ABSTRACT
Doublet discrimination is usually based on pulse analysis of light scatter parameters. A combination of two pulse parameters (Area, A; Height, H; or Width, W) can be used to discriminate a pulse originated in a single cell from a pulse originated from cells stuck together. Fluorescence signals can be also used to discriminate aggregates, being essential to identify cells in the G2/M phase from doublets in the G0/G1 phase in cell cycle/DNA applications. The most used method combines FSC-A versus FSC-H, whereas other strategies combine FSC-H versus FSC-W, SSC-H versus SSC-A and SSC-H versus SSC-W. However, when studying activated or proliferating cells, scatter discrimination can be difficult. In this study, we have compared the use of light scattering with fluorescence measurement techniques for successful doublet discrimination for single cells. Effective use of FSC and SSC height, area and width are commonly used to eliminate aggregates. However, fluorescence-based methods using viable DNA stains provide a good compromise between performance and accurate manual gating methods, especially for highly concentrated cell products and pathological specimens. Viable DNA dyes, such as Vybrant™ DyeCycle™ Violet stain or Hoechst 33342, can be used to detect nucleated cells in blood and in bone marrow, or to discriminate cell aggregates and debris based on no-lyse no-wash assays, where scatter degradation is a dominant component of the measured data, which increases with event rate.
Subject(s)
DNA , Flow Cytometry/methods , Cell Cycle , DNA/analysisABSTRACT
Flow cytometry (FCM) enumeration of peripheral blood dendritic cells (PBDCs) is a minimally invasive procedure extremely useful for immunological studies. Numbers of PBDCs vary depending on age, lifestyle, or in pathologies like cancer, leukemia or immunodeficiencies. Conventional methods for PBDC identification by FCM involve red blood cell lysis using either formaldehyde or ammonium chloride-based solutions. This specific procedure has been widely reported to cause a detrimental effect as well as an artifactual detection of target populations. Alternatively, minimal sample perturbation assays that avoid the use of erythrolytic solutions with centrifugation steps and preserve the native cellular state are simpler and more robust than conventional methods. In this study, we aimed to evaluate how conventional FCM assays can alter dendritic cell (DC) counting when compared with minimal sample perturbation protocols, in terms of absolute cell counting, percentage and stain index (SI) of PBDC subsets. We evaluated the use of three different erythrolytic solutions (CyLyse, OptiLyse C, and Pharm Lyse) on a series of n = 20 peripheral blood specimens for conventional and plasmacytoid DCs detection as well as for leukocyte and basophil detection. Our results showed a significant reduction of leukocytes and specifically, of DCs and basophils in terms of absolute number when using erythrolytic solutions. In conclusion, our study shows that PBDC counting is heavily affected when lysing solutions are used, indicating that these stellate-shaped populations appear to be more labile.
Subject(s)
Blood Cells , Erythrocytes , Flow Cytometry/methods , Cell Count , Dendritic CellsABSTRACT
The challenges associated with analyzing rare cells are dependent on a series of factors, which usually require large numbers of cells per sample for successful resolution. Among these is determining the minimum number of total events needed to be acquired as defined by the expected frequency of the target cell population. The choice of markers that identify the target population, as well as the event rate and the number of aborted events/second, will also determine the statistically significant detection of rare cell events. Sample preparation is another important but often overlooked factor in rare cell analysis, and in this study we examine Poisson theory and methods to determine the effect of sample manipulation on rare cell detection. After verifying the applicability of this theory, we have evaluated the potential impact of red cell lysis on rare cell analysis, and how cell rarity can be underestimated or overestimated based on erythrolytic sensitivity or resistance of healthy leukocytes and pathological rare cells.
Subject(s)
Erythrocytes , Leukocytes , Cell Death , Specimen Handling , Flow CytometryABSTRACT
Margareth Ndomondo-Sigonda outlines future challenges for the East African Medicines Regulatory Harmonization initiative.
Subject(s)
Community Medicine/legislation & jurisprudence , Community Medicine/trends , Drug and Narcotic Control/legislation & jurisprudence , Drug and Narcotic Control/trends , Pharmacovigilance , Africa, Eastern/epidemiology , Clinical Trials as Topic/legislation & jurisprudence , Clinical Trials as Topic/standards , Community Medicine/standards , Forecasting , HumansABSTRACT
Hiiti Sillo and colleagues reveal how the East African Community's Medicines Regulatory Harmonization initiative improves access to important medicines in Africa.
