How do we leverage data through replication and warehousing to meet blood collection and transfusion service needs.

BACKGROUND: With the widespread adoption of Blood Establishment Computer Systems and other Blood Collection and Transfusion Service (BCTS) clinical information systems (CIS), electronic blood donor, product, and patient data are now routinely required for clinical, regulatory, operational, and quality needs. That data are often not readily accessible for such secondary use within CIS databases, particularly for applications with significant data availability requirements such as machine learning and artificial intelligence. Data replication provides one avenue by which CIS data can be made more readily available. STUDY DESIGN AND METHODS: Members of the AABB's Information Systems Committee along with institutional information technology colleagues provided a multi-institutional viewpoint on data replication through the lens of BCTS specific use cases. Case studies of informatics offerings leveraging such technologies were also elicited. RESULTS: Six distinct use cases describe the potential role of data replication including the creation of data warehouses for frontline laboratory staff. Specific BCTS examples for each use case are presented to highlight the value of data replication, including visualization of critical inventory (O red blood cells, HLA-compatible platelets) and utilization analytics for patient blood management. Two case studies describe the approach to implement such technologies to (1) optimize staffing via laboratory workload reporting and (2) improve access to blood via antigen-negative blood product location services. DISCUSSION: Data replication and warehousing can empower BCTS analytic offerings not otherwise natively available through one's CIS to improve patient care and laboratory operations.

On return to school: disorder symptoms, stress at home and evaluations of school COVID-19 measures.

Background: Social restrictions during the COVID-19 pandemic had a major impact on the mental health of children. Yet, analyses on the mental health of younger children in the later course of the pandemic are scarce. The present study assessed 8- to 11-year-olds' internalising disorder symptoms during the last three weeks, from the third week of February through to the first week of March, of the 2021 national lockdown.Method: One hundred and forty-five pupils, including a subset of keyworkers' children who had face-toface schooling, completed the validated Revised Child Anxiety and Depression scales, items on COVID-related stress at home, and evaluations of home-learning and school's measures for reopening.Results: Symptoms increased with age in months and/or number of siblings. Girls reported more symptoms and home stress than boys did. Pupils who had face-to-face schooling were more satisfied with school measures and less satisfied with home learning compared with those who only had home learning. Hierarchical regression analyses corroborated the contributions of sociodemographic characteristics and found that home stress and school measures evaluations were associated most with major depression, generalised anxiety, and social phobia.Conclusion: Findings can contribute to mental health practice by promoting school communications and family and educator awareness of stressors, vulnerabilities and symptoms to boost pupils' readiness for school returns.

A Case of Fatal Clostridium perfringens Sepsis with Massive Hemolysis in the Setting of a Coincidental Platelet Transfusion.

A 62-year-old woman with acute myeloid leukemia (AML) died of shock and massive hemolysis shortly after receiving two platelet transfusions at a routine clinic visit. Subsequent investigation into what was initially believed to be an acute hemolytic transfusion reaction secondary to platelet transfusions revealed that the patient died of Clostridium perfringens sepsis leading to massive hemolysis. Further investigation ruled out bacterially-contaminated platelets since a patient blood sample from 2 days prior had Clostridium species. The unusual findings and management considerations for this oncology patient are reviewed and compared with previously reported cases of C. perfringens transfusion-transmitted infections. Oncology patients may be especially susceptible to unusual presentations involving unusual pathogens.

Integrating Somatic and Germline Next-Generation Sequencing Into Routine Clinical Oncology Practice.

Next-generation sequencing (NGS) is rapidly expanding into routine oncology practice. Genetic variations in both the cancer and inherited genomes are informative for hereditary cancer risk, prognosis, and treatment strategies. Herein, we focus on the clinical perspective of integrating NGS results into patient care to assist with therapeutic decision making. Five key considerations are addressed for operationalization of NGS testing and application of results to patient care as follows: (1) NGS test ordering and workflow design; (2) result reporting, curation, and storage; (3) clinical consultation services that provide test interpretations and identify opportunities for molecularly guided therapy; (4) presentation of genetic information within the electronic health record; and (5) education of providers and patients. Several of these key considerations center on informatics tools that support NGS test ordering and referencing back to the results for therapeutic purposes. Clinical decision support tools embedded within the electronic health record can assist with NGS test utilization and identifying opportunities for targeted therapy including clinical trial eligibility. Challenges for project and change management in operationalizing NGS-supported, evidence-based patient care in the context of current information technology systems with appropriate clinical data standards are discussed, and solutions for overcoming barriers are provided.

Sex-biased response to and brain cell infection by SARS-CoV-2 in a highly susceptible human ACE2 transgenic model

The COVID-19 pandemic is caused by SARS-CoV-2 infection. Human angiotensin-converting enzyme II (hACE2) has been identified as the receptor enabling SARS-CoV-2 host entry. To establish a mouse model for COVID-19, we generated transgenic mouse lines using the (HS4)2-pCAG-hACE2-HA-(HS4)2 transgene cassette, which expresses HA-tagged hACE2 under control of the CAG promoter and is flanked by HS4 insulators. Expression levels of the hACE2 transgene are respectively higher in lung, brain and kidney of our CAG-hACE2 transgenic mice and relatively lower in duodenum, heart and liver. The CAG-hACE2 mice are highly susceptibility to SARS-CoV-2 infection, with 100 PFU of SARS-CoV-2 being sufficient to induce 87.5% mortality at 9 days post-infection and resulting in a sole (female) survivor. Mortality was 100% at the higher titer of 1000 PFU. At lower viral titers, we also found that female mice exposed to SARS-CoV-2 infection suffered much less weight loss than male mice, implying sex-biased responses to SARS-CoV-2 infection. We subjected neuronal cultures to SARS-CoV-2 pseudovirus infection to ascertain the susceptibilities of neurons and astrocytes. Moreover, we observed that expression of SARS-CoV-2 Spike protein alters the synaptic responses of cultured neurons. Our transgenic mice may serve as a model for severe COVID-19 and sex-biased responses to SARS-CoV-2 infection, aiding in the development of vaccines and therapeutic treatments for this disease.

Reactive oxygen species modulate macrophage immunosuppressive phenotype through the up-regulation of PD-L1.

The combination of immune checkpoint blockade with chemotherapy is currently under investigation as a promising strategy for the treatment of triple negative breast cancer (TNBC). Tumor-associated macrophages (TAMs) are the most prominent component of the breast cancer microenvironment because they influence tumor progression and the response to therapies. Here we show that macrophages acquire an immunosuppressive phenotype and increase the expression of programmed death ligand-1 (PD-L1) when treated with reactive oxygen species (ROS) inducers such as the glutathione synthesis inhibitor, buthionine sulphoximine (BSO), and paclitaxel. Mechanistically, these agents cause accumulation of ROS that in turn activate NF-κB signaling to promote PD-L1 transcription and the release of immunosuppressive chemokines. Systemic in vivo administration of paclitaxel promotes PD-L1 accumulation on the surface of TAMS in a mouse model of TNBC, consistent with in vitro results. Combinatorial treatment with paclitaxel and an anti-mouse PD-L1 blocking antibody significantly improved the therapeutic efficacy of paclitaxel by reducing tumor burden and increasing the number of tumor-associated cytotoxic T cells. Our results provide a strong rationale for the use of anti-PD-L1 blockade in the treatment of TNBC patients. Furthermore, interrogation of chemotherapy-induced PD-L1 expression in TAMs is warranted to define appropriate patient selection in the use of PD-L1 blockade.

AhR controls redox homeostasis and shapes the tumor microenvironment in BRCA1-associated breast cancer.

Cancer cells have higher reactive oxygen species (ROS) than normal cells, due to genetic and metabolic alterations. An emerging scenario is that cancer cells increase ROS to activate protumorigenic signaling while activating antioxidant pathways to maintain redox homeostasis. Here we show that, in basal-like and BRCA1-related breast cancer (BC), ROS levels correlate with the expression and activity of the transcription factor aryl hydrocarbon receptor (AhR). Mechanistically, ROS triggers AhR nuclear accumulation and activation to promote the transcription of both antioxidant enzymes and the epidermal growth factor receptor (EGFR) ligand, amphiregulin (AREG). In a mouse model of BRCA1-related BC, cancer-associated AhR and AREG control tumor growth and production of chemokines to attract monocytes and activate proangiogenic function of macrophages in the tumor microenvironment. Interestingly, the expression of these chemokines as well as infiltration of monocyte-lineage cells (monocyte and macrophages) positively correlated with ROS levels in basal-like BC. These data support the existence of a coordinated link between cancer-intrinsic ROS regulation and the features of tumor microenvironment. Therapeutically, chemical inhibition of AhR activity sensitizes human BC models to Erlotinib, a selective EGFR tyrosine kinase inhibitor, suggesting a promising combinatorial anticancer effect of AhR and EGFR pathway inhibition. Thus, AhR represents an attractive target to inhibit redox homeostasis and modulate the tumor promoting microenvironment of basal-like and BRCA1-associated BC.

