Titration Protocol Research Articles

Implementation strategies for inpatient continuous glucose monitoring-based diabetes management: a systematic review.

Continuous glucose monitoring (CGM) provides real-time glucose data that has revolutionised outpatient diabetes care; however, its impact on inpatient care remains limited, likely due to the lack of standardised CGM-based insulin titration protocols, implementation strategies, and proper familiarity with the technology among others. A systematic literature search was conducted on October 15, 2024, using PubMed and Embase, without a restriction on publication date. The search focused on CGM-based insulin titration protocols and related implementation strategies in non-intensive care unit (non-ICU) settings. This systematic review was registered with PROSPERO (RD42024596819). A total of 7,625 references were screened. Nine protocols for inpatient CGM-based insulin titration and related implementation strategies were identified. Six protocols recommended a weight-based basal-bolus insulin regimen. Insulin titration on basal and bolus insulin was mostly done daily based on either clinical discretion or clearly defined insulin titration protocols. All protocols employed a hybrid approach, utilising both CGM and finger prick glucose testing to guide glucose management. Diabetes-trained staff oversaw CGM-based insulin titration and glucose management in five protocols. CGM-alarm settings varied widely, with hyperglycaemic alarm thresholds between >13.9 and >22.2 mmol/l, and hypoglycaemia alarm thresholds between <3.9 and <5.0 mmol/l. We observed considerable variation in the detail and clarity provided by the reviewed protocols. This highlights the need for standardised operational protocols for CGM-based insulin titration and related implementation strategies to implement CGM effectively in non-ICU settings.

Protocol for recombinant rabies virus titration by quantitative PCR.

Preparing high-titer virus and performing accurate titer determination are criticalto subsequent experiments. However, not all applied recombinant rabies viruses, such as the L-deleted virus,1 are equipped with fluorescent proteins for titration by fluorescence-activated cell sorting (FACS). Here, we present a quantitative reverse-transcription PCR (RT-qPCR) approach for titrating recombinant rabies virus. We describe steps for preparing standards for RT-qPCR, rabies virus genome RNA extraction, and reverse transcription of virus RNA. We then detail procedures for RT-qPCR for titration and stereotaxic rabies virus injection for titer verification.

NORSE secondary to anti-GAD65 antibody-positive encephalitis treated with novel adjunctive rapid titrationVNS protocol.

New Onset Refractory Status Epilepticus (NORSE) is a rare and severe condition characterized by refractory seizures in individuals without a prior history of epilepsy. This case report describes a 37-year-old woman diagnosed with anti-glutamic acid decarboxylase 65 (anti-GAD65) antibody-positive encephalitis-related NORSE. Her seizures were refractory to multiple interventions, including anti-seizure medications, anesthetics, immunotherapies, a ketogenic diet, and electroconvulsive therapy. Seizures recurred twice during the tapering of anesthetic medications. However, after 32 days of treatment, the seizures were successfully controlled. To maintain seizure control and facilitate the weaning of anesthetics, a Vagus Nerve Stimulator (VNS) was implanted using a novel rapid titration protocol. This allowed for the successful tapering of anesthetics by day 50, with no recurrence of seizures. At her 9-month follow-up, the patient remained seizure-free and had an improved quality of life. This case highlights that early initiation of immunosuppressive treatment may lead to a favorable prognosis. The novel application of VNS therapy assisted seizure control in NORSE, thus encouraging further research investigating the potential role of VNS in this condition. PLAIN LANGUAGE SUMMARY: New Onset Refractory Status Epilepticus (NORSE) is a rare and severe condition characterized by relentless seizures in individuals without a prior epilepsy history. This report shares the case of a 37-year-old woman with NORSE, associated with a high anti-glutamic acid decarboxylase 65 antibody titer. Her seizures were super-refractory, requiring multiple anti-seizure medications, anesthetics, immunotherapies, a ketogenic diet, and electroconvulsive therapy. Seizures recurred twice during the tapering of anesthetic medications. However, by hospital day 32, the seizures were successfully controlled with these interventions. To further stabilize seizure control and enable the successful discontinuation of anesthetics, a Vagus Nerve Stimulator (VNS) was implanted. The patient had no further seizures and gradually recovered back to her pre-disease baseline. This case suggests that a novel rapid VNS titration protocol could be a promising treatment option for NORSE, warranting further investigation.

Early Results of Implementing Rapid Methadone Titration for Hospitalized Patients: A Case Series.

