Autoagglutination Research Articles

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

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.

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.

Severe haemolytic disease of newborn with possibility of blocked D phenomenon:A Case Report

Anti-D is the most common antibody causing severe hemolytic disease of foetus and newborn (HDFN).The mothers who have very high titres of anti-D,antibodies can coat and block D antigens on red blood cells (RBCs) of the newborn.This blocking phenomenon prevents the agglutination of RBCs with IgM type of anti-D antisera.This results in false negative results.Here, we report the case of 1 day old baby born to a multiparous female with Rh Negative blood group suffering from severe HDFN.Mother and baby’s samples were sent to our blood centre for the advanced immunohaematological analysis.On detailed immunohaematological work up, possibility of blocked D phenomenon was considered. key-words: Haemolytic disease of foetus and new born, Blocked D Phenomenon, Maternal anti-D antibody.

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.

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

Abstract LB129: Preclinical development of a bispecific antibody-trap selectively targeting CD38 and CD47 for treating hematologic malignancies

Abstract CD38 and CD47 are co-expressed in various hematologic malignancies including multiple myeloma (MM), B-cell non-Hodgkin lymphoma (NHL), B-cell acute lymphoblastic leukemia (ALL), T-cell ALL and B-cell chronic lymphocytic leukemia (CLL). We present, herein, several CD38 antibody - CD47 ligand trap bispecific antibodies (BsAbs) in an ADCC-enhanced IgG1 form that harbor high CD38 binding affinity, low CD47 binding affinity, along with negligible affinity to red blood cells (RBCs) and no induction of RBCs agglutination. Using flow cytometry and Biolayer interferometry (BLI), we confirmed that our BsAbs can simultaneously bind to both CD38 and CD47, and that binding to either antigen first does not prevent binding to the other antigen subsequently. On CD47+/CD38low Jurkat (T leukemic) cells, our BsAbs reveal less blocking effect on the CD47/SIRPα interaction than a monospecific CD47 ligand trap molecule based on which our BsAbs were designed; conversely, on CD38/CD47 double-positive Reh (pro-B ALL) cells, our BsAbs reveal 200-500-fold greater blocking effect on the CD47/SIRPα interaction than the same monospecific CD47 ligand trap molecule, indicating that the inhibition of CD47/SIRPα by our BsAbs is depended on the engagement of CD38 on tumor cells. On CD47+/CD38+ Daudi (B NHL) lymphoma cells and L-1236 (B Hodgkin) lymphoma cells, four of our BsAbs inhibited CD38 enzymatic activity, which is responsible for immune-suppressive adenosine production in tumor microenvironment. Our BsAbs presented potent dose-dependent antibody-dependent cellular cytotoxicity (ADCC) against CD47+/CD38+ Raji (B NHL) and NCI-H929 (B plasmacytoma myeloma) cells, while revealing negligible ADCC activity against CD47+/CD38− cells, indicating low off-tumor toxicity. Likewise, our BsAbs also harbor potent antibody-dependent cellular phagocytosis (ADCP) activity against Raji NHL cells, varied degree of complement-dependent cytotoxicity (CDC) activity and direct apoptosis induction against Daudi NHL cells. In a mouse xenograft model harboring NCI-H929 multiple myeloma, our BsAbs produced more potent tumor growth inhibition, than monospecific anti-CD47 ligand trap, anti-CD38 (isatuximab) or combination of isatuximab and anti-CD47 ligand trap, at the same molecular dose per body weight. Notably, clone IMM5605-12C10 eradicated all tumors and achieved 100% complete response rate in a group of six mice. In summary, simultaneous targeting CD38 and CD47 using our CD38 antibody - CD47 ligand trap bispecific design in an ADCC-enhanced IgG1 form presents a novel multimodal approach for treating hematologic malignancies that are resistant to anti-CD38 antibodies. Citation Format: Wenzhi Tian, Song Li, Dianze Chen, Yanan Yang, Huiqin Guo, Dandan Liu, Ruliang Zhang, Fan Zhang. Preclinical development of a bispecific antibody-trap selectively targeting CD38 and CD47 for treating hematologic malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB129.

