Galectin-4 Antimicrobial Activity Primarily Occurs Through its C-Terminal Domain.

Although immune tolerance evolved to reduce reactivity with self, it creates a gap in the adaptive immune response against microbes that decorate themselves in self-like antigens. This is particularly apparent with carbohydrate-based blood group antigens, wherein microbes can envelope themselves in blood group structures similar to human cells. In this study, we demonstrate that the innate immune lectin, galectin-4 (Gal-4), exhibits strain-specific binding and killing behavior towards microbes that display blood group-like antigens. Examination of binding preferences using a combination of microarrays populated with ABO(H) glycans and a variety of microbial strains, including those that express blood group-like antigens, demonstrated that Gal-4 binds mammalian and microbial antigens that have features of blood group and mammalian-like structures. Although Gal-4 was thought to exist as a monomer that achieves functional bivalency through its two linked carbohydrate recognition domains, our data demonstrate that Gal-4 forms dimers and that differences in the intrinsic ability of each domain to dimerize likely influences binding affinity. While each Gal-4 domain exhibited blood group-binding activity, the C-terminal domain (Gal-4C) exhibited dimeric properties, while the N-terminal domain (Gal-4N) failed to similarly display dimeric activity. Gal-4C not only exhibited the ability to dimerize but also possessed higher affinity toward ABO(H) blood group antigens and microbes expressing glycans with blood group-like features. Furthermore, when compared to Gal-4N, Gal-4C exhibited more potent antimicrobial activity. Even in the context of the full-length protein, where Gal-4N is functionally bivalent by virtue of Gal-4C dimerization, Gal-4C continued to display higher antimicrobial activity. These results demonstrate that Gal-4 exists as a dimer and exhibits its antimicrobial activity primarily through its C-terminal domain. In doing so, these data provide important insight into key features of Gal-4 responsible for its innate immune activity against molecular mimicry.

QMrebind: incorporating quantum mechanical force field reparameterization at the ligand binding site for improved drug-target kinetics through milestoning simulations.

Understanding the interaction of ligands with biomolecules is an integral component of drug discovery and development. Challenges for computing thermodynamic and kinetic quantities for pharmaceutically relevant receptor-ligand complexes include the size and flexibility of the ligands, large-scale conformational rearrangements of the receptor, accurate force field parameters, simulation efficiency, and sufficient sampling associated with rare events. Our recently developed multiscale milestoning simulation approach, SEEKR2 (Simulation Enabled Estimation of Kinetic Rates v.2), has demonstrated success in predicting unbinding (koff) kinetics by employing molecular dynamics (MD) simulations in regions closer to the binding site. The MD region is further subdivided into smaller Voronoi tessellations to improve the simulation efficiency and parallelization. To date, all MD simulations are run using general molecular mechanics (MM) force fields. The accuracy of calculations can be further improved by incorporating quantum mechanical (QM) methods into generating system-specific force fields through reparameterizing ligand partial charges in the bound state. The force field reparameterization process modifies the potential energy landscape of the bimolecular complex, enabling a more accurate representation of the intermolecular interactions and polarization effects at the bound state. We present QMrebind (Quantum Mechanical force field reparameterization at the receptor-ligand binding site), an ORCA-based software that facilitates reparameterizing the potential energy function within the phase space representing the bound state in a receptor-ligand complex. With SEEKR2 koff estimates and experimentally determined kinetic rates, we compare and interpret the receptor-ligand unbinding kinetics obtained using the newly reparameterized force fields for model host-guest systems and HSP90-inhibitor complexes. This method provides an opportunity to achieve higher accuracy in predicting receptor-ligand koff rate constants.

Integrated Immunopeptidomic and Proteomic Analysis of COVID-19 lung biopsies.

Introduction: Severe respiratory illness is the most prominent manifestation of patients infected with SARS-CoV-2, and yet the molecular mechanisms underlying severe lung disease in COVID-19 affected patients still require elucidation. Human leukocyte antigen class I (HLA-I) expression is crucial for antigen presentation and the host's response to SARS-CoV-2. Methods: To gain insights into the immune response and molecular pathways involved in severe lung disease, we performed immunopeptidomic and proteomic analyses of lung tissues recovered at four COVID-19 autopsy and six non-COVID-19 transplants. Results: We found signals of tissue injury and regeneration in lung fibroblast and alveolar type I/II cells, resulting in the production of highly immunogenic self-antigens within the lungs of COVID-19 patients. We also identified immune activation of the M2c macrophage as the primary source of HLA-I presentation and immunogenicity in this context. Additionally, we identified 28 lung signatures that can serve as early plasma markers for predicting infection and severe COVID-19 disease. These protein signatures were predominantly expressed in macrophages and epithelial cells and were associated with complement and coagulation cascades. Discussion: Our findings emphasize the significant role of macrophage-mediated immunity in the development of severe lung disease in COVID-19 patients.