With the increase in illicit fentanyl use in the USA, hospitals face challenges managing opioid withdrawal and opioid use disorder (OUD). To improve opioid withdrawal and OUD treatment among hospitalized patients with daily fentanyl use, we developed a rapid methadone titration (RMT) protocol. We describe development, implementation, and outcomes during the first 12weeks. We analyzed electronic health record data of hospitalizations seen by the Addiction Consult Team (ACT) for methadone initiation between 9/11/23 and 12/3/23. Adults aged 18-64 reporting daily fentanyl use, desiring methadone, and without end-stage organ damage or critical illness were RMT eligible. We characterized patients who received RMT, abstracting demographic and clinical characteristics, adverse events graded by the Common Terminology Criteria for Adverse Events(CTCAE), and methadone and additional full agonist opioid (FAO) dosing. Our primary outcome was adverse events. Secondary outcomes included median time to 100mg of methadone, FAO dosing, and self-directed discharge. ACT assessed 55 hospitalizations representing 47 patients for RMT eligibility. Among these, 19 (34.5%) hospitalizations representing 17 patients were eligible for and received RMT. Four (21.2%) hospitalizations that received RMT had sedation events, and all were mild or moderate grade by CTCAE. Hospitalizations achieved a median methadone dose of 100mg by day 6, with FAO doses peaking on day 5. One (5.3%) hospitalization had a self-directed discharge. With careful patient selection and ACT evaluation, a RMT protocol for hospitalized patients with fentanyl use disorder experienced few adverse events other than mild-moderate sedation, even among those receiving FAO and those with concurrent substance use disorders.

Unfractionated heparin monitoring by anti-factor Xa versus activated partial thromboplastin time strategies during venoarterial extracorporeal life support.

No clear guidelines exist for unfractionated heparin (UFH) monitoring in adult patients on veno-arterial extracorporeal life support (VA-ECLS) for refractory cardiogenic shock. In this study, we sought to compare outcomes between anti-factor Xa (FXa) and activated partial thromboplastin time (aPTT) strategies for UFH monitoring during VA-ECLS. This is a single-center, retrospective review of VA-ECLS patients who received UFH in the cardiothoracic intensive care unit between July 2019 and November 2023. Standard protocol for UFH titration was aPTT goal of 45-60 sec (n = 52) before September 2021, then transitioned to FXa goal of 0.1-0.2 U/mL (n = 50) thereafter. Inverse probability of treatment weighting was used to balance baseline differences between cohorts. In adjusted analyses, 89.3% of FXa patients and 76.0% of aPTT patients achieved goal range for their respective assay. Total UFH duration (4.0 vs 4.0 days, p = .239) and maximum weight-adjusted UFH dose (9.3 vs 9.4 U/hr/kg, p = .823) remained comparable between adjusted FXa and aPTT cohorts. Moreover, in-hospital mortality (50.3% vs 33.9%, p = .133), major bleeding events (20.6% vs 11.2%, p = .292), and thromboembolic events (30.1% vs 30.1%, p = .998) were not significantly different. Extracorporeal circuit thrombosis and cannula site bleeding were the most frequent events in both groups. Multivariate logistic regression found the FXa strategy was not a significant risk factor for the composite outcome of major bleeding or thromboembolism (OR [95% CI]: 1.539 [0.575, 4.116], p = .393). In adult VA-ECLS patients at our institution, bleeding and thromboembolic complications occurred at a similar rate regardless of which UFH monitoring strategy was utilized. Further studies in larger and more institutionally diverse cohorts are warranted.

Dual guided heparin monitoring with anti-Xa and APTT during micro-axial flow pump support for cardiogenic shock

Abstract Background Precise management of unfractionated heparin (UFH) is key to avoid coagulopathic complications in patients on percutaneous mechanical circulatory support (pMCS) for cardiogenic shock (CS). While the activated partial thromboplastin time (APTT) is commonly used for this purpose, it can be affected by various laboratory factors during critical illness. Alternatively, the anti-Xa test directly measures UFH activity. We proposed a parallel anti-Xa/APTT-based protocol for UFH titration during pMCS. (1) Purpose We aimed to investigate the correlation between parallel anti-Xa/APTT measurements in a CS population on pMCS. We also evaluated mortality correlation in samples exhibiting prolonged APTT and in-range anti-Xa levels, based on fibrinogen levels and INR. Methods We assessed all CS patients with isolated left-sided ImpellaTM-support (&amp;gt;24h) and UFH anticoagulation monitoring per protocol at two pMCS-institutions from April-2017 to June-2022. Overall correlation between parallel anti-Xa/APTT samples was assessed by Pearson correlation coefficient (with ≤0.7 considered to confer a weak correlation). Anti-Xa levels were defined as 0.20-0.30IU/mL or 0.31-0.50IU/mL, with corresponding APTT values of 40-55s and 55-80s, respectively. Samples with prolonged APTT as compared to the in-range anti-Xa were stratified based on fibrinogen (&amp;lt;1.5g/dL or ≥ 1.5g/dL) and INR (&amp;lt;1.5 or ≥1.5) on the same day as the index sample. Mortality of patients with normal fibrinogen ànd INR was then compared to those with abnormal fibrinogen and/or INR via a Chi² test. Results Ninety-three ImpellaTM-runs (77 men, 83%) with a median age of 56 years (IQR 46-66) were analyzed. Median support was 6 days (IQR 4-12). In 2447 parallel anti-Xa/APTT samples, the Pearson correlation coefficient was 0.50 (p&amp;lt;0.001). Among the 1914 in-range anti-Xa samples, APPT was shortened in 460 (24%), in-range in 986 (52%) and prolonged in 468 (24%) samples. In the 59 patients with samples with prolonged APTT, two patients (with 80 samples) were excluded due to absence of fibrinogen and/or INR measurements on the same day. The mortality in patients with prolonged APTT but normal fibrinogen levels and normal INR (N=29; APTT prolongation due to FXI/FXII-consumption) was significantly lower compared to the remaining 28 patients who showed samples with reduced fibrinogen levels and/or prolonged INR (suffering from concomitant conditions) (10% vs. 32%; p=0.043). Conclusion We highlight a weak correlation between anti-Xa and APTT in an anti-Xa guided UFH anticoagulated CS population during left-sided ImpellaTM support. Patients with APTT-prolongation due to concomitant conditions with INR prolongation and/or fibrinogen shortage are at highest mortality risk. The APTT/Anti-Xa coefficient can serve as an independent risk factor whilst managing critically CS patients.

A Novel Monoclonal Antibody Against a Modified Vaccinia Ankara (MVA) Envelope Protein as a Tool for MVA Virus Titration by Flow Cytometry.

Modified vaccinia Ankara (MVA) virus is a widely used vaccine platform, making accurate titration essential for vaccination studies. However, the current plaque forming unit (PFU) assay, the standard for MVA titration, is prone to observer bias and other limitations that affect accuracy and precision. To address these challenges, we developed a new flow cytometry-based quantification method using a highly specific monoclonal antibody (mAb) for the detection of MVA-infected cells, as a more accurate titration assay. Through previous work, we serendipitously identified three MVA-specific hybridoma antibody clones, which we characterized through ELISA, immunoblot, and flow cytometry, confirming their specificity for MVA. Sequencing confirmed that each antibody was monoclonal, and mass spectrometry results revealed that all mAbs target the MVA cell surface binding protein (CSBP, MVA105L). We next optimized the titration protocol using the most effective mAb, 33C7 by refining culture conditions and staining protocols to enhance sensitivity and minimize background. Our optimized method demonstrated superior sensitivity, reliability, and reduced processing time when compared with the traditional PFU assay, establishing it as a more accurate and efficient approach for MVA titration.

Quantification of Adeno-Associated Viral Genomes in Purified Vector Samples by Digital Droplet Polymerase Chain Reaction.

Adeno-associated virus (AAV) is a non-pathogenic virus used as a delivery vehicle to transfer therapeutic genes into patients. Accurate quantification of AAV genome copy number in vector preparations is essential for bioprocess optimization and dosage calculation in both preclinical and clinical studies of AAV-based gene therapy products. Currently, a consensus protocol for AAV viral genome titration is lacking. Here, we present a digital droplet PCR (dd_PCR) protocol for the quantification of viral genomes in purified vector samples. Samples are treated with DNase I to eliminate unpackaged contaminant DNA. DNase-treated samples are then mixed with an appropriate primer-probe set (designed according to the target AAV genome) and PCR reagents, and then loaded into a droplet generator. The prepared droplets are transferred into a PCR plate, where PCR amplification is performed and analyzed. The viral genome titer is calculated based on the concentration (copies/µL), accounting for sample dilutions. A successful measurement shows a clear separation of the positive and negative droplet clouds, has at least 10,000 accepted droplets, shows a value between 10 copies/µL and 10,000 copies/µL, and has a coefficient of variation (CV) between repeats lower than 20%. Reliable viral genome titration will aid in the development of safe and effective AAV-based gene therapy products.

Comparative study of the propagation and plaque titration conditions for human coronavirus OC43 as a surrogate for SARS-CoV-2.

The ongoing COVID-19 pandemic is threatening human health globally. The development of effective drugs and vaccines against SARS-CoV-2 is hindered by the limited access to high-biosafety-level facilities. Although human coronavirus (HCoV) OC43, a low-pathogenic endemic human coronavirus, has been used as a surrogate virus for SARS-CoV-2 research, a standard technique for HCoV-OC43 culture and plaque titration has not been established. Our objective was to establish optimized culture and titration protocols for HCoV-OC43. The growth kinetics and permissibility to HCoV-OC43 infection of seven different cell lines were examined concurrently at two different temperatures, 33°C and 37°C. Cell lines exhibiting a cytopathic effect (CPE) were selected for plaque titration. No significant difference in the rate of cell growth was observed at the two temperatures tested. Interestingly, HCoV-OC43 was found not to be a high-temperature-sensitive virus, since it grew well at 37°C. Although RD, LLC-MK2, MRC-5, and HCT-8 cell lines supported virus growth with an obvious cytopathic effect and a high yield of virus after two days of infection, only RD cells were suitable for producing countable plaques. The incubation of the cells with 1.2% low-viscosity Avicel as an overlay medium at 37°C for 4 days appeared to promote clearer and sharper plaque morphology. However, further optimization of the plaque titration protocol is still required due to the continued observation of plaque size variation and hazy zones. We propose a cost-effective protocol for HCoV-OC43 culture and plaque titration that can be implemented at a standard conventional temperature without the need for additional special equipment.

Isoflurane Titration Improves Detection of Hippocampal Lactate by 1H-MRS

Abstract Lactate has increasingly been recognized as both an important fuel source and signaling molecule within the brain. Alterations in brain lactate levels are associated with various neurological diseases. Thus, there is great interest in the in vivo detection and measurement of cerebral lactate levels in animals used for investigation of normal brain function and models of disease. Proton magnetic resonance spectroscopy (1H-MRS) is a non-invasive technique used to measure lactate and other metabolites within the brain. However, lactate can be difficult to detect with conventional 1H-MRS due to its low abundance and spectral overlap with lipids. In addition, volatile anesthetics used during image acquisition increase lactate production, potentially masking any subtle physiological changes in lactate levels. Here we made use of a transgenic mouse model in which expression of lactate dehydrogenase A (Ldha), the rate-limiting enzyme of lactate production, was induced within cortical and hippocampal neurons. Unexpectedly, 1H-MRS analysis under typical isoflurane-induced anesthesia of 4% induction followed by 1.6-2% maintenance, revealed no significant elevation of hippocampal lactate levels in neuronal Ldha induction mice compared to control mice. In contrast, 1H-MRS analysis using an isoflurane titration protocol in which mice were sequentially exposed to 1.6%, 2%, then finally 3% isoflurane, revealed significantly higher hippocampal lactate levels in Ldha transgenic mice compared to controls. In addition, significantly fewer mice were required to detect differences in lactate levels using the isoflurane titration protocol compared to conventional isoflurane-induced anesthesia. Our findings highlight the importance of controlling for the effects of anesthesia when detecting changes in hippocampal lactate levels in vivo and offer a novel protocol for enhanced cerebral lactate detection.

Heart Failure With Reduced Ejection Fraction Polypill Implementation Strategy in India: A Convergent Parallel Mixed Methods Study.

A polypill-based implementation strategy has been proposed to increase rates of guideline-directed medical therapy (GDMT) in patients with heart failure with reduced ejection fraction. This has the potential to improve mortality and morbidity in India and undertreated populations globally. We conducted a convergent parallel mixed methods study integrating quantitative data from stakeholder surveys using modified implementation science outcome measures and qualitative data from key informant in-depth interviews. Our objective was to explore physician, nurse, pharmacist, and patient perspectives on a HFrEF polypill implementation strategy in India from January 2021 to April 2021. Quantitative and qualitative data were integrated to develop an Implementation Research Logic Model. Among 69 respondents to the stakeholder survey, there was moderate acceptability (mean [SD] 3.8 [1.0]), appropriateness (3.6 [1.0]), and feasibility (3.7 [1.0]) of HFrEF polypill implementation strategy. Participants in the key-informant in-depth interviews (n = 20) highlighted numerous relative advantages of the HFrEF polypill innovation including potential to simplify medication regimens and improve patient adherence. Key relative disadvantages elucidated, include concerns about side effects and interruption of multiple GDMT medications due to polypill discontinuation for side effects or hospitalizations. Based on this data, the proposed implementation strategies in the Implementation Research Logic Model include 1) HFrEF polypills, 2) HFrEF polypill initiation, titration, and maintenance protocols, and 3) HFrEF polypill laboratory monitoring protocols for safety which we postulate will lead to desired clinical and implementation outcomes through multiple mechanisms including increased medication adherence to a single pill. This study demonstrates that a HFrEF polypill-based implementation strategy is considered acceptable, feasible, and appropriate among healthcare providers in India. We identified contextually relevant determinants, strategies, mechanism, and outcomes outlined in an Implementation Research Logic Model to inform future research to improve heart failure care in South Asia.

Nurse-led diuretic titration via a point-of-care urinary sodium sensor in patients with acute decompensated heart failure (EASY-HF): A single-centre, randomized, open-label study.

Early evaluation of the natriuretic response is recommended to guide diuretic therapy in acute decompensated heart failure (ADHF). However, its implementation in daily practice is hampered by implementation barriers and increased time constraints. The Readily Available Urinary Sodium Analysis in Patients with Acute Decompensated Heart Failure (EASY-HF) study assessed the feasibility, efficacy and safety of a nurse-led urinary sodium-based diuretic titration protocol with the use of a point-of-care urinary sodium sensor. The EASY-HF study was a single-centre, randomized, open-label study comparing diuretic management at the treating physician's discretion as standard of care (SOC) with a nurse-led natriuresis-guided protocol in patients with ADHF. The LAQUAtwin Sodium Meter (HORIBA) was used as point-of-care sensor to measure urine sodium concentration. The primary endpoint was natriuresis after 48 h. Secondary endpoints included safety profile and user-friendliness of both the protocol and the point-of-care sensor. Sixty patients were randomized towards SOC (n = 30) versus protocolized care (n = 30). The mean age was 80 ± 8 years, 25% were women and median N-terminal pro-B-type natriuretic peptide was 4667 (2667-7709) ng/L. Natriuresis after 48 h was significantly higher in the protocolized versus SOC group (820 ± 279 vs. 657 ± 273 mmol; p = 0.027). Pre-defined safety endpoints were similar among both groups. The sensor-based protocol was evaluated as easy to use by the nursing staff, and preferred over urinary collections. A nurse-led diuretic titration protocol via a point-of-care urinary sodium sensor was feasible, safe and resulted in an increased natriuresis in ADHF compared to SOC.

Protocol-driven approach to guideline-directed medical therapy optimization for heart failure: A real-world application to recovery

The objective of our study was to evaluate the real-world effects of an aggressive, personalized protocol for guideline-directed medical therapy (GDMT) titration in patients with heart failure (HF) with reduced ejection fraction (HFrEF). We conducted a two-center retrospective cohort study. Patients with HFrEF who presented to a HF clinic from January 2020 to December 2022 were placed on a GDMT protocol. 180 patients were included in the study. Mean GDMT score significantly increased from 4.7 to 5.9 (p < 0.001) between initial and final visits. Mean left ventricular ejection fraction (LVEF) significantly increased from 28 % to 33 % (+5 %, p < 0.001). 27 (15.7 %) of the 172 patients with complete New York Heart Association (NYHA) classification data had improvement by at least 1 class, while 2 (1.2 %) patients had worsening NYHA classification. 140 (77.8 %) patients had no unplanned hospitalizations between visits. 21 (11.7 %) patients had an unplanned hospitalization for acute HF during the study period with a mean time from first clinic visit to hospitalization of 183 days (range: 13–821 days). 2 (1.1 %) patients were hospitalized due to GDMT-associated adverse drug events (i.e. hypotension, hyperkalemia). 7 (3.9 %) patients died during the study period, which was lower than the predicted 1-year death rate for our cohort (12.3 %) using the MAGGIC score. In conclusion, an aggressive, personalized protocol for GDMT titration in patients with HFrEF led to significant improvements in LVEF, NYHA classification, hospitalization, and mortality in a real-world setting. This protocol may help serve as a road map to lessen the gap between clinical knowledge and practice surrounding optimization of GDMT and move HFrEF patients toward a path to recovery.

