SLC44A2 knockout K562 cell line, confirming that the COST antigens are carried on this protein. In addition, using a panel of K562 cells expressing HNA-3a or 3b antigens developed by CRISPR/Cas9 genome editing, we confirmed that anti-Cs a recognizes the HNA-3a antigen. Overall, our results confirmed that HNA-3 and COST antigens have the same molecular basis. As a result, the Cs a and Cs b red cell antigens can now be included in the CTL2 blood group system, encoded by the SLC44A2 gene. This new finding could be of medical and biological value because anti-HNA-3a may be responsible for severe cases of neonatal neutropenia and transfusion-related acute lung injury (TRALI). Current testing for HNA-3a antibodies includes the granulocyte agglutination test and granulocyteindirect immunofluorescence test, as recommended by the ISBT Working Party on Granulocyte Immunobiology. These classical methods, however, are cumbersome because they require fresh granulocytes that have to be prepared daily and used immediately. Consequently, screening for HNA-3a antibodies is restricted to a few expert laboratories. Therefore, we propose to use the simple indirect antiglobulin red cell agglutination test (IAT) and Cs(a-) RBCs to screen for this antibody in the plasma of donors and patients. Finally, given that the starting point for this project was the study of a potential association of the HNA-3 polymorphism with clinical and biological features in SCA patients, we found that the HNA-3b allele was significantly associated with a higher monocyte and platelet count (p&amp;lt;0.02) and the occurrence of leg ulcer (p=0.01), suggesting an important role of the SLC44A2 protein in the SCD physiopathology.

Improved in vitro Hemagglutination Assays Utilizing P-Type and Type 1 Uropathogenic Escherichia coli to Evaluate Bacterial Anti-Adhesion Activity of Cranberry Products

Cranberries have a long history of use in the prevention of urinary tract infections. Cranberry products vary in proanthocyanidin content, a compound implicated in preventing the adhesion of uropathogenic Escherichia coli (E. coli) to uroepithelial cells. Testing is routinely done by cranberry product formulators to evaluate in vitro bacterial anti-adhesion bioactivity, shelf-life, and potential efficacy of cranberry products for consumer use to maintain urinary tract health. Hemagglutination assays evaluate the anti-adhesion bioactivity of cranberry products by determining how effectively the products prevent agglutination of specific red blood cells with E. coli expressing P-type and Type 1 fimbriae. The current study sought to improve upon an established anti-adhesion assay method by expanding the number of E. coli strains used to broaden potential in vivo efficacy implications and presenting results using photomicrographic data to improve accuracy and build databases on products that are routinely tested. Different lots of cranberry powder ingredient and two formulated products were tested independently for anti-adhesion activity using the established method and the improved method. Positive harmonization of results on the same samples using rigorous controls was achieved and provides the substantiation needed for the cranberry industry to utilize the improved, rapid in vitro testing method to standardize cranberry products for sufficient anti-adhesion bioactivity and maintain consumer confidence.

Leptin-mediated ERK Signaling Pathway Promotes the Transformation of Rat Alveolar Type II Epithelial Cells Induced by Yunnan Tin Mine Dust

Currently, a significant number of miners are involved in mining operations at the Gejiu tin mine in Yunnan. This occupational setting is associated with exposure to dust particles, heavy metals, polycyclic aromatic hydrocarbons, and radioactive radon, thereby significantly elevating the risk of lung cancer. This study aims to investigate the involvement of leptin-mediated extracellular regulated protein kinase (ERK) signaling pathway in the malignant transformation of rat alveolar type II epithelial cells induced by Yunnan tin mine dust. Immortalized rat alveolar cells type II (RLE-6TN) cells were infected with Yunnan tin mine dust at a concentration of 200 μg/mL for nine consecutive generations to establish the infected cell model, which was named R₂₀₀ cells. The cells were cultured normally, named as R cells. The expression of leptin receptor in both cell groups was detected using the Western blot method. The optimal concentration of leptin and mitogen-activated protein kinase kinase (MEK) inhibitor (U0126) on R₂₀₀ cells was determined using the MTT method. Starting from the 20th generation, the cells in the R group were co-cultured with leptin, while the cells in the R₂₀₀ group were co-cultured with the MEK inhibitor U0126. The morphological alterations of the cells in each group were visualized utilizing hematoxylin-eosin staining. Additionally, concanavalin A (ConA) was utilized to detect any morphological differences, and an anchorage-independent growth assay was conducted to assess the malignant transformation of the cells. The changes in the ERK signaling pathway in epithelial cells after the action of leptin were detected using the Western blot method. Both the cells in the R group and R₂₀₀ group express leptin receptor OB-R. Compared to the R₂₀₀ group, the concentration of leptin at 100 ng/mL shows the most significant pro-proliferation effect. The proliferation of R₂₀₀ cells infected with the virus is inhibited by 30 μmol/L U0126, and a statistically significant divergence was seen when compared to the control group (P<0.05). Starting from the 25th generation, the cell morphology of the leptin-induced R₂₀₀ group (R₂₀₀L group) underwent changes, leading to malignant transformation observed at the 30th generation. The characteristics of malignant transformation became evident by the 40th generation in the R₂₀₀L group. In contrast, the other groups showed agglutination of P40 cells, and the speed of cell aggregation increased with an increase in ConA concentration. Notably, the R₂₀₀L group exhibited faster cell aggregation compared to the U0126-induced R₂₀₀ (R₂₀₀LU) group. Additionally, the cells in the R₂₀₀L group were capable of forming clones starting from P30, with a colony formation rate of 2.25‰±0.5‰. However, no clonal colonies were observed in the R₂₀₀LU group and R₂₀₀ group. The expression of phosphorylated extracellular signal-regulated kinase (pERK) was enhanced in cells of the R₂₀₀L group. However, when the cells in the R₂₀₀L group were treated with U0126, a blocking agent, the phosphorylation level of pERK decreased. Leptin can promote the malignant transformation of lung epithelial cells infected by mine dust, and the ERK signaling pathway may be necessary for the transformation of alveolar type II epithelial cells induced by Yunnan tin mine dust.