Blood Demand Forecasting and Supply Management: An Analytical Assessment of Key Studies Utilizing Novel Computational Techniques.

Use of data-driven methodologies in enhancing blood transfusion practices is rising, leveraging big data, machine learning, and optimization techniques to improve demand forecasting and supply chain management. This review used a narrative approach to identify, evaluate, and synthesize key studies that considered novel computational techniques for blood demand forecasting and inventory management through a search of PubMed and Web of Sciences databases for studies published from January 01, 2016, to March 30, 2023. The studies were analyzed for their utilization of various techniques, and their strengths, limitations, and areas for improvement. Seven key studies were identified. The studies focused on different blood components using various computational methods, such as regression, machine learning, hybrid models, and time series models, across different locations and time periods. Key variables used for demand forecasting were largely derived from electronic health record data, including clinical related predictors such as laboratory test results and hospital census by location. Each study offered unique strengths and valuable insights into the use of data-driven methods in blood bank management. Common limitations were unknown generalizability to other healthcare settings or blood components, need for field-specific performance measures, lack of ABO compatibility consideration, and ethical challenges in resource allocation. While data-driven research in blood demand forecasting and management has progressed, limitations persist and further exploration is needed. Understanding these innovative, interdisciplinary methods and their complexities can help refine inventory strategies and address healthcare challenges more effectively, leading to more robust, accurate models to enhance blood management across diverse healthcare scenarios.

Impact of allele-specific anti-human leukocyte antigen class I antibodies on organ allocation.

Technological advances in the field of histocompatibility have allowed us to define anti-human leukocyte antigen (HLA) antibody specificity at the allelic level. However, how allele-specific antibodies affect organ allocation is poorly studied. We examined allelic specificities of class I HLA antibodies in 6726 consecutive serum samples from 2953 transplant candidates and evaluated their impact on the corresponding crossmatch and organ allocation. Out of 17 class I HLA antigens represented by >1 allele in the LABScreen single antigen bead assay, 12 had potential allele-specific reactivity. Taking advantage of our unbiased cohort of deceased donor-candidate testing (123,135 complement-dependent cytotoxicity crossmatches between 2014 and 2017), we estimated that the presence of allele-specific antibody detected using a single antigen bead assay (median fluorescence intensity, >3000) against only the rare allele was a poor predictor of a positive complement-dependent cytotoxicity crossmatch, with a positive predictive value of 0% to 7%, compared with 52.5% in allele-concordant class I HLA antibodies against A or B locus antigens. Further, we confirmed allele-specific reactivity using flow crossmatch in 3 scenarios: A11:01/A11:02, A68:01/A68:02, and B44:02/B44:03. Our results suggest that allele-specific antibodies may unnecessarily exclude transplant candidates (up to 10%) from organ offers by overcalling unacceptable antigens; incorporation of selective reactivity pattern in allocation may promote precision matching and more equitable allocation.

Selectivity and Ranking of Tight-Binding JAK-STAT Inhibitors Using Markovian Milestoning with Voronoi Tessellations.

Janus kinases (JAK), a group of proteins in the nonreceptor tyrosine kinase (NRTKs) family, play a crucial role in growth, survival, and angiogenesis. They are activated by cytokines through the Janus kinase-signal transducer and activator of a transcription (JAK-STAT) signaling pathway. JAK-STAT signaling pathways have significant roles in the regulation of cell division, apoptosis, and immunity. Identification of the V617F mutation in the Janus homology 2 (JH2) domain of JAK2 leading to myeloproliferative disorders has stimulated great interest in the drug discovery community to develop JAK2-specific inhibitors. However, such inhibitors should be selective toward JAK2 over other JAKs and display an extended residence time. Recently, novel JAK2/STAT5 axis inhibitors (N-(1H-pyrazol-3-yl)pyrimidin-2-amino derivatives) have displayed extended residence times (hours or longer) on target and adequate selectivity excluding JAK3. To facilitate a deeper understanding of the kinase-inhibitor interactions and advance the development of such inhibitors, we utilize a multiscale Markovian milestoning with Voronoi tessellations (MMVT) approach within the Simulation-Enabled Estimation of Kinetic Rates v.2 (SEEKR2) program to rank order these inhibitors based on their kinetic properties and further explain the selectivity of JAK2 inhibitors over JAK3. Our approach investigates the kinetic and thermodynamic properties of JAK-inhibitor complexes in a user-friendly, fast, efficient, and accurate manner compared to other brute force and hybrid-enhanced sampling approaches.