Subject(s)
Community Medicine/standards , Drug and Narcotic Control , Health Services Accessibility/standards , Patient Care Planning/standards , Africa, Eastern/epidemiology , Community Medicine/legislation & jurisprudence , Community Medicine/trends , Drug and Narcotic Control/legislation & jurisprudence , Drug and Narcotic Control/trends , Health Services Accessibility/legislation & jurisprudence , Health Services Accessibility/trends , Humans , Patient Care Planning/legislation & jurisprudence , Patient Care Planning/trendsABSTRACT
Jane H. Mashingia and colleagues reveal the progress made to date for the East African Community Medicines Regulatory Harmonization initiative.
Subject(s)
Community Medicine/trends , Drug Approval , Drug and Narcotic Control/trends , Health Services Accessibility/trends , Africa, Eastern/epidemiology , Community Medicine/legislation & jurisprudence , Drug Approval/legislation & jurisprudence , Drug and Narcotic Control/legislation & jurisprudence , Health Services Accessibility/legislation & jurisprudence , Humans , Time FactorsABSTRACT
Access to quality-assured medical products improves health and save lives. However, one third of the world's population lacks timely access to quality-assured medicines while estimates indicate that at least 10% of medicine in low- and middle-income countries (LMICs) are substandard or falsified (SF), costing approximately US$ 31 billion annually. National regulatory authorities are the key government institutions that promote access to quality-assured medicines and combat SF medical products but despite progress, regulatory capacity in LMICs is still insufficient. Continued and increased investment in regulatory system strengthening (RSS) is needed. We have therefore reviewed existing global normative documents and resources and engaged with our networks of global partners and stakeholders to identify three critical challenges being faced by NRAs in LMICs that are limiting access to medical products and impeding detection of and response to SF medicines. The challenges are; implementing value-added regulatory practices that best utilize available resources, a lack of timely access to new, quality medical products, and limited evidence-based data to support post-marketing regulatory actions. To address these challenges, we have identified seven focused strategies; advancing and leveraging convergence and reliance initiatives, institutionalizing sustainability, utilizing risk-based approaches for resource allocation, strengthening registration efficiency and timeliness, strengthening inspection capacity and effectiveness, developing and implementing risk-based post-marketing quality surveillance systems, and strengthening regulatory management of manufacturing variations. These proposed solutions are underpinned by 13 focused recommendations, which we believe, if financed, technically supported and implemented, will lead to stronger health system and as a consequence, positive health outcomes.
Subject(s)
Developing Countries , Drugs, Essential/supply & distribution , Global Health/economics , Health Priorities , Humans , Legislation, DrugABSTRACT
Increased failure rates due to metallic wear particle-associated adverse local tissue reactions (ALTR) is a significant clinical problem in resurfacing and total hip arthroplasty. Retrieved periprosthetic tissue of 53 cases with corrosion/conventional metallic wear particles from 285 revision operations for ALTR was selected for nano-analyses. Three major classes of hip implants associated with ALTR, metal-on-metal hip resurfacing arthroplasty (MoM HRA) and large head total hip replacement (MoM LHTHA) and non-metal-on-metal dual modular neck total hip replacement (Non-MoM DMNTHA) were included. The size, shape, distribution, element composition, and crystal structure of the metal particles were analyzed by conventional histological examination and electron microscopy with analytic tools of 2D X-ray energy dispersive spectrometry and X-ray diffraction. Distinct differences in size, shape, and element composition of the metallic particles were detected in each implant class which correlate with the histological features of severity of ALTR and variability in implant performance.