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.

The CD47-Blocking Immune Checkpoint Inhibitor Maplirpacept Does Not Interfere with Blood Transfusion Compatibility Testing

CD47 is a transmembrane glycoprotein that delivers an anti-phagocytic (“do-not-eat”) signal by binding to signal regulatory protein α (SIRPα) on the surface of macrophages. Many tumors express high levels of CD47 as a means to escape macrophage-mediated immune surveillance and blockade of the CD47-SIRPα pathway has emerged as a novel therapeutic strategy with demonstrated anti-tumor activity both in vitro and in vivo (Petrova et al. 2017; Weiskopf 2017). Maplirpacept (PF-07901801, TTI-622) is a soluble human SIRPαFc fusion protein with an IgG4 domain which acts as an innate immune checkpoint inhibitor that prevents CD47-SIRPα binding, engages activating Fcγ receptors on macrophages, and promotes phagocytosis of malignant cells (Lin et al. AACR 2018). Maplirpacept is currently in clinical development to treat patients with ovarian cancer, multiple myeloma, acute myeloid leukemia and lymphoma. Additionally, CD47 is expressed on red blood cells (RBCs) and plays a role in the natural clearance of aged RBCs by splenic macrophages in vivo (Oldenborg et al. 2000). CD47 expression on RBCs was shown to interfere with pre-transfusion laboratory testing in patients treated with the anti-CD47 mAb, Hu5F9-G4 (Velliquette et al. 2019). Unlike anti-CD47 mAbs, minimal binding to human RBCs is observed with Maplirpacept (Lin et al. AACR 2018). In this study we investigated whether the minimal binding of Maplirpacept to human RBCs precludes interference with routine pre-transfusion laboratory tests. RBC suspensions from healthy donors representing all 8 ABO-Rh blood groups were spiked with Maplirpacept at 0.6, 1.2 or 2 mg/ml or anti-CD47 (clone Hu5F9). Blood typing was performed according to the manufacture's protocol (Seraclone, Biorad) and observed for the presence or absence of agglutination. For cross matching experiments, plasma was prepared from blood of healthy donors and spiked with Maplirpacept or anti-CD47 at the same concentrations used for the blood typing experiments. RBCs from two healthy donors were mixed with plasma and an indirect antiglobulin test (IAT) was performed according to the manufacture's protocol (Coombs Anti-IgG, Biorad). Using RBCs from healthy donors representing the 8 major ABO-Rh blood groups, we now show that the addition of Maplirpacept does not cause agglutination of test RBCs and produces the expected blood typing result in a standard forward blood typing test. No interference was observed at the highest concentration tested. However, addition of an anti-CD47 mAb caused pan-agglutination as well as false positive blood typing results (Table 1). In a standard IAT that mimics the in vitro compatibility cross matching test performed prior to a blood transfusion, the anti-CD47 mAb gave false positive results while Maplirpacept produced results identical to the control serum condition (Table 2). This data is consistent with a study demonstrating interference by Hu5F9 in routine pre-transfusion blood testing (Velliquette et al. 2019). Additionally, no interference was observed when Maplirpacept was added to conditions which produced agglutination in a standard IAT across a range of blood types. Interference with RBC panel testing has important consequences in transfusion medicine as the presence of irregular antibodies can complicate the selection of suitable RBC units for treated patients. Here we demonstrate that interference with blood compatibility testing is dependent upon the type of CD47-blocking agent. Decoy receptors such as Maplirpacept, unlike anti-CD47 mAb, do not interfere with blood group serologic testing, which may confer a significant clinical advantage by negating the need for additional approaches to prevent interference.

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.

A portable point-of-care testing device for forward blood typing with hemophilia diagnosis.