ABO Genotyping finds more A2 to B kidney transplant opportunities than lectin-based subtyping.

ABO compatibility is important for kidney transplantation, with longer waitlist times for blood group B kidney transplant candidates. However, kidneys from non-A1 (eg, A2) subtype donors, which express less A antigen, can be safely transplanted into group B recipients. ABO subtyping is routinely performed using anti-A1 lectin, but DNA-based genotyping is also possible. Here, we compare lectin and genotyping testing. Lectin and genotype subtyping was performed on 554 group A deceased donor samples at 2 transplant laboratories. The findings were supported by 2 additional data sets of 210 group A living kidney donors and 124 samples with unclear lectin testing sent to a reference laboratory. In deceased donors, genotyping found 65% more A2 donors than lectin testing, most with weak lectin reactivity, a finding supported in living donors and samples sent for reference testing. DNA sequencing and flow cytometry showed that the discordances were because of several factors, including transfusion, small variability in A antigen levels, and rare ABO∗A2.06 and ABO∗A2.16 sequences. Although lectin testing is the current standard for transplantation subtyping, genotyping is accurate and could increase A2 kidney transplant opportunities for group B candidates, a difference that should reduce group B wait times and improve transplant equity.

Genome screening, reporting, and genetic counseling for healthy populations.

Rapid advancements of genome sequencing (GS) technologies have enhanced our understanding of the relationship between genes and human disease. To incorporate genomic information into the practice of medicine, new processes for the analysis, reporting, and communication of GS data are needed. Blood samples were collected from adults with a PCR-confirmed SARS-CoV-2 (COVID-19) diagnosis (target N = 1500). GS was performed. Data were filtered and analyzed using custom pipelines and gene panels. We developed unique patient-facing materials, including an online intake survey, group counseling presentation, and consultation letters in addition to a comprehensive GS report. The final report includes results generated from GS data: (1) monogenic disease risks; (2) carrier status; (3) pharmacogenomic variants; (4) polygenic risk scores for common conditions; (5) HLA genotype; (6) genetic ancestry; (7) blood group; and, (8) COVID-19 viral lineage. Participants complete pre-test genetic counseling and confirm preferences for secondary findings before receiving results. Counseling and referrals are initiated for clinically significant findings. We developed a genetic counseling, reporting, and return of results framework that integrates GS information across multiple areas of human health, presenting possibilities for the clinical application of comprehensive GS data in healthy individuals.

Genome Reporting for Healthy Populations-Pipeline for Genomic Screening from the GENCOV COVID-19 Study.

Genome sequencing holds the promise for great public health benefits. It is currently being used in the context of rare disease diagnosis and novel gene identification, but also has the potential to identify genetic disease risk factors in healthy individuals. Genome sequencing technologies are currently being used to identify genetic factors that may influence variability in symptom severity and immune response among patients infected by SARS-CoV-2. The GENCOV study aims to look at the relationship between genetic, serological, and biochemical factors and variability of SARS-CoV-2 symptom severity, and to evaluate the utility of returning genome screening results to study participants. Study participants select which results they wish to receive with a decision aid. Medically actionable information for diagnosis, disease risk estimation, disease prevention, and patient management are provided in a comprehensive genome report. Using a combination of bioinformatics software and custom tools, this article describes a pipeline for the analysis and reporting of genetic results to individuals with COVID-19, including HLA genotyping, large-scale continental ancestry estimation, and pharmacogenomic analysis to determine metabolizer status and drug response. In addition, this pipeline includes reporting of medically actionable conditions from comprehensive gene panels for Cardiology, Neurology, Metabolism, Hereditary Cancer, and Hereditary Kidney, and carrier screening for reproductive planning. Incorporated into the genome report are polygenic risk scores for six diseases-coronary artery disease; atrial fibrillation; type-2 diabetes; and breast, prostate, and colon cancer-as well as blood group genotyping analysis for ABO and Rh blood types and genotyping for other antigens of clinical relevance. The genome report summarizes the findings of these analyses in a way that extensively communicates clinically relevant results to patients and their physicians. © 2022 Wiley Periodicals LLC. Basic Protocol 1: HLA genotyping and disease association Basic Protocol 2: Large-scale continental ancestry estimation Basic Protocol 3: Dosage recommendations for pharmacogenomic gene variants associated with drug response Support Protocol: System setup.

