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OBJECTIVES: Clinical laboratory results are required for critical medical decisions, underscoring the importance of quality results. As part of total quality management, external quality assessment (EQA) is a vital component to ensure laboratory accuracy. The goal of this survey was to evaluate the current status of global laboratory quality systems and assess the need for implementation, expansion, or harmonization of EQA programs (EQAP) for Clinical Chemistry and Laboratory Medicine. METHODS: The International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) Task Force on Global Laboratory Quality (TF-GLQ) conducted a survey of IFCC full and affiliate members (n=110) on laboratory quality practice. A total of 41 (37.3%) countries representing all IFCC regions except North America provided responses about EQA availability and practices. RESULTS: All 41 countries perform EQA, 38 reported that their laboratories had EQA policies and procedures, and 39 further act/evaluate unacceptable EQA results. 39 countries indicated they have international and/or national EQAP and 30 use alternative performance assessments. EQA frequency varied among countries. Generally, an EQAP provided the EQA materials (40/41) with four countries indicating that they did not have an EQAP in their country. CONCLUSIONS: Globally, most laboratories participate in an EQAP and have defined quality procedures for EQA. There remain gaps in EQA material availability and implementation of EQA as a part of a total laboratory quality system. This survey highlights the need for education, training, and harmonization and will guide efforts of the IFCC TF-GLQ in identifying areas for enhancing global laboratory quality practices.
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Química Clínica , Laboratorios , Humanos , Encuestas y Cuestionarios , Gestión de la Calidad Total , Garantía de la Calidad de Atención de SaludRESUMEN
OBJECTIVES: The trueness and precision of clinical laboratory results are ensured through total quality management systems (TQM), which primarily include internal quality control (IQC) practices. However, quality practices vary globally. To understand the current global state of IQC practice and IQC management in relation to TQM the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) Task Force on Global Laboratory Quality (TF-GLQ) conducted a survey of IFCC member countries on IQC practices and management. METHODS: The survey included 16 questions regarding IQC and laboratory TQM practices and was distributed to IFCC full and affiliate member countries (n=110). A total of 46 (41.8â¯%) responses were received from all regions except North America. RESULTS: Of the responding countries, 78.3â¯% (n=36) had legislative regulations or accreditation requirements governing medical laboratory quality standards. However, implementation was not mandatory in 46.7â¯% (n=21) of responding countries. IQC practices varied considerably with 57.1â¯% (n=28) of respondents indicating that they run 2 levels of IQC, 66.7â¯% (n=24) indicating they run IQC every 24â¯h and 66.7â¯% (n=28) using assay manufacturer IQC material sources. Only 29.3â¯% (n=12) of respondents indicated that every medical laboratory in their country has written IQC policies and procedures. By contrast, 97.6â¯% (n=40) of responding countries indicated they take corrective action and result remediation in the event of IQC failure. CONCLUSIONS: The variability in TQM and IQC practices highlights the need for more formal programs and education to standardize and improve TQM in medical laboratories.
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Laboratorios , Gestión de la Calidad Total , Humanos , Control de Calidad , Encuestas y CuestionariosRESUMEN
Assembly of signaling molecules into micrometer-sized clusters is driven by multivalent protein-protein interactions, such as those found within the nephrin-Nck (Nck1 or Nck2) complex. Phosphorylation on multiple tyrosine residues within the tail of the nephrin transmembrane receptor induces recruitment of the cytoplasmic adaptor protein Nck, which binds via its triple SH3 domains to various effectors, leading to actin assembly. The physiological consequences of nephrin clustering are not well understood. Here, we demonstrate that nephrin phosphorylation regulates the formation of membrane clusters in podocytes. We also reveal a connection between clustering and endocytosis, which appears to be driven by threshold levels of nephrin tyrosine phosphorylation and Nck SH3 domain signaling. Finally, we expose an in vivo correlation between transient changes in nephrin tyrosine phosphorylation, nephrin localization and integrity of the glomerular filtration barrier during podocyte injury. Altogether, our results suggest that nephrin phosphorylation determines the composition of effector proteins within clusters to dynamically regulate nephrin turnover and podocyte health.