This paper presents a portable point-of-care testing (POCT) device to conduct simultaneous and on-site tests of ABO and Rh(D) forward blood typing and hemophilia diagnosis using only a small amount of human whole blood sample. The POCT device consisted of a spinning module, a measuring circuit, an interdigitated electrode (IDE) for hemophilia diagnosis, and three disposable microfluidic chips for bioassays with anti-A, anti-B, and anti-D, respectively, and measurement of the concentration of factor VIII. Agglutination will occur if red blood cells (RBCs) are exposed to the corresponding antibody. To evaluate the degree of RBC agglutination, a linear sweep voltage, ranging from - 0.5 to + 0.5V, was applied to the electrodes of the microfluidic chip and the resulting current was measured. For different levels of agglutination, the measured I-V curves were explicitly discriminated, providing five clinical levels from non-agglutination (level 0) to strong agglutination (level 4). The quantitative norm obtained from cubic fitting function of each I-V curve served as the criterion to represent this agglutination level. The ABO blood type was determined by both agglutination levels of the blood sample reacting with anti-A and anti-B. The degree of agglutination with anti-D gave the Rh(D) type. Moreover, the concentration of factor VIII was detected for the determination of hemophilia. Without requiring expensive equipment, this POCT device is especially suitable for usage in emergency or natural disasters to provide quantitative testing in rescue and relief operations.

Hemagglutination Assay via Optical Density Characterization in 3D Microtrap Chips.

Hemagglutination assay has been used for blood typing and detecting viruses, thus applicable for the diagnosis of infectious diseases, including COVID-19. Therefore, the development of microfluidic devices for fast detection of hemagglutination is on-demand for point-of-care diagnosis. Here, we present a way to detect hemagglutination in 3D microfluidic devices via optical absorbance (optical density, OD) characterization. 3D printing is a powerful way to build microfluidic structures for diagnostic devices. However, mixing liquid in microfluidic chips is difficult due to laminar flow, which hampers practical applications such as antigen-antibody mixing. To overcome the issue, we fabricated 3D microfluidic chips with embedded microchannel and microwell structures to induce hemagglutination between red blood cells (RBCs) and antibodies. We named it a 3D microtrap chip. We also established an automated measurement system which is an integral part of diagnostic devices. To do this, we developed a novel way to identify RBC agglutination and non-agglutination via the OD difference. By adapting a 3D-printed aperture to the microtrap chip, we obtained a pure absorbance signal from the microchannels by eliminating the background brightness of the microtrap chip. By investigating the underlying optical physics, we provide a 3D device platform for detecting hemagglutination.

SERS of Human Red Blood Cells in Non-Resonant Conditions: Benefits, Limitations, and Complementary Tools (CytoViva and GFAAS)

Herein, Raman and surface-enhanced Raman spectroscopies (SERS) were successfully employed to establish the chemical interactions of citrate-capped silver nanoparticles (AgNPs, 10–15 nm) with human red blood cells (RBCs). The Raman/SERS spectra offered spectral evidence for the cellular uptake of AgNPs and the subsequent change in the conformation of the most abundant component, hemoglobin (Hb), from oxyhemoglobin to deoxyhemoglobin. The spectral characterization of AgNPs’ interactions with other RBC biomarkers (membrane proteins and lipids) was impeded by the dominant Hb bands, even for non-resonant Hb conditions. CytoViva hyperspectral imaging and graphite furnace atomic absorption spectroscopy (GFAAS) served as complementary tools to effectively address the challenges related to a single excitation line (632.8 nm) and the resolution of the confocal Raman microscope (0.5–1.0 µm). CytoViva confirmed the RBC-AgNP interactions through hyperspectral signatures and facilitated the label-free localization of AgNPs extracellularly and intracellularly. Irreversible agglutination of RBCs was noted after 24 h of exposure, raising concerns about the toxicity of AgNPs of biocompatible citrate coatings. GFAAS validated the Raman/SERS results by quantifying the proportion of AgNPs absorbed by RBCs, which was significant (~48% AgNPs by mass), mostly at the membrane (60% RBCs), and size dependent (no large AgNPs or AgNP-aggregates in RBCs, after 12–24 h).