International Society of Blood Transfusion Working Party on Red Cell Immunogenetics and Blood Group Terminology Report of Basel and three virtual business meetings: Update on blood group systems.

BACKGROUND AND OBJECTIVES: Under the ISBT, the Working Party (WP) for Red Cell Immunogenetics and Blood Group Terminology is charged with ratifying blood group systems, antigens and alleles. This report presents the outcomes from four WP business meetings, one located in Basel in 2019 and three held as virtual meetings during the COVID-19 pandemic in 2020 and 2021. MATERIALS AND METHODS: As in previous meetings, matters pertaining to blood group antigen nomenclature were discussed. New blood group systems and antigens were approved and named according to the serologic, genetic, biochemical and cell biological evidence presented. RESULTS: Seven new blood group systems, KANNO (defined numerically as ISBT 037), SID (038), CTL2 (039), PEL (040), MAM (041), EMM (042) and ABCC1 (043) were ratified. Two (039 and 043) were de novo discoveries, and the remainder comprised reported antigens where the causal genes were previously unknown. A further 15 blood group antigens were added to the existing blood group systems: MNS (002), RH (004), LU (005), DI (010), SC (013), GE (020), KN (022), JMH (026) and RHAG (030). CONCLUSION: The ISBT now recognizes 378 antigens, of which 345 are clustered within 43 blood group systems while 33 still have an unknown genetic basis. The ongoing discovery of new blood group systems and antigens underscores the diverse and complex biology of the red cell membrane. The WP continues to update the blood group antigen tables and the allele nomenclature tables. These can be found on the ISBT website (http://www.isbtweb.org/working-parties/red-cell-immunogenetics-and-blood-group-terminology/).

Reversal of viral and epigenetic HLA class I repression in Merkel cell carcinoma.

Cancers avoid immune surveillance through an array of mechanisms, including perturbation of HLA class I antigen presentation. Merkel cell carcinoma (MCC) is an aggressive, HLA-I-low, neuroendocrine carcinoma of the skin often caused by the Merkel cell polyomavirus (MCPyV). Through the characterization of 11 newly generated MCC patient-derived cell lines, we identified transcriptional suppression of several class I antigen presentation genes. To systematically identify regulators of HLA-I loss in MCC, we performed parallel, genome-scale, gain- and loss-of-function screens in a patient-derived MCPyV-positive cell line and identified MYCL and the non-canonical Polycomb repressive complex 1.1 (PRC1.1) as HLA-I repressors. We observed physical interaction of MYCL with the MCPyV small T viral antigen, supporting a mechanism of virally mediated HLA-I suppression. We further identify the PRC1.1 component USP7 as a pharmacologic target to restore HLA-I expression in MCC.

Innate immune Galectin-7 specifically targets microbes that decorate themselves in blood group-like antigens.

Adaptive immunity can target a nearly infinite range of antigens, yet it is tempered by tolerogenic mechanisms that limit autoimmunity. Such immunological tolerance, however, creates a gap in adaptive immunity against microbes decorated with self-like antigens as a form of molecular mimicry. Our results demonstrate that the innate immune lectin galectin-7 (Gal-7) binds a variety of distinct microbes, all of which share features of blood group-like antigens. Gal-7 binding to each blood group expressing microbe, including strains of Escherichia coli, Klebsiella pneumoniae, Providencia alcalifaciens, and Streptococcus pneumoniae, results in loss of microbial viability. Although Gal-7 also binds red blood cells (RBCs), this interaction does not alter RBC membrane integrity. These results demonstrate that Gal-7 recognizes a diverse range of microbes, each of which use molecular mimicry while failing to induce host cell injury, and thus may provide an innate form of immunity against molecular mimicry.

Recommendation for validation and quality assurance of non-invasive prenatal testing for foetal blood groups and implications for IVD risk classification according to EU regulations.

BACKGROUND AND OBJECTIVES: Non-invasive assays for predicting foetal blood group status in pregnancy serve as valuable clinical tools in the management of pregnancies at risk of detrimental consequences due to blood group antigen incompatibility. To secure clinical applicability, assays for non-invasive prenatal testing of foetal blood groups need to follow strict rules for validation and quality assurance. Here, we present a multi-national position paper with specific recommendations for validation and quality assurance for such assays and discuss their risk classification according to EU regulations. MATERIALS AND METHODS: We reviewed the literature covering validation for in-vitro diagnostic (IVD) assays in general and for non-invasive foetal RHD genotyping in particular. Recommendations were based on the result of discussions between co-authors. RESULTS: In relation to Annex VIII of the In-Vitro-Diagnostic Medical Device Regulation 2017/746 of the European Parliament and the Council, assays for non-invasive prenatal testing of foetal blood groups are risk class D devices. In our opinion, screening for targeted anti-D prophylaxis for non-immunized RhD negative women should be placed under risk class C. To ensure high quality of non-invasive foetal blood group assays within and beyond the European Union, we present specific recommendations for validation and quality assurance in terms of analytical detection limit, range and linearity, precision, robustness, pre-analytics and use of controls in routine testing. With respect to immunized women, different requirements for validation and IVD risk classification are discussed. CONCLUSION: These recommendations should be followed to ensure appropriate assay performance and applicability for clinical use of both commercial and in-house assays.

Ultrasonic Doppler-guided catheterization of the dorsal pedal artery in dogs and cats under general anesthesia.

BACKGROUND AND OBJECTIVE: Noninvasive blood pressure measurements are less accurate than direct measurements but arterial catheterization for direct blood pressure monitoring can be challenging especially in patients with a nonpalpable pulse. The purpose of this study is to describe a novel technique of locating the dorsal pedal arterial pulse using a portable ultrasonic Doppler for arterial catheter placement. KEY FINDINGS: Using the described technique in anesthetized dogs and cats with strong (n = 13) to weak dorsal pedal pulses (n = 6), the overall success rate of arterial puncture was 70% (19/27) with successful catheterization in 53% (10/19) of those. The overall success rate of arterial catheterization was 37% (10/27). The pulse strength did not significantly affect the success rate. Neither were patient weight, blood pressure, heart rate, and premedication significantly different between the 2 groups. SIGNIFICANCE: This study describes a novel technique for locating the dorsal pedal artery for arterial catheterization. Having another technique of pulse detection may increase the likelihood of arterial catheter placement for direct blood pressure monitoring and aid in arterial puncture for obtaining a sample for blood gas analysis in critical patients.

PIGG defines the Emm blood group system.

Emm is a high incidence red cell antigen with eight previously reported Emm- probands. Anti-Emm appears to be naturally occurring yet responsible for a clinically significant acute hemolytic transfusion reaction. Previous work suggests that Emm is located on a GPI-anchored protein, but the antigenic epitope and genetic basis have been elusive. We investigated samples from a South Asian Indian family with two Emm- brothers by whole genome sequencing (WGS). Additionally, samples from four unrelated Emm- individuals were investigated for variants in the candidate gene. Filtering for homozygous variants found in the Emm- brothers and by gnomAD frequency of < 0.001 resulted in 1818 variants with one of high impact; a 2-bp deletion causing a frameshift and premature stop codon in PIGG [NM_001127178.3:c.2624_2625delTA, p.(Leu875*), rs771819481]. PIGG encodes for a transferase, GPI-ethanolaminephosphate transferase II, which adds ethanolamine phosphate (EtNP) to the second mannose in a GPI-anchor. The four additional unrelated Emm- individuals had various PIGG mutations; deletion of Exons 2-3, deletion of Exons 7-9, insertion/deletion (indel) in Exon 3, and new stop codon in Exon 5. The Emm- phenotype is associated with a rare deficiency of PIGG, potentially defining a new Emm blood group system composed of EtNP bound to mannose, part of the GPI-anchor. The results are consistent with the known PI-linked association of the Emm antigen, and may explain the production of the antibody in the absence of RBC transfusion. Any association with neurologic phenotypes requires further research.

Utility of Routine Computed Tomography Angiogram Screening for Blunt Carotid Injury in Isolated Mandible Fractures.

PURPOSE: Determine the incidence of blunt carotid injury (BCAI) in the blunt trauma induced isolated mandible fracture population and determine the utility of computed tomographic angiography (CTA) screening for BCAI in this population. METHODS: A retrospective data review was performed on patients presenting to Boston Medical Center from January 2008 to January 2019 with a diagnosis of mandible fracture. Population selected utilizing ICD-9 and ICD-10 diagnosis codes for mandible fracture and BCAI. Excluded populations were pediatric (less than 18 years) and penetrating carotid injuries. RESULTS: A total of 1,508 mandible fractures were identified, with 73% (n = 1,103) being isolated injuries. Five BCAIs were identified, and of these, 1 was associated with an isolated mandible fracture (incidence <0.01%). One of 110 isolated mandible fractures screened for BCAI with the use of CTA was positive (incidence 0.9%). CONCLUSIONS: BCAI is a rare finding in isolated mandible fractures. The inclusion of this population as an independent risk factor for BCAI should be questioned. Routine screening with CTA is not warranted.

USP13 interacts with cohesin and regulates its ubiquitination in human cells.

Cohesin is a multiprotein ring complex that regulates 3D genome organization, sister chromatid cohesion, gene expression, and DNA repair. Cohesin is known to be ubiquitinated, although the mechanism, regulation, and effects of cohesin ubiquitination remain poorly defined. We previously used gene editing to introduce a dual epitope tag into the endogenous allele of each of 11 known components of cohesin in human HCT116 cells. Here we report that mass spectrometry analysis of dual-affinity purifications identified the USP13 deubiquitinase as a novel cohesin-interacting protein. Subsequent immunoprecipitation/Western blots confirmed the endogenous interaction in HCT116, 293T, HeLa, and RPE-hTERT cells; demonstrated that the interaction occurs specifically in the soluble nuclear fraction (not in the chromatin); requires the ubiquitin-binding domains (UBA1/2) of USP13; and occurs preferentially during DNA replication. Reciprocal dual-affinity purification of endogenous USP13 followed by mass spectrometry demonstrated that cohesin is its primary interactor in the nucleus. Ectopic expression and CRISPR knockout of USP13 showed that USP13 is paradoxically required for both deubiquitination and ubiquitination of cohesin subunits in human cells. USP13 was dispensable for sister chromatid cohesion in HCT116 and HeLa cells, whereas it was required for the dissociation of cohesin from chromatin as cells transit through mitosis. Together these results identify USP13 as a new cohesin-interacting protein that regulates the ubiquitination of cohesin and its cell cycle regulated interaction with chromatin.

A multicenter observational study investigating care errors, staffing levels, and workload in small animal intensive care units.

OBJECTIVE: To investigate associations among care errors, staffing, and workload in small animal ICUs. DESIGN: Multicenter observational cohort study conducted between January 2017 and September 2018. SETTING: Three small animal teaching hospital ICUs. ANIMALS: None. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Data on patient numbers, illness severity (assesed via the acute patient physiologic and laboratory evaluation [APPLE] score), care burden, staffing levels, technician experience/education level, and care errors were collected at each study site. Care errors were categorized as major (unanticipated arrest or death; patient endangerment through IV line, arterial catheter, chest tube or other invasive device mismanagement, or errors in drug calculation/administration) or minor. Median patient:technician ratio was 4.3 (range: 1-18). Median patient illness severity was 15.1 (4.7-27.1) APPLE score units. A total of 221 major and 3,317 minor errors were observed over the study period. The odds of a major error increased by an average of 11% (odds ratio [OR] = 1.11; 95% confidence interval [CI], 1.02-1.20; P = 0.012) for each 1 patient increase in the patient:technician ratio after averaging by ICU location. The major error incident rate ratio was 2.53 (95% CI, 1.84-3.54; P < 0.001) for patient:technician ratios of >4.0 compared with ≤4.0. The odds of a major error increased by 0.5% per total unit APPLE score increase (OR = 1.005; 95% CI, 1.002-1.007; P < 0.001). The major error incident rate ratio was 1.71 (95% CI, 1.30-2.25; P < 0.001) for APPLEfast :technician ratios of >73 compared with ≤73. The odds of a major error decreased by 2% (OR = 0.98; 95% CI, 0.97-0.99; P = 0.01) for each year increase in total technician years of ICU work experience. CONCLUSIONS: Substantial reductions in major care errors may be achieved by maintaining ICU patient:technician ratios at ≤4. Technician experience and total unit burden of patient illness severity are also associated with error incidence, and should be taken into consideration when scheduling staff.