The complications of long time treatment of insulin therapy in type-2 diabetes patients: A Review

Diabetes is a chronic metabolic disease characterized by elevated blood sugar levels. It can also mean long-term problems with the metabolism of carbohydrates caused by insulin deficiency, which leads to hyperglycemia and glycosuria. Weight gain is a common side effect of insulin therapy and other oral antidiabetic medications for diabetic patients, which can hinder their ability to meet strict glycemic targets and potentially reduce treatment success. Insulin-related weight gain is attributed to anabolic effects, appetite increases, and reduced glycosuria. Insulin therapy has been linked to increased cardiovascular risk and mortality in type 2 diabetes patients, as well as inflammation, atherosclerosis, hypertension, dyslipidemia, heart failure, and arrhythmias. Large-scale evaluations suggest that insulin therapy has a poorer short- and long-term safety profile than other anti-T2D therapies. To treat diabetes, a variety of insulin formulations are available, each acting at a distinct pace. Whether they are long-acting, intermediate-acting, or rapid-acting, there are similar adverse effects to be mindful of. Weight gain, injection site responses, and hypoglycemia (low blood glucose) are common side effects of insulin. Lipodystrophy, or improper fat distribution, and limb edema are uncommon adverse effects of insulin. When it comes to insulin introduction, titration, and follow-up treatment, doctors can manage patients with type 2 diabetes mellitus more effectively by employing simple algorithms. Primary care doctors and other medical professionals can manage patients with type 2 diabetes mellitus more easily by using streamlined insulin introduction and titration protocols.

Standard Versus Rapid Inpatient Methadone Titration for Pregnant Patients With Opioid Use Disorder: A Retrospective Cohort Study.

Our study evaluated if rapid inpatient titration of methadone for pregnant patients with opioid use disorder (OUD) improved outcomes without increasing the risk for overdose. This is a retrospective cohort study of pregnant patients admitted for inpatient methadone titration from January 2020 to June 2022. Outcomes were compared between standard versus rapid titration protocols. Standard titration involved an initial methadone dose with additional doses every 6 hours if clinical opiate withdrawal score (COWS) is >9. Rapid titration involved an initial methadone dose with additional doses every 4 hours if COWS is >9. The primary outcome was time required to achieve stable dose. Secondary outcomes included elopement prior to achieving stable dose, methadone-related readmission, opioid overdose, and final dose. There were 97 patients in the standard titration (STP) and 97 patients in the rapid titration (RTP) groups. Demographic characteristics and substance use history did not differ between the 2 groups. Time to stable dose did not differ between the 2 groups (RTP, 5.0 days ±4.0; STP, 4.0 days ±3.0; P = 0.08). Patients in the rapid titration group were less likely to elope from the hospital prior to stabilization (RTP 23.0% vs STP 37.9%, P = 0.03) and had fewer methadone-related readmissions ( P < 0.001). One patient (1.0%) in the RTP group required naloxone treatment while inpatient for concern for overdose, while none did in the STP group ( P = 0.32). There was no difference in median final stable dose between the 2 groups ( P = 0.07). Rapid titration of methadone for pregnant patients with OUD was associated with decreased medical elopement and methadone-related readmission, without increasing the risk for overdose.

Increasing the Volume of Delivered Enteral Feeds Using a Volume-Based Feeding Protocol in a Neuroscience Intensive Care Unit.

Iatrogenic malnutrition is a significant burden to patients, clinicians, and health care systems. Compared with well-nourished patients, underfed patients (those who receive less than 80% of their daily energy requirement) have more adverse outcomes related to nutritional status. Volume-based protocols allow for catch-up titrations, are consistently superior to rate-based protocols, and can be implemented in most settings. This project was conducted in an 8-bed neuroscience intensive care unit in which up to 41% of patients who required enteral feeding were underfed. This quality improvement clinical practice change project used a before-and-after design to evaluate (1) the effect of implementing a volume-based feeding protocol on the delivery of enteral feeds and (2) the effect of a nutrition-based project on staff members' attitudes regarding nutrition in critical care. The effectiveness of a volume-based feeding titration protocol was compared with that of a rate-based feeding protocol for achieving delivery of at least 80% of prescribed nutrition per 24-hour period. Staff members' attitudes were assessed using a survey before and after the project. During 241 enteral feeding days (n = 40 patients), the percentage of delivered enteral feeding volume and the percentage of days patients received at least 80% of the prescribed volume increased after volume-based feeding was implemented. After project implementation, 74 staff members reported increased emphasis on nutrition delivery in their practice and a higher level of agreement that nutrition is a priority when caring for critically ill patients. Using a volume-based feeding protocol with supplemental staff education resulted in improved delivery of prescribed enteral feeding.

Differential impact of sex on regulation of skeletal muscle mitochondrial function and protein homeostasis by hypoxia-inducible factor-1α in normoxia.

Hypoxia-inducible factor (HIF)-1α is continuously synthesized and degraded in normoxia. During hypoxia, HIF1α stabilization restricts cellular/mitochondrial oxygen utilization. Cellular stressors can stabilize HIF1α even during normoxia. However, less is known about HIF1α function(s) and sex-specific effects during normoxia in the basal state. Since skeletal muscle is the largest protein store in mammals and protein homeostasis has high energy demands, we determined HIF1α function at baseline during normoxia in skeletal muscle. Untargeted multiomics data analyses were followed by experimental validation in differentiated murine myotubes with loss/gain of function and skeletal muscle from mice without/with post-natal muscle-specific Hif1a deletion (Hif1amsd). Mitochondrial oxygen consumption studies using substrate, uncoupler, inhibitor, titration protocols; targeted metabolite quantification by gas chromatography-mass spectrometry; and post-mitotic senescence markers using biochemical assays were performed. Multiomics analyses showed enrichment in mitochondrial and cell cycle regulatory pathways in Hif1a deleted cells/tissue. Experimentally, mitochondrial oxidative functions and ATP content were higher with less mitochondrial free radical generation with Hif1a deletion. Deletion of Hif1a also resulted in higher concentrations of TCA cycle intermediates and HIF2α proteins in myotubes. Overall responses to Hif1amsd were similar in male and female mice, but changes in complex II function, maximum respiration, Sirt3 and HIF1β protein expression and muscle fibre diameter were sex-dependent. Adaptive responses to hypoxia are mediated by stabilization of constantly synthesized HIF1α. Despite rapid degradation, the presence of HIF1α during normoxia contributes to lower mitochondrial oxidative efficiency and greater post-mitotic senescence in skeletal muscle. In vivo responses to HIF1α in skeletal muscle were differentially impacted by sex. KEY POINTS: Hypoxia-inducible factor -1α (HIF1α), a critical transcription factor, undergoes continuous synthesis and proteolysis, enabling rapid adaptive responses to hypoxia by reducing mitochondrial oxygen consumption. In mammals, skeletal muscle is the largest protein store which is determined by a balance between protein synthesis and breakdown and is sensitive to mitochondrial oxidative function. To investigate the functional consequences of transient HIF1α expression during normoxia in the basal state, myotubes and skeletal muscle from male and female mice with HIF1α knockout were studied using complementary multiomics, biochemical and metabolite assays. HIF1α knockout altered the electron transport chain, mitochondrial oxidative function, signalling molecules for protein homeostasis, and post-mitotic senescence markers, some of which were differentially impacted by sex. The cost of rapid adaptive responses mediated by HIF1α is lower mitochondrial oxidative efficiency and post-mitotic senescence during normoxia.

Immuno-Hematologic Complexity of ABO-Incompatible Allogeneic HSC Transplantation.

ABO incompatibility is not considered a contraindication for hematopoietic stem cell transplantation (HSCT). Approximately 30% of transplants from related donors and up to 50% of transplants from unrelated donors are ABO incompatible. Immuno-hematologic investigations allow to estimate donor/recipient ABO mismatch and anti-A/B isohemagglutinin (IHA) titration in the pre-HSCT phase. Immediate hemolysis or delayed complications (passenger lymphocyte syndrome and pure red cell aplasia) can occur post HSCT. Some preventive measures take into consideration either decision-making algorithms based on the recipient's IHA titration or clinical protocols for the removal/reduction of IHAs through plasma exchange or immunoadsorption procedures. Product manipulation through red blood cell (RBC) and/or plasma depletion can also be taken into account. Currently, the best approach in the management of ABO-incompatible transplant is not defined in expert consensus documents or with solid evidence. In addition, the methods for IHA titration are not standardized. A transfusion strategy must consider both the donor's and recipient's blood group systems until the RBC engraftment catches on and ABO conversion (forward and reverse typing) is confirmed on two consecutive and independent samples. Therefore, ABO incompatibility in HSCT represents a demanding immuno-hematologic challenge and requires all necessary preventive measures, including the appropriate selection of ABO blood components for transfusion.