Thirty-five years of CD4 T-cell counting in HIV infection: From flow cytometry in the lab to point-of-care testing in the field.

CD4 T-cell counting was introduced in clinical laboratories shortly after the discovery of the human immune deficiency virus (HIV) in the early eighties. In western clinical laboratories, improvements in the CD4 T-cell counting methods were mainly driven by progress in the field of flow cytometry and immunology. In contrast, the development of dedicated CD4 T-cell counting technologies were needs driven. When antiretroviral treatment (ART) was made available on a large scale by international Acquired Immune Deficiency Syndrome (AIDS) relief programs to HIV+ patients living in low income countries in 2003, there was a distinct need for simplified and affordable CD4 T-cell counting technologies. The first decade of 2000, several compact flow cytometers appeared on the market, mainly to the benefit of low income countries with limited resources. More recently, however, portable point-of-care (POC) CD4 T-cell counting devices have been developed especially to improve access to affordable monitoring of HIV+ patients in low income countries. The accuracy of these POC instruments is not yet very well documented as many are still under development and clinical validation but preliminary evidence is encouraging. The new HIV treatment guidelines released by the World Health Organization in 2016 give CD4 T-cell counting a less central role in the management of HIV infection. It is, therefore, to be expected that CD4 T-cell counting will be phased out as a tool to assess eligibility of HIV+ patients for ART in the future. However, CD4 T-cell counting will remain a valuable tool for directing treatment against opportunistic infections. © 2016 International Clinical Cytometry Society.

Endoscopic duodenal-jejunal bypass liner rapidly improves plasma parameters of nonalcoholic fatty liver disease.

Bariatric surgery reduces nonalcoholic fatty liver disease (NAFLD). We investigated the effects of duodenal-jejunal bypass liner (DJBL), nonsurgical bariatric device, on plasma parameters of NAFLD. Seventeen obese subjects with type 2 diabetes received the DJBL for 24 weeks. Before, during, and after DJBL implantation, we determined plasma levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), γ-glutamyltransferase (γ-GT), albumin, caspase-cleaved cytokeratin-18 (CK-18), and liver fatty acid-binding protein (L-FABP). At baseline, subjects had increased levels of AST (35 ± 4 IU/L), ALT (54 ± 5 IU/L), and γ-GT (66 ± 14 IU/L), compared with healthy individuals; subjects' mean concentrations of caspase-cleaved CK-18 and L-FABP were 214.4 ± 35.6 U/L and 29.3 ± 2.6 ng/mL, respectively. Three months after implantation of DJBL, all NAFLD-related parameters had decreased from baseline (AST, 28 ± 3 IU/L; ALT, 32 ± 2 IU/L; γ-GT, 44 ±7 IU/L; caspase-cleaved CK-18, 140.6 ± 16.3U/L; and L-FABP, 18.2 ± 1.5 ng/mL; all P < .05). After 6 months, levels of ALT and γ-GT had further decreased (ALT, 28 ± 2 IU/L and γ-GT, 35 ± 5 IU/L), whereas levels of AST, caspase-cleaved CK-18, and L-FABP had stabilized (P = not significant). Six months after DJBLs were removed, levels of ALT (37 ± 3 IU/L), γ-GT (42 ± 5 IU/L), and caspase-cleaved CK-18 (124.5 ± 12.5U/L) were still reduced (P < .05), whereas AST and L-FABP had returned to near baseline levels (P = not significant). Therefore, in obese subjects, DJBL reduces plasma parameters of NAFLD. ClinicalTrials.gov, Number: NCT00985114.

Beneficial effects of sitostanol on the attenuated immune function in asthma patients: results of an in vitro approach.