Subject(s)
Arthroplasty, Replacement, Hip/adverse effects , Hip Prosthesis/adverse effects , Metals/adverse effects , Nanoparticles/adverse effects , Corrosion , Female , Humans , Male , Middle Aged , Nanoparticles/ultrastructure , Prosthesis Design , Prosthesis Failure/adverse effectsABSTRACT
Understanding the geographic role of different species of mosquito vectors and vertebrate hosts in West Nile virus (WNV) transmission cycles can facilitate the development and implementation of targeted surveillance and control measures. This study examined the relationship between WNV-antibody rates in birds and mosquito infection rates and bloodfeeding patterns in east-central Illinois. The earliest detection of WNV-RNA by reverse transcription-polymerase chain reaction TaqMan was from Culex restuans; however, amplification typically coincided with an increase in abundance of Cx. pipiens. Trap type influenced annual estimates of infection rates in Culex species, as well as estimation of blood meal source. Bird species with the highest WNV-antibody rates (i.e., Mourning Doves [Zenaida macroura], Northern Cardinals [Cardinalis cardinalis], American Robins [Turdus migratorius], and House Sparrows [Passer domesticus]) were also the common species found in Culex blood meals. Although antibody rates were not directly proportional to estimated avian abundance, the apparent availability of mammal species did influence proportion of mammal to bird blood meals. Antibody prevalence in the American Robin was lower than expected based on the strong attraction of Culex to American Robins for blood meals. Age-related differences in serology were evident, antibody rates increased in older groups of robins and sparrows, whereas 1st-year hatch and older adults of Mourning Doves and Northern Cardinals had equally high rates of antibody-positive serum samples. The vector and host interactions observed in east-central Illinois (Champaign County), an urban area surrounded by agriculture, are compared to studies in the densely population areas of southern Cook County.
Subject(s)
Bird Diseases/virology , Culex/virology , Insect Vectors/virology , West Nile Fever/transmission , West Nile Fever/veterinary , West Nile virus/isolation & purification , Animals , Antibodies, Viral/blood , Bird Diseases/blood , Birds , Culex/physiology , Feeding Behavior , Humans , Illinois/epidemiology , Insect Vectors/physiology , Population Density , Prevalence , Reverse Transcriptase Polymerase Chain Reaction/veterinary , Seroepidemiologic Studies , Species Specificity , West Nile Fever/epidemiology , West Nile Fever/virologyABSTRACT
Children of young and socially disadvantaged parents are more likely to experience adverse outcomes. In response to this, a unique young families' project in Swansea, UK, was created, which drew together a team of multi-agency professionals, to support people aged 16-24 from 17 weeks of pregnancy throughout 1,001 days of the child's life. The aim of the JIGSO (the Welsh word for Jigsaw) project is for young people to reach their potential as parents and to break the cycle of health and social inequality. This evaluation analysed routinely collected data held by the project from January 2017 to December 2018 exploring health and social outcomes, including smoking and alcohol use in pregnancy, breastfeeding, maternal diet and social services outcomes. Outcomes were compared to local and national averages, where available. Data relating to parenting knowledge and skills were available via records of 10-point Likert scales, one collected at the start of the JIGSO involvement and one around 4-6 months later. Findings showed higher than average levels of breastfeeding initiation and lower smoking and alcohol use in pregnancy. Parents also reported enhanced knowledge and confidence in their child care skills, as well as improved family relationships. Parents with high levels of engagement with JIGSO also appeared to have positive outcomes with Social Services (their child's name was removed from child protection register or their case was closed to social services). This was a post-hoc evaluation, not an intervention study or trial, and thus findings must be interpreted with caution. Despite this, the findings are promising and more prospective research exploring similar services is required.
Subject(s)
Parenting , Parents , Adolescent , Child, Preschool , Female , Humans , Pregnancy , Prospective Studies , Social Support , United KingdomABSTRACT
PURPOSE: COVID-19 brought about unprecedented challenges to healthcare, with nuclear medicine (NM) being no exception. The British Nuclear Medicine Society (BNMS) COVID-19 survey assessed the impact of the first wave of pandemic on NM services in the UK. With COVID-19 resurge compounded by seasonal winter pressures, we reflect and share lessons learnt from the first wave of pandemic to guide future strategy. METHODS: A questionnaire consisting of 34 questions was sent out to all BNMS members over 2 weeks in May 2020, to evaluate the impact of 'lockdown'. RESULTS: One hundred thirty-eight members (92 sites) from a multidisciplinary background responded. There was a 65% reduction across all services; 97.6% of respondents reported some reduction in diagnostic procedures and 71.3% reduction in therapies; 85% worked with a reduced workforce. The North East of England, Greater London and South East and Wessex were most affected by staff absences. The North East reported the highest number of COVID-19 positive staff; London reported the greatest lack of testing. The reported time required to clear the backlog was 1-12 months. Seventy-one percent of participants used BNMS COVID-19 guidance. CONCLUSION: The first wave caused a major disruption of NM service delivery and impacted on the workforce. The departmental strategies should tailor services to evolving local and regional differences in prevalence of COVID-19. A blanket shutdown of services with a 'one size fits all' strategy would likely have a severe impact on future delivery of NM and health services in general. Timely testing of staff and patients remains of paramount importance.
Subject(s)
COVID-19/epidemiology , Nuclear Medicine/statistics & numerical data , Humans , Pandemics/prevention & control , Pandemics/statistics & numerical data , Personal Protective Equipment/supply & distribution , United Kingdom/epidemiology , Workforce/statistics & numerical dataABSTRACT
National regulatory authorities (NRAs) are the gatekeepers of the supply chain of medical products, and they have a mandate to ensure the quality, safety and efficacy of medicines, vaccines, blood, and blood products, medical devices, including diagnostics and traditional, or herbal medicines. However, the majority of the world's regulators are still struggling to reach a level of maturity, whereby they have a stable, well-functioning and integrated regulatory system. The World Health Organization (WHO) has developed a Global Benchmarking Tool (GBT) as part of its five-step capacity building program to assist NRAs, using the tool, they can benchmark their own strengths and areas of weakness, and then engage in a formal benchmarking process together with WHO and international experts in order to formulate an effective and workable institutional development plan. The GBT is comprehensive across the entire product life cycle and allows benchmarking to be customized to the needs of the NRA. It has evolved from decades of experience using a variety of benchmarking tools, within WHO and other stakeholder organizations. By the end of December 2019, 26 countries had undergone formal benchmarking, and a further 54 countries had used the GBT to conduct self-benchmarking exercises assisted by WHO.
ABSTRACT
[This corrects the article DOI: 10.18632/oncotarget.12497.].
ABSTRACT
BACKGROUND: A 40-year-old man presented to an emergency department with slurred speech, diplopia and agitation several hours after cocaine use. His level of consciousness rapidly dropped in the hours following presentation. INVESTIGATIONS: Physical examination, CT scan of the head, cerebral angiography. DIAGNOSIS: Cocaine-induced basilar artery thrombosis. MANAGEMENT: Intra-arterial thombolysis, mechanical clot aspiration, intravenous abciximab, neurointensive care support, rehabilitation at a specialist stroke unit.
Subject(s)
Basilar Artery/pathology , Cocaine/adverse effects , Stroke/chemically induced , Thrombosis/chemically induced , Vasoconstrictor Agents/adverse effects , Adult , Brain Infarction/complications , Cerebellar Diseases/complications , Cerebral Angiography , Fibrinolytic Agents/therapeutic use , Humans , Male , Stroke/pathology , Stroke/physiopathology , Thrombolytic Therapy , Thrombosis/pathology , Thrombosis/physiopathology , Tomography, X-Ray ComputedABSTRACT
The concept of integrated care has been advocated for many years to address some of the challenges faced by the NHS. This report examines the experiences of respiratory healthcare specialists working in an integrated role. Twelve qualitative telephone interviews were undertaken with a range of integrated respiratory specialists and their teams working in both hospitals and the community. A descriptive and thematic approach to data analysis was adopted. Participants were very enthusiastic about their roles and saw themselves as ambassadors for this new way of working. Several key themes were identified from the analysis which participants identified as barriers or enablers to the successful undertaking of an integrated respiratory specialist role. These included the participants' previous work experience and background, the range of multi-disciplinary expertise within or needed for the team, the structure of the team leadership and the measurement of outcomes to evaluate the team. Participants identified the need for clear job descriptions and roles, shared training and standards and appropriate outcome evaluation. More research is needed to understand how these new ways of working are developing and how they can be evaluated.
ABSTRACT
Distortions of the normal bi-concave disc shape for red blood cells (RBCs) appear in a number of pathologies resulting from defects in cell membrane skeletal architecture, erythrocyte ageing, and mechanical damage. We present here the potential of acoustic cytometry for developing new approaches to light-scattering based evaluation of red blood cell disorders and of the effects of storage and ageing on changes or damage to RBCs membranes. These approaches could be used to immediately evaluate the quality of erythrocytes prior to blood donation and following transfusion. They could also be applied to studying RBC health in diseases and other pathologies, such as artificial heart valve hemolysis, thermal damage or osmotic fragility. Abnormal distributions of erythrocytes can typically be detected after just 30 to 45 seconds of acquisition time using 1-2 µL starting blood volumes.
Subject(s)
Erythrocytes , Flow Cytometry/methods , Sound , Annexin A5 , Blood Preservation/methods , Erythrocyte Aging , Erythrocyte Deformability , Erythrocyte Indices , Erythrocyte Membrane/ultrastructure , Erythrocyte Transfusion , Erythrocytes/ultrastructure , Flow Cytometry/instrumentation , Fluorescent Dyes , Hemolysis , Humans , Hydrodynamics , Light , Membrane Lipids/blood , Phosphatidylserines/blood , Scattering, Radiation , Spherocytes/ultrastructure , Spherocytosis, Hereditary/bloodABSTRACT
With the aim to detect candidate malignant primitive progenitor populations, we modified an original alkaline phosphatase (ALP) stem cell detection method based on the identification of alkaline phosphatase fluorescent cells in combination with flow cytometry immunophenotyping. Over a period of one year, we have been using this technique to study its activity in patients with leukemia and lymphoma, showing that changes in the alkaline phosphatase levels can be used to detect rare populations of highly refractory malignant cells. By screening different blood cancers, we have observed that this activity is not always restricted to CD34+ leukemic cells, and can be overexpressed in CD34 negative leukemia. We have verified that this method gives accurate and reproducible measurements and our preliminary results suggest that CD34+/ALPhigh cells appear to sustain leukemogenesis over time.
Subject(s)
Alkaline Phosphatase/metabolism , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Enzyme Assays/methods , Neoplastic Stem Cells/enzymology , Precursor Cell Lymphoblastic Leukemia-Lymphoma/diagnosis , Adult , Aged , Antigens, CD34/metabolism , Antigens, Neoplasm/metabolism , Biomarkers, Tumor/metabolism , Bone Marrow/enzymology , Bone Marrow/pathology , Cell Transformation, Neoplastic/metabolism , Child , Drug Resistance, Neoplasm , Early Detection of Cancer/methods , Enzyme Assays/instrumentation , Female , Flow Cytometry/instrumentation , Flow Cytometry/methods , Gene Rearrangement , Histone-Lysine N-Methyltransferase/genetics , Humans , Immunophenotyping/instrumentation , Immunophenotyping/methods , Male , Myeloid-Lymphoid Leukemia Protein/genetics , Neoplasm, Residual , Precursor Cell Lymphoblastic Leukemia-Lymphoma/drug therapy , Precursor Cell Lymphoblastic Leukemia-Lymphoma/enzymology , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , Proto-Oncogene Proteins c-myc/genetics , Reproducibility of ResultsABSTRACT
INTRODUCTION: Studies are needed to understand the role of CD34 expressing cells with regard to efficient engraftment, especially in the adjuvant treatment of cancer. MATERIALS AND METHODS: In this study we have used a modified method in our laboratory for routinely counting CD34+ cells. Unlysed whole blood samples were stained with the DNA-selective and cell membrane-permeant Vibrant DyeCycle Violet stain. RESULTS: CD34+ cells exhibit a consistent and differential Vybrant Dye Cycle Violet staining pattern. Based on their different DCV intensity, we classified these subpopulations as CD34+/DCV(high) and CD34+/DCV(low) cells. In general, DCV(high) cells are about 12-times brighter than DCV(low) cells. CONCLUSION: DCV staining may be used to discriminate subsets of CD34+ cells similarly to other methods which have previously defined different functional properties that can be related to the characterization, resolution, and purification of primitive hematopoietic stem cells in combination with specific useful markers for multicolor flow cytometric measurements.