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Podocitos , Tirosina , Análisis por Conglomerados , Endocitosis , Proteínas de la Membrana , Proteínas Oncogénicas/metabolismo , Fosforilación , Podocitos/metabolismo , Tirosina/metabolismoRESUMEN
BACKGROUND: Pancreatic autoantibodies (PABs) are detected in patients with inflammatory bowel disease (IBD). Their prevalence is higher in Crohn's disease (CrD) than in ulcerative colitis (UC). Glycoprotein 2 (GP2) and, more recently, CUB and zona pellucida-like domain-containing protein 1 (CUZD1) have been identified as target autoantigens of PAB. The clinical utility of CUZD1 autoantibodies has only recently been assessed by indirect immunofluorescence (IIF) assays. In this study, we developed and validated novel immunoassays for the detection of CUZD1 autoantibodies. METHODS: Recombinant CUZD1 protein was utilized as a solid-phase antigen for the development of two immunoassays for the detection of IgG and IgA CUZD1 autoantibodies. Serum samples from 100 patients with CrD, 100 patients with UC, 129 patients assessed for various autoimmune diseases (vADs) and 50 control individuals were analyzed. RESULTS: Two immunofluorometric assays for the detection of IgG and IgA CUZD1-specific antibodies were developed. CUZD1 autoantibodies were detected in 12.5% (25/200) IBD patients, including 16% of patients with CrD and in 9% of patients with UC (CrD vs. UC, p<0.05), compared with 3.1% (4/129) patients suspected of having vADs (CrD vs. ADs, p<0.05; UC vs. ADs, p=0.08). CUZD1 autoantibody positivity was not found to be related to disease location, age of disease onset or disease phenotype. CONCLUSIONS: This is the first study to describe novel IgA and IgG CUZD1 autoantibody enzyme-linked immunosorbent assay. These immunoassays agree well with standard IIF techniques and can be utilized in multicenter studies to investigate the diagnostic and clinical utility of CUZD1 autoantibodies.
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Autoanticuerpos/sangre , Técnica del Anticuerpo Fluorescente Indirecta , Enfermedades Inflamatorias del Intestino/diagnóstico , Proteínas de la Membrana/inmunología , Autoanticuerpos/inmunología , Colitis Ulcerosa/diagnóstico , Enfermedad de Crohn/diagnóstico , Ensayo de Inmunoadsorción Enzimática , Humanos , Inmunoglobulina A/sangre , Inmunoglobulina G/sangre , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
These are exciting times for cancer immunotherapy. After many years of disappointing results, the tide has finally changed and immunotherapy has become a clinically validated treatment for many cancers. Immunotherapeutic strategies include cancer vaccines, oncolytic viruses, adoptive transfer of ex vivo activated T and natural killer cells, and administration of antibodies or recombinant proteins that either costimulate cells or block the so-called immune checkpoint pathways. The recent success of several immunotherapeutic regimes, such as monoclonal antibody blocking of cytotoxic T lymphocyte-associated protein 4 (CTLA-4) and programmed cell death protein 1 (PD1), has boosted the development of this treatment modality, with the consequence that new therapeutic targets and schemes which combine various immunological agents are now being described at a breathtaking pace. In this review, we outline some of the main strategies in cancer immunotherapy (cancer vaccines, adoptive cellular immunotherapy, immune checkpoint blockade, and oncolytic viruses) and discuss the progress in the synergistic design of immune-targeting combination therapies.
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Anticuerpos Monoclonales/uso terapéutico , Antígeno CTLA-4/antagonistas & inhibidores , Vacunas contra el Cáncer/uso terapéutico , Inmunoterapia , Neoplasias/tratamiento farmacológico , Biomarcadores de Tumor , Terapia Combinada , Humanos , Inmunoterapia/métodos , Neoplasias/inmunologíaRESUMEN
In pancreatic cancer, the incidence and mortality curves coincide. One major reason for this high mortality rate in pancreatic ductal adenocarcinoma (PDAC) patients is the dearth of effective diagnostic, prognostic, and disease-monitoring biomarkers. Unfortunately, existing tumor markers, as well as current imaging modalities, are not sufficiently sensitive and/or specific for early-stage diagnosis. There is, therefore, an urgent need for improved serum markers of the disease. Herein, we performed Orbitrap® mass spectrometry proteomic analysis of four PDAC tissues and their adjacent benign tissues and identified a total of 2190 nonredundant proteins. Sixteen promising candidates were selected for further scrutiny using a systematic scoring algorithm. Our preliminary serum verification of the top four candidates (DSP, LAMC2, GP73, and DSG2) in 20 patients diagnosed with pancreatic cancer and 20 with benign pancreatic cysts, showed a significant (p < 0.05) elevation of LAMC2 in pancreatic cancer serum. Extensive validation of LAMC2 in healthy, benign, and PDAC sera from geographically diverse cohorts (n = 425) (Japan, Europe, and USA) demonstrated a significant increase in levels in early-stage PDAC compared with benign diseases. The sensitivity of LAMC2 was comparable to CA19.9 in all data sets, with an AUC value greater than 0.85 in discriminating healthy patients from early-stage PDAC patients. LAMC2 exhibited diagnostic complementarity with CA19.9 by showing significant (p < 0.001 in two out of three cohorts) elevation in PDAC patients with clinically low CA19.9 levels.
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Biomarcadores de Tumor/metabolismo , Carcinoma Ductal Pancreático/metabolismo , Laminina/metabolismo , Neoplasias Pancreáticas/metabolismo , Anciano , Femenino , Humanos , Masculino , Persona de Mediana Edad , ProteómicaRESUMEN
BACKGROUND: By using proteomics and bioinformatics, we have previously identified a group of highly pancreas-specific proteins as candidate pancreatic ductal adenocarcinoma (PDAC) biomarkers. With the use of commercially available ELISAs, the performance of some of these candidates was initially evaluated in a relatively small serum cohort (n = 100 samples). This phase revealed that CUB and zona pellucida-like domains protein 1 (CUZD1) may represent a new, promising PDAC biomarker. METHODS: We performed detailed experiments to investigate the specificity of the commercial CUZD1 ELISA assay. CUZD1 was expressed in house in both bacteria and yeast expression systems. Recombinant CUZD1 and biological samples containing CUZD1, as well as commercial CUZD1 ELISA standards, were analyzed by Western blot, size exclusion HPLC, and mass spectrometry (LC-MS Orbitrap). RESULTS: We confirmed that instead of CUZD1, the commercial assay is recognizing a nonhomologous, known cancer antigen [cancer antigen 125 (CA125)]. CONCLUSIONS: We conclude that poor characterization of commercial ELISA assays is a factor that could lead to false biomarker discovery. To our knowledge, this is the first report documenting that a commercial ELISA marketed for one analyte (CUZD1) may, in fact, recognize a different, nonhomologous antigen (CA125).
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Antígeno Ca-125/sangre , Carcinoma Ductal Pancreático/sangre , Ensayo de Inmunoadsorción Enzimática/normas , Proteínas de la Membrana/sangre , Neoplasias Pancreáticas/sangre , Calibración , Línea Celular Tumoral , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , Detección Precoz del Cáncer , Ensayo de Inmunoadsorción Enzimática/métodos , Reacciones Falso Negativas , Humanos , Proteínas de la Membrana/genética , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Espectrometría de Masas en TándemRESUMEN
BACKGROUND: Despite stewardship efforts, laboratory testing overuse persists across medicine. OBJECTIVES: To understand laboratory stewardship perceptions and testing patterns at a tertiary care pediatric hospital so that we could identify potential improvement opportunities. METHODS: An electronic survey exploring laboratory stewardship was sent to all pediatric medicine resident and staff physicians. Laboratory testing data were also assessed for patterns of testing and overuse. RESULTS: The survey response rate was 54% (43/80). The results indicated good familiarity with stewardship but poor familiarity with testing specifics (eg, cost). A mobile reference application was the most preferred quality improvement intervention, and online modules were the least desired. Overuse was apparent, with as many as 53% of laboratory tests being repeated within 7 days and only half of repeated tests subsequently yielding abnormal results. CONCLUSIONS: Altogether, the data we collated demonstrated poor understanding of laboratory stewardship and substantial repeat testing with few abnormal results. These study findings suggest that laboratory stewardship is lacking at our center, and that multiple improvement opportunities exist.
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Detecting dyslipidemia early is important because atherosclerosis originates in childhood and early treatment can improve outcomes. In 2022, the Canadian Cardiovascular Society (CCS) and Canadian Pediatric Cardiology Association (CPCA) published a clinical practice update to detect, evaluate, and manage pediatric dyslipidemia. However, guidance on its translation into clinical laboratories is lacking. The Canadian Society of Clinical Chemists Working Group on Reference Interval Harmonization Lipid Team aims to assist guideline implementation and promote harmonized pediatric lipid reporting across Canada. The 2022 CCS/CPCA clinical practice update, 2011 National Heart, Lung, and Blood Institute integrated guidelines, and new data analysis (Canadian pediatric reference values from the Canadian Laboratory Initiative on Pediatric Reference Intervals [CALIPER] and retrospective patient data from large community laboratories) were incorporated to develop 5 key recommendations. These include recommendations to: (1) offer nonfasting and fasting lipid testing; (2) offer a lipid panel including total cholesterol, low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), non-HDL-C, and triglycerides, with apolipoprotein B and lipoprotein(a) available as individually orderable tests; (3) flag total cholesterol, LDL-C, and non-HDL-C results ≥ 95th percentile, and HDL-C results < 10th percentile, as recommended by CCS/CPCA/National Heart, Lung, and Blood Institute and validated by CALIPER, and flag apolipoprotein B and nonfasting triglyceride results ≥ 95th percentile on the basis of CALIPER, and do not flag Lp(a) results but mention the adult cutoff in the interpretive comments; (4) implement interpretive comments listed in the current report; and (5) implement the National Institutes of Health LDL-C equation. The Canadian Society of Clinical Chemists Working Group on Reference Interval Harmonization Lipid Team will support clinical laboratories to implement these recommendations using knowledge translation strategies. Harmonizing pediatric lipid reporting across Canadian clinical laboratories will optimize clinical decision-making and improve cardiovascular risk management in youth.
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Dislipidemias , Lípidos , Humanos , Canadá , Niño , Lípidos/sangre , Dislipidemias/diagnóstico , Dislipidemias/sangre , Sociedades Médicas , Valores de ReferenciaRESUMEN
BACKGROUND: Protein cancer biomarkers serve multiple clinical purposes, both early and late, during disease progression. The search for new and better biomarkers has become an integral component of contemporary cancer research. However, the number of new biomarkers cleared by the US Food and Drug Administration has declined substantially over the last 10 years, raising concerns regarding the efficiency of the biomarker-development pipeline. CONTENT: We describe different clinical uses of cancer biomarkers and their performance requirements. We also present examples of protein cancer biomarkers currently in clinical use and their limitations. The major barriers that candidate biomarkers need to overcome to reach the clinic are addressed. Finally, the long and arduous journey of a protein cancer biomarker from the bench to the clinic is outlined with an example. SUMMARY: The journey of a protein biomarker from the bench to the clinic is long and challenging. Every step needs to be meticulously planned and executed to succeed. The history of clinically useful biomarkers suggests that at least a decade is required for the transition of a marker from the bench to the bedside. Therefore, it may be too early to expect that the new technological advances will catalyze the anticipated biomarker revolution any time soon.
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Biomarcadores de Tumor/análisis , Neoplasias/diagnóstico , Diagnóstico Precoz , HumanosRESUMEN
BACKGROUND: The identification of new serum biomarkers with high sensitivity and specificity is an important priority in pancreatic cancer research. Through an extensive proteomics analysis of pancreatic cancer cell lines and pancreatic juice, we previously generated a list of candidate pancreatic cancer biomarkers. The present study details further validation of four of our previously identified candidates: regenerating islet-derived 1 beta (REG1B), syncollin (SYCN), anterior gradient homolog 2 protein (AGR2), and lysyl oxidase-like 2 (LOXL2). METHODS: The candidate biomarkers were validated using enzyme-linked immunosorbent assays in two sample sets of serum/plasma comprising a total of 432 samples (Sample Set A: pancreatic ductal adenocarcinoma (PDAC, n = 100), healthy (n = 92); Sample Set B: PDAC (n = 82), benign (n = 41), disease-free (n = 47), other cancers (n = 70)). Biomarker performance in distinguishing PDAC from each control group was assessed individually in the two sample sets. Subsequently, multiparametric modeling was applied to assess the ability of all possible two and three marker panels in distinguishing PDAC from disease-free controls. The models were generated using sample set B, and then validated in Sample Set A. RESULTS: Individually, all markers were significantly elevated in PDAC compared to healthy controls in at least one sample set (p ≤ 0.01). SYCN, REG1B and AGR2 were also significantly elevated in PDAC compared to benign controls (p ≤ 0.01), and AGR2 was significantly elevated in PDAC compared to other cancers (p < 0.01). CA19.9 was also assessed. Individually, CA19.9 showed the greatest area under the curve (AUC) in receiver operating characteristic (ROC) