Massive Transfusion Activations in Non-Trauma Patients.

INTRODUCTION: Massive transfusion activations (MTAs) are commonly used in the care of the trauma patient. However, MTA for trauma patients constitutes only a small fraction of MTA at our institution. The aim of this study was to characterize MTA in non-trauma patients to better understand how this strategy is employed at a larger tertiary hospital. METHODS: All MTA involving non-trauma patients from January 2017 to April 2019 were reviewed. Patients with unclear indications for MTA were excluded. Data collected included patient demographics, reason for MTA, transfusion ratios, use of adjunctive antifibrinolytics, use of viscoelastic testing, and vasopressor administration at the time of MTA. RESULTS: There were 328 patients and 353 MTA identified over the study period. The mean age was 52.0 years and 40.9% were male. Patients were most commonly under the care of a medical service (55.2%), while 25.3% were obstetric patients and 19.5% were surgical patients. Compliance with 1:1:1 transfusion ratios was low. Concomitant vasopressor use was high (70.8%), while antifibrinolytic agents (13.0%) and viscoelastic testing (19.0%) were used less commonly. The overall mortality of the study population was 56.1%. CONCLUSIONS: Massive transfusion activations are frequently used in non-trauma patients. There was a low rate of adherence to 1:1:1 transfusion ratios as well as utilization of adjuncts and tools that could allow for targeted resuscitation. Understanding practice patterns relating to MTA may allow for an opportunity for improvement.

Evidence of premature lymphocyte aging in people with low anti-spike antibody levels after BNT162b2 vaccination.

SARS-CoV-2 vaccines have unquestionably blunted the overall impact of the COVID-19 pandemic, but host factors such as age, sex, obesity, and other co-morbidities can affect vaccine efficacy. We identified individuals in a relatively healthy population of healthcare workers (CORALE study cohort) who had unexpectedly low peak anti-spike receptor binding domain (S-RBD) antibody levels after receiving the BNT162b2 vaccine. Compared to matched controls, "low responders" had fewer spike-specific antibody-producing B cells after the second and third/booster doses. Moreover, their spike-specific T cell receptor (TCR) repertoire had less depth and their CD4+ and CD8+T cell responses to spike peptide stimulation were less robust. Single cell transcriptomic evaluation of peripheral blood mononuclear cells revealed activation of aging pathways in low responder B and CD4+T cells that could underlie their attenuated anti-S-RBD antibody production. Premature lymphocyte aging may therefore contribute to a less effective humoral response and could reduce vaccination efficacy.

Implementation of a low-titer stored whole blood transfusion program for civilian trauma patients: Early experience and logistical challenges.

INTRODUCTION: Cold-stored low titer group O whole blood (LTOWB) is increasingly utilized in the initial resuscitation of exsanguinating trauma patients. We report on our early experience with LTOWB, focusing on logistics, implementation challenges, and outcomes. METHODS: In February, 2019, LTOWB was incorporated into the massive transfusion protocol (MTP) activated for trauma patients in the emergency department (ED.) Up to 4 units of LTOWB were included in the MTP cooler, depending on availability, and were transfused prior to transfusion of any other blood products from the MTP cooler. Demographics, injury characteristics, and outcomes were obtained, and the logistics of LTOWB availability were reviewed. RESULTS: Over a 12-month period, MTP was activated for 74 trauma patients. Of those, 38 (51%) MTP included at least one unit of LTOWB, with 19/38 (50%) including 4 LTOWB units. A total of 177 units of LTOWB were purchased during the study period, and of those, 74 (42%) expired before use. Patients who received LTOWB had a similar mortality compared to those who received component therapy (39% vs. 47%; Odds Ratio [95% CI]: 0.7 [0.3, 2.0]; p = 0.72,) however, they were able to achieve a significantly higher plasma:pRBC ratio during the duration of MTP activation (mean [SD] 0.8 [0.2] vs. 0.4 [0.4]; mean difference [95% CI]: 0.4 [0.2, 0.5]; p < 0.01.) CONCLUSIONS: Our early experience with LTOWB transfusion demonstrates feasibility, but also highlights challenges with inventory management. These findings triggered changes to our protocol aiming at minimizing wastage. The use of LTOWB may yield a higher plasma:pRBC ratio early during the resuscitation period. Further investigation is required to explore whether this may yield a survival advantage. LEVEL OF EVIDENCE: III (Therapeutic/Care Management).

Diagnosing heparin-induced thrombocytopenia in mechanical circulatory support device patients.

BACKGROUND: Mechanical circulatory support device (MCSD) patients with positive heparin-induced thrombocytopenia (HIT) screening pose a unique challenge, as clinicians must make rapid decisions about their anticoagulation and whether they can safely undergo cardiopulmonary bypass. We identified screening practices at our institution and other institutions nationwide that differed from American Society of Hematology (ASH) guidelines. This discovery prompted a data review to confirm the applicability of guidelines to this unique population and to highlight complications of "gestalt" screening. METHODS: Our study included MCSD patients with HIT testing from April 2014 to August 2020. We evaluated 510 PF4 IgG ELISA results. RESULTS: HIT was confirmed in 4.2% of patients. There was an increased prevalence of HIT in patients with nondurable (5.3%) vs durable devices (2.9%) or those in the preimplantation setting (1.3%), however this difference was not statistically significant (p = 0.26). None of the patients with a low probability 4T Score had HIT. All patients with a high probability 4T Score and PF4 immunoassay OD >2.0 had HIT. False positive results occurred in 22% of assays ordered for patients with a low probability 4T Score. Twelve patients with a low probability 4T Score and a false positive immunoassay were switched to a direct thrombin inhibitor (DTI) while awaiting confirmatory results. Two patients experienced clinically significant bleeding after conversion to a DTI. An organ was refused in one patient with false positive HIT screening. CONCLUSIONS: Our findings demonstrate that an opportunity exists to improve clinical outcomes by re-emphasizing the utility of established guidelines and highlighting their safe use in the MCSD patient population.

Blood group O convalescent plasma donations have significantly lower levels of SARS-CoV-2 IgG antibodies compared to blood group A donations.

BACKGROUND: COVID-19 convalescent plasma (CCP) is plasma collected from individuals who have recovered from SARS-CoV-2 infection. The FDA Emergency Use Authorization restricts use of CCP to high-titer units only. The purpose of this study was to determine if donor ABO blood group was associated with SARS-CoV-2 antibody response, and subsequent qualification as high-titer CCP. METHODS: All CCP donations collected from April 21, 2020 to September 1, 2020 were included. The Abbott ARCHITECT semi-quantitative chemiluminescent microparticle immunoassay was used to assess IgG antibodies to the nucleocapsid protein of SARS-CoV-2. Units with a S/C value ≥4.5 were considered high titer. RESULTS: A total of 232 CCP donations were evaluated. There were no significant differences in the distribution of sex, age, and interval from symptom resolution to donation by ABO blood group. The mean SARS-CoV-2 IgG antibody S/C value was significantly lower in blood group O donations (3.6), compared to blood group A (5.0) donations (p < .001). There was no difference in antibody response between the other blood group pairings. Blood group O donations resulted in a lower percentage of high-titer units (35%), compared to blood group A (60%), B (58%), and AB (65%) donations. CONCLUSION: Blood group O donations were found to have significantly lower levels of SARS-CoV-2 IgG nucleocapsid antibodies compared to blood group A donations and were less likely to produce CCP units that qualified as high titer. These findings may aid donor recruitment to promote availability of high-titer CCP to meet patient needs.

SARS-CoV-2 specific antibody and neutralization assays reveal the wide range of the humoral immune response to virus.

Development of antibody protection during SARS-CoV-2 infection is a pressing question for public health and for vaccine development. We developed highly sensitive SARS-CoV-2-specific antibody and neutralization assays. SARS-CoV-2 Spike protein or Nucleocapsid protein specific IgG antibodies at titers more than 1:100,000 were detectable in all PCR+ subjects (n = 115) and were absent in the negative controls. Other isotype antibodies (IgA, IgG1-4) were also detected. SARS-CoV-2 neutralization was determined in COVID-19 and convalescent plasma at up to 10,000-fold dilution, using Spike protein pseudotyped lentiviruses, which were also blocked by neutralizing antibodies (NAbs). Hospitalized patients had up to 3000-fold higher antibody and neutralization titers compared to outpatients or convalescent plasma donors. Interestingly, some COVID-19 patients also possessed NAbs against SARS-CoV Spike protein pseudovirus. Together these results demonstrate the high specificity and sensitivity of our assays, which may impact understanding the quality or duration of the antibody response during COVID-19 and in determining the effectiveness of potential vaccines.

Altered type 1 interferon responses in alloimmunized and nonalloimmunized patients with sickle cell disease.

Patients with sickle cell disease (SCD) have a high prevalence of RBC alloimmunization. However, underlying mechanisms are poorly understood. Given that proinflammatory type 1 interferons (IFNα/ß) and interferon stimulated genes (ISGs) promote alloimmunization in mice, we hypothesized that IFNα/ß may contribute to the increased frequency of alloimmunization in patients with SCD. To investigate this, expression of ISGs in blood leukocytes and peripheral blood mononuclear cells (PBMCs) of previously transfused SCD patients with or without alloimmunization and race-matched healthy controls were quantified, and IFNα/ß gene scores were calculated. IFNα/ß gene scores of SCD leukocytes and plasma cytokines were elevated, compared to controls (gene score, p < 0.01). Upon stimulation with IFNß, isolated PBMCs from patients with SCD had elevated ISGs and IFNα/ß gene scores (p < 0.05), compared to stimulated PBMCs from controls. However, IFNß-stimulated and unstimulated ISG expression did not significantly differ between alloimmunized and non-alloimmunized patients. These findings indicate that patients with SCD express an IFNα/ß gene signature, and larger studies are needed to fully determine its role in alloimmunization. Further, illustration of altered IFNα/ß responses in SCD has potential implications for IFNα/ß-mediated viral immunity, responses to IFNα/ß-based therapies, and other sequelae of SCD.

Comparison of the platelet activation status of single-donor platelets obtained with two different cell separator technologies.

BACKGROUND: The microparticle content (MP%) of apheresis platelets-a marker of platelet activation-is influenced by donor factors and by external stressors during collection and storage. This study assessed the impact of apheresis technology and other factors on the activation status (MP%) of single-donor apheresis platelets. STUDY DESIGN AND METHODS: Data from six US hospitals that screened platelets by measuring MP% through dynamic light scattering (ThromboLUX) were retrospectively analyzed. Relative risks (RRs) were derived from univariate and multivariable regression models, with activation rate (MP% ≥15% for plasma-stored platelets; ≥10% for platelet additive solution [PAS]-stored platelets) and MP% as outcomes. Apheresis platform (Trima Accel vs Amicus), storage medium (plasma vs PAS), pathogen reduction, storage time, and testing location were used as predictors. RESULTS: Data were obtained from 7511 platelet units collected using Trima (from 16 suppliers, all stored in plasma, 20.0% were pathogen-reduced) and 2456 collected using Amicus (from four different collection facilities of one supplier, 65.0% plasma-stored, 35.0% PAS-stored, none pathogen-reduced). Overall, 30.0% of Trima platelets were activated compared to 45.6% of Amicus platelets (P < .0001). Multivariable analysis identified apheresis platform as significantly associated with platelet activation, with a lower activation rate for Trima than Amicus (RR: 0.641, 95% confidence interval [CI]: 0.578; 0.711, P < .0001) and a 6.901% (95% CI: 5.926; 7.876, P < .0001) absolute reduction in MP%, when adjusting for the other variables. CONCLUSION: Trima-collected platelets were significantly less likely to be activated than Amicus-collected platelets, irrespective of the storage medium, the use of pathogen reduction, storage time, and testing site.

Novel SARS-CoV-2 specific antibody and neutralization assays reveal wide range of humoral immune response during COVID-19.

Development of antibody protection during SARS-CoV-2 infection is a pressing question for public health and for vaccine development. We developed highly sensitive SARS-CoV-2-specific antibody and neutralization assays. SARS-CoV-2 Spike protein or Nucleocapsid protein specific IgG antibodies at titers more than 1:100,000 were detectable in all PCR+ subjects (n=115) and were absent in the negative controls. Other isotype antibodies (IgA, IgG1-4) were also detected. SARS-CoV-2 neutralization was determined in COVID-19 and convalescent plasma at up to 10,000-fold dilution, using Spike protein pseudotyped lentiviruses, which were also blocked by neutralizing antibodies (NAbs). Hospitalized patients had up to 3000-fold higher antibody and neutralization titers compared to outpatients or convalescent plasma donors. Interestingly, some COVID-19 patients also possessed NAbs against SARS-CoV Spike protein pseudovirus. Together these results demonstrate the high specificity and sensitivity of our assays, which may impact understanding the quality or duration of the antibody response during COVID-19 and in determining the effectiveness of potential vaccines.

Probable transfusion transmission of West Nile virus from an apheresis platelet that screened non-reactive by individual donor-nucleic acid testing.

BACKGROUND: Despite West Nile virus (WNV) blood donation screening using nucleic acid testing (NAT), donors with low viral loads not detected by mini-pool-NAT have led to transfusion transmitted (TT)-WNV infection. We describe a probable case of fatal TT-WNV infection from an individual donor (ID)-NAT non-reactive apheresis platelet donation. STUDY DESIGN AND METHODS: An apheresis platelet donation was WNV ID-NAT reactive and prior donations from the same donor were investigated. A WNV ID-NAT non-reactive apheresis platelet unit collected 26 days earlier was transfused during heart transplantation to a patient who subsequently developed WNV neuroinvasive disease and expired. The source of the recipient's WNV infection was investigated. RESULTS: Twenty-six days after collection of the suspect platelet unit, a donation from the same donor was WNV ID-NAT reactive and WNV IgM and IgG positive. In addition to the suspect platelet unit, the heart transplant recipient who developed WNV infection received 17 blood components from 24 donors. Serologic testing performed on 11 of the remaining 24 donors (46%) was WNV IgM negative. Pre-transplant recipient and heart donor samples tested WNV RNA and IgM negative. CONCLUSION: A probable case of fatal neuroinvasive TT-WNV was linked to an infectious apheresis platelet unit undetected by WNV ID-NAT. It is hypothesized that the suspect unit was collected early in the viremic period when viral RNA was below the limit-of-detection of the ID-NAT assay. Implementation of ID-NAT screening of blood donors has not entirely eliminated the risk of TT-WNV infections, which may best be addressed by pathogen inactivation technologies.

RHD genotyping of serologic RhD-negative blood donors in a hospital-based blood donor center.

BACKGROUND: Serologic RhD-negative blood donors are tested by a method known to detect weak D antigen expression. Serology does not detect all red blood cells with RhD expression and RHD genotyping has been used to identify variant RHD alleles, which may lead to some RhD expression. The aim of this study was to determine the frequency of RHD variant alleles in serologic RhD-negative blood donors at a hospital-based donor center in Los Angeles. STUDY DESIGN AND METHODS: RHD genotyping of serologic RhD-negative blood donors over a 20-month period was performed using the Immucor RHD BeadChip assay. DNA sequencing was performed when the RHD BeadChip assay failed to assign a genotype. For RHD variants known or suspected to result in RhD expression, recipients of previous blood donations were investigated for alloimmunization. RESULTS: RHD genotyping was performed in 1174 RhD-negative blood donors, and 1122 were genotyped for RHCE variants. Eleven donors (0.94%) harbored mutations predicted to yield RhD expression. The predicted phenotypes were, in decreasing frequency, DEL, partial, and weak D phenotypes. Anti-D was not detected in 16 patients who had received blood from these donors after an average follow up of 182 days. CONCLUSION: Genotyping can be used to identify donors with the potential to sensitize RhD-negative recipients. In this limited study, 0.94% of serologic RhD-negative blood donors were found to have variant RHD alleles that might cause alloimmunization in RhD-negative recipients. To our knowledge, a study of this nature has not been reported in the United States.

Systematic genetic dissection of chitin degradation and uptake in Vibrio cholerae.

Vibrio cholerae is a natural resident of the aquatic environment, where a common nutrient is the chitinous exoskeletons of microscopic crustaceans. Chitin utilization requires chitinases, which degrade this insoluble polymer into soluble chitin oligosaccharides. These oligosaccharides also serve as an inducing cue for natural transformation in Vibrio species. There are 7 predicted endochitinase-like genes in the V. cholerae genome. Here, we systematically dissect the contribution of each gene to growth on chitin as well as induction of natural transformation. Specifically, we created a strain that lacks all 7 putative chitinases and from this strain, generated a panel of strains where each expresses a single chitinase. We also generated expression plasmids to ectopically express all 7 chitinases in our chitinase deficient strain. Through this analysis, we found that low levels of chitinase activity are sufficient for natural transformation, while growth on insoluble chitin as a sole carbon source requires more robust and concerted chitinase activity. We also assessed the role that the three uptake systems for the chitin degradation products GlcNAc, (GlcNAc)2 and (GlcN)2 , play in chitin utilization and competence induction. Cumulatively, this study provides mechanistic details for how this pathogen utilizes chitin to thrive and evolve in its environmental reservoir.

The nucleoid occlusion protein SlmA is a direct transcriptional activator of chitobiose utilization in Vibrio cholerae.

Chitin utilization by the cholera pathogen Vibrio cholerae is required for its persistence and evolution via horizontal gene transfer in the marine environment. Genes involved in the uptake and catabolism of the chitin disaccharide chitobiose are encoded by the chb operon. The orphan sensor kinase ChiS is critical for regulation of this locus, however, the mechanisms downstream of ChiS activation that result in expression of the chb operon are poorly understood. Using an unbiased transposon mutant screen, we uncover that the nucleoid occlusion protein SlmA is a regulator of the chb operon. SlmA has not previously been implicated in gene regulation. Also, SlmA is a member of the TetR family of proteins, which are generally transcriptional repressors. In vitro, we find that SlmA binds directly to the chb operon promoter, and in vivo, we show that this interaction is required for transcriptional activation of this locus and for chitobiose utilization. Using point mutations that disrupt distinct functions of SlmA, we find that DNA-binding, but not nucleoid occlusion, is critical for transcriptional activation. This study identifies a novel role for SlmA as a transcriptional regulator in V. cholerae in addition to its established role as a cell division licensing factor.

Multiplex Genome Editing by Natural Transformation (MuGENT) for Synthetic Biology in Vibrio natriegens.

Vibrio natriegens has recently emerged as an alternative to Escherichia coli for molecular biology and biotechnology, but low-efficiency genetic tools hamper its development. Here, we uncover how to induce natural competence in V. natriegens and describe methods for multiplex genome editing by natural transformation (MuGENT). MuGENT promotes integration of multiple genome edits at high-efficiency on unprecedented time scales. Also, this method allows for generating highly complex mutant populations, which can be exploited for metabolic engineering efforts. As a proof-of-concept, we attempted to enhance production of the value added chemical poly-ß-hydroxybutyrate (PHB) in V. natriegens by targeting the expression of nine genes involved in PHB biosynthesis via MuGENT. Within 1 week, we isolated edited strains that produced ∼100 times more PHB than the parent isolate and ∼3.3 times more than a rationally designed strain. Thus, the methods described here should extend the utility of this species for diverse academic and industrial applications.

Multi-Center Evaluation of the Automated Immunohematology Instrument, the ORTHO VISION Analyzer.

BACKGROUND: ORTHO VISION Analyzer (Vision), is an immunohematology instrument using ID-MT gel card technology with digital image processing. It has a continuous, random sample access with STAT priority processing. The efficiency and ease of operation of Vision was evaluated at 5 medical centers. METHODS: De-identified patient samples were tested on the ORTHO ProVue Analyzer (ProVue) and repeated on the Vision mimicking the daily workload pattern. Turnaround times (TAT) were collected and compared. Operators rated key features of the analyzer on a scale of 1 to 5. RESULTS: A total of 507 samples were tested on both instruments at the 5 trial sites. The mean TAT (SD) were 31.6 minutes (5.5) with Vision and 35.7 minutes (8.4) with ProVue, which renders a 12% reduction. Type and screens were performed on 381 samples; the mean TAT (SD) was 32.2 minutes (4.5) with Vision and 37.0 minutes (7.4) with ProVue. Antibody identification with eleven panel cells was performed on 134 samples on Vision; TAT (SD) was 43.2 minutes (8.3). The installation, training, configuration, maintenance and validation processes are all streamlined to provide a short implementation time. The average rating of main functions by the operators was 4.1 to 4.8. Opportunities for improvement, such as flexibility with editing QC results, maintenance schedule, and printing options were identified. The capabilities to perform serial dilutions, to accept pediatric tubes, and review results by e-Connectivity are enhancements over the ProVue. CONCLUSIONS: Vision provides shorter TAT compared to ProVue. Every site described a positive experience using Vision.

Systematic genetic dissection of PTS in Vibrio cholerae uncovers a novel glucose transporter and a limited role for PTS during infection of a mammalian host.

A common mechanism for high affinity carbohydrate uptake in microbial species is the phosphoenolpyruvate-dependent phosphotransferase system (PTS). This system consists of a shared component, EI, which is required for all PTS transport, and numerous carbohydrate uptake transporters. In Vibrio cholerae, there are 13 distinct PTS transporters. Due to genetic redundancy within this system, the carbohydrate specificity of each of these transporters is not currently defined. Here, using multiplex genome editing by natural transformation (MuGENT), we systematically dissect PTS transport in V. cholerae. Specifically, we generated a mutant strain that lacks all 13 PTS transporters, and from this strain, we created a panel of mutants where each expresses a single transporter. Using this panel, we have largely defined the carbohydrate specificities of each PTS transporter. In addition, this analysis uncovered a novel glucose transporter. We have further defined the mechanism of this transporter and characterized its regulation. Using our 13 PTS transporter mutant, we also provide the first clear evidence that carbohydrate transport by the PTS is not essential during infection in an infant mouse model of cholera. In summary, this study shows how multiplex genome editing can be used to rapidly dissect complex biological systems and genetic redundancy in microbial systems.

Successful reduction of plasma free-hemoglobin using therapeutic plasma exchange: A case report.

Massive intravascular hemolysis may overwhelm hemoglobin (Hgb) clearance mechanisms leading to accumulation of excess plasma free-Hgb and subsequent acute kidney injury. We present the case of a 44-year-old male with cardiac failure necessitating placement of a subcutaneous left ventricular assist device. Following insertion, the patient developed mechanical hemolysis and an acute decline in renal function. Three therapeutic plasma exchange procedures were performed resulting in a dramatic decrease in plasma free-Hgb levels and stabilization of renal function. This demonstrates that therapeutic plasma exchange can be used to decrease plasma free-Hgb in cases of intravascular hemolysis, possibly protecting the patient from hemoglobinuric acute kidney injury.

Decreased Threshold of Aggregation to Low-Dose Epinephrine is Evidence of Platelet Hyperaggregability in Patients with Thrombosis.

Sticky platelet syndrome has been described as a hereditary thrombophilic condition. The aim of this study is to identify the presence of platelet hyperaggregability in patients who have experienced thrombosis. Light-transmittance platelet aggregometry was used to assess for spontaneous platelet aggregation, aggregation in response to full and low-dose (LD) epinephrine (Epi) and adenosine diphosphate, as well as arachidonic acid, and identify a distinct pattern of platelet hyperaggregability. Light-transmittance platelet aggregometry results were correlated with PFA-100® (Dade-Behring, Marburg, Germany) results, when available. An exaggerated response to LD Epi was found in 68% of patients with thrombosis compared to only 36% of healthy controls (P=0.034). Patients with thrombosis, either arterial or venous, demonstrated an exaggerated response to LD Epi nearly twice as frequently as healthy controls, even without significant family history of thrombophilia or other known risk factors for thrombosis. This suggests that platelet hyperaggregability may be multifactorial in nature and not necessarily hereditary.