Heparin monitoring in ImpellaTM-supported cardiogenic shock patients: the benefit of parallel dual testing

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Fonds Wetenschappelijk Onderzoek Flanders. Background Coagulopathic complications often jeopardize the outcome of critically ill patients in cardiogenic shock (CS) during percutaneous mechanical circulatory support (pMCS). Therefore, meticulous management of heparin therapy is mandatory to ensure appropriate anticoagulation management. In this context, the most convenient and most frequently used test is the activated partial thromboplastin time (APTT). However, the APTT-test can be prolonged or shortened independent of the patient's anticoagulation status due to various confounding factors during critical illness (e.g., sepsis, liver failure, factor XI/XII consumption). Alternatively, the anti-Xa assay directly measures heparin activity. Therefore, to optimize heparin anticoagulation during critical illness, an anti-Xa-guided protocol for heparin titration combined with parallel APTT measurements to assess both bleeding and thrombotic risk has recently been proposed. (1) Purpose We aimed to investigate the correlation between parallel anti-Xa and APTT-measurements in an anti-Xa-guided heparin anticoagulated CS population during left-sided ImpellaTM support. Methods Between April-2017 and June-2022, all CS patients with isolated left-sided ImpellaTM-support (&amp;gt;24h) and heparin anticoagulation monitored by anti-Xa with APTT in parallel were assessed at two high-volume tertiary institutions. Anti-Xa was considered in range between 0.20-0.30IU/mL or 0.31-0.50IU/mL based on the patient’s profile (per center protocol, underlying atrial fibrillation or venous thromboembolism). In range APTT values were considered 40-55s and 55-80s respectively. Correlation between parallel anti-Xa and APTT values was assessed by Pearson correlation. A Pearson correlation coefficient of 0.7 or lower was considered to confer a weak correlation. Results Ninety-three ImpellaTM-patient runs (77 men, 83%) with a median age of 56 years (IQR 46-66) were analyzed. Median support duration was 6 days (IQR 4-12). In total, 2447 parallel anti-Xa/APTT samples were analyzed, showing an overall Pearson correlation coefficient of 0.50 (p&amp;lt;0.001) (Fig. 1A). For samples with in-range anti-Xa (N=1914), matching APPT was shortened in 460 (24%), in-range in 986 (52%) and prolonged in 468 (24%) samples (Fig. 1B). Conclusions We highlight a weak correlation between anti-Xa and APTT in an anti-Xa guided heparin anticoagulated CS population during left-sided ImpellaTM support. Heparin titration based on APTT alone would result in over- (24%) or undertitration (24%) in 48% of our cases, due to confounding factors influencing the APTT-assay in critically ill, exposing these patients to coagulopathy associated problems. Therefore, we strongly advocate a combined (anti-Xa/APTT) testing anticoagulation monitoring strategy to adequately manage the coagulation state of this critically ill population. Future trials will investigate whether this approach will effectively reduce the high coagulopathy associated morbidity and mortality on pMCS.

Assessment of Key Factors Impacting Variability in AAV Vector Genome Titration by Digital PCR.

Recombinant adeno-associated virus (rAAV) has emerged as a prominent vector for in vivo gene therapy, owing to its distinct advantages. Accurate determination of the rAAV genome titer is crucial for ensuring the safe and effective administration of clinical doses. The evolution of the rAAV genome titer assay from quantitative PCR (qPCR) to digital PCR (dPCR) has enhanced accuracy and precision, yet practical challenges persist. This study systematically investigated the impact of various operational factors on genome titration in a single-factor manner, aiming to address potential sources of variability in the quantitative determination process. Our findings revealed that a pretreatment procedure without genome extraction exhibits superior precision compared with titration with genome extraction. Additionally, notable variations in titration results across different brands of dPCR instruments were documented, with relative standard deviation (RSD) reaching 23.47% for AAV5 and 11.57% for AAV8. Notably, optimal operations about DNase I digestion were identified; we thought treatment time exceeding 30 min was necessary, and there was no need for thermal inactivation after digestion. And we highlighted that thermal capsid disruption before serial dilution substantially affected AAV genome titers, causing a greater than ten-fold decrease. Conversely, this study found that additive components of dilution buffer are not significant contributors to titration variations. Furthermore, we found that repeated freeze-thaw cycles significantly compromised AAV genome titers. In conclusion, a comprehensive dPCR titration protocol, incorporating insights from these impact factors, was proposed and successfully tested across multiple serotypes of AAV. The results demonstrate acceptable variations, with the RSD consistently below 5.00% for all tested AAV samples. This study provides valuable insights to reduce variability and improve the reproducibility of AAV genome titration using dPCR.