BACKGROUND: In vitro and animal studies have suggested that plant sterols and stanols increase cytokine production by T-helper-1 cells. This may be beneficial for patient groups characterized by a T-helper-2 dominant immune response, e.g. asthma patients. (1) to evaluate whether sitostanol induces a T-helper-1 shift in peripheral blood mononuclear cells (PBMCs) from asthma patients, and (2) to unravel the role of regulatory T-cells in this respect. METHODOLOGY/PRINCIPAL FINDINGS: PBMCs from 10 asthma patients and 10 healthy subjects were isolated and incubated with 1.2 µM sitostanol, while stimulated with 5 µg/ml PHA. Similar amounts of cholesterol were used to determine whether effects were specific for plant stanols or for sterols in general. Changes in cytokine production were measured using antibody arrays and ELISAs. Changes in regulatory T-cell population size were measured by flow cytometry, using intracellular Foxp3 staining. Sitostanol increased production of IFNγ by 6.5% and IL-2 by 6.0% compared to cholesterol (p<0.01). No changes in IL-4 and IL-13 were found. Interestingly, this effect was only present in PBMCs from asthma patients. The number of Foxp3+ cells tended to increase and their activity, measured by IL-10 production, increased after sitostanol treatment in PBMCs from asthma patients compared to controls by 32.3% (p = 0.077) and 13.3% (p<0.05), respectively. CONCLUSIONS/SIGNIFICANCE: Altogether, the sitostanol-induced Thelper-1 shift in PBMCs from asthma patients and the stimulating effects of sitostanol on Treg cell numbers and activity indicate a possible novel approach for plant stanol ester enriched functional foods in the amelioration of asthmatic symptoms. Functional effects, however, require further evaluation.

Complex febrile seizures followed by complete recovery in an infant with high-titer 2009 pandemic influenza A (H1N1) virus infection.

We describe a 2009 H1N1 virus infection with a high viral load in a previously healthy infant who presented with complex febrile seizures and improved on oseltamivir without neurologic sequelae. Febrile seizures may be a complication in young children experiencing infection with high viral loads of 2009 H1N1 influenza virus.

Implementation of a closed-loop reporting system for critical values and clinical communication in compliance with goals of the joint commission.

BACKGROUND: Current practices of reporting critical laboratory values make it challenging to measure and assess the timeliness of receipt by the treating physician as required by The Joint Commission's 2008 National Patient Safety Goals. METHODS: A multidisciplinary team of laboratorians, clinicians, and information technology experts developed an electronic ALERTS system that reports critical values via the laboratory and hospital information systems to alphanumeric pagers of clinicians and ensures failsafe notification, instant documentation, automatic tracking, escalation, and reporting of critical value alerts. A method for automated acknowledgment of message receipt was incorporated into the system design. RESULTS: The ALERTS system has been applied to inpatients and eliminated approximately 9000 phone calls a year made by medical technologists. Although a small number of phone calls were still made as a result of pages not acknowledged by clinicians within 10 min, they were made by telephone operators, who either contacted the same physician who was initially paged by the automated system or identified and contacted alternate physicians or the patient's nurse. Overall, documentation of physician acknowledgment of receipt in the electronic medical record increased to 95% of critical values over 9 months, while the median time decreased to <3 min. CONCLUSIONS: We improved laboratory efficiency and physician communication by developing an electronic system for reporting of critical values that is in compliance with The Joint Commission's goals.

Affordable CD4 T-cell enumeration for resource-limited regions: a status report for 2008.

BACKGROUND: The global struggle with human immunodeficiency virus (HIV) and the battle to develop affordable CD4 T-cell counting technology are both unfulfilled goals in 2008. The need for such instrumentation is more critical now as implementation of antiretroviral therapy (ART) is in progress in many resource limited regions. Major scaling-up efforts in rural situations are difficult to implement without laboratory infrastructure. CD4 T-cell counting is especially critical when trying to reach individuals with HIV to have them enrolled in ART as soon as they qualify for treatment based on CD4 count. METHOD: This review covers both the chronological evolution and the scientific milestones of technological development of affordable immunophenotyping. It is more focused on flow cytometry but does consider the potential contribution by digital image cytometry. RESULTS: Thus far flow cytometry offered only modest progress toward affordable immunophenotyping. A list with desirable features is offered for side by side comparison. Digital image cytometry has yet to show its enormous affordable market potential. CONCLUSIONS: It is possible to develop truly affordable, portable flow cytometry but it is not here yet. There are some hopeful signs as there are innovative and practical technical components appearing at regular intervals. However, so far the technical breakthroughs have been fragmented efforts without any attempts to consider intercorporate collaboration to optimize critical mass and synergy. The smaller players in the industry have made some progress toward meeting the monumental needs in Africa and Asia. Digital image cytometry may well be the ultimate winner in the affordable technology race.

Rapid detection and identification of the bacterium Pantoea stewartii in maize by TaqMan real-time PCR assay targeting the cpsD gene.

AIMS: The development and evaluation of a sensitive and specific TaqMan real-time polymerase chain reaction (PCR) for the detection and identification of Pantoea stewartii on maize. METHODS AND RESULTS: A TaqMan-based real-time PCR assay targeting the cpsD gene enabling specific detection of P. stewartii in maize leaves and seeds was developed. Under optimal conditions, the selected primers and probe were specific for the detection of all 14 reference P. stewartii strains by real-time PCR. The 32 non-Panteoa and eight other Pantoea strains tested negative. The TaqMan PCR assay detected 1 pg of purified DNA and 10(4)P. stewartii colony forming units per millilitre (10 cells per reaction) in pure cultures consisting of 92.0% intact (viable) cells. Direct processing of leaf lesions and seeds by the real-time PCR detected 10 and 50 P. stewartii cells per reaction respectively. TaqMan real-time PCR results were validated by dilution plating of macerates and PCR-based subcloning followed by DNA sequencing. CONCLUSIONS: The real-time PCR assay described is a rapid, reliable and more sensitive tool for the detection of P. stewartii. SIGNIFICANCE AND IMPACT OF THE STUDY: This real-time PCR assay would avoid false-negative results and reduce the time required for certifying maize seed shipments.

Selection of lymphocyte gating protocol has an impact on the level of reliability of T-cell subsets in aging specimens.

BACKGROUND: In the past decade, human immunodeficiency virus (HIV) lymphocyte immunophenotyping has evolved significantly. New fluorochromes, new multicolor reagents, enhanced instruments, and the capacity to provide absolute cell counts using the single-platform technique have all contributed to the reliability of T-cell subset measurements. In this study, four gating protocols were evaluated to select the most robust method for T-cell subset enumeration. METHODS: Peripheral blood specimens from 21 HIV(+) and 20 HIV(-) individuals were monitored up to 96 h. Aliquots of specimens were stored at room temperature and analyzed at 6 (baseline), 48, 72, and 96 h. Aliquots were stained with CD45-fluorescein isothiocyanate (FITC)/CD3PC5/CD4RD1/CD8ECD. Data analysis was performed with all four gating protocols. RESULTS: Only with fresh blood did all protocols provide similar results. From samples that were 48 h old, the choice of gating strategy had a dramatic impact on immunophenotyping results. The largest deviations from baseline values occurred at 96 h and gating protocols that included dual light scatter gates provided the greatest shift of T-cell subset values over time. The gating protocols that were based exclusively on cell lineage-specific gates gave the most robust T-cell values up to 96 h. CONCLUSION: By selecting the appropriate gating protocol, the temporal integrity of specimens can be extended up to 4 days.

Enumeration of absolute cell counts using immunophenotypic techniques.

Absolute counting of cells or cell subsets has a number of significant clinical applications: monitoring the disease status of HIV-infected patients, enumerating residual white blood cells in leukoreduced blood products, and assessing immunodeficiency in a variety of situations. The single-platform method (flow cytometry alone) has emerged as the method of choice for absolute cell enumeration. This technology counts only the cells of interest in a precisely determined blood volume. Exact cell identification is accomplished by a logical electronic gating algorithm capable of identifying lineage-specific immunofluorescent markers. Exclusion of unwanted cells is automatic. This extensive and detailed unit presents protocols for both volumetric and flow-rate determination of residual white blood cells and of leukocyte subsets.

Overview and application of suspension array technology.

A cell-based solid-phase immunoassay usually is called immunophenotyping when performed on a flow cytometer. Using the same principles, there is a new flow cytometric application available with the suspension array technology. The significant difference is that the immunologic reaction does not occur on the surface of leukocytes, but rather on the surface of plastic fluorospheres. This is a multiplexed solid-phase flow cytometry-based technology with some clinically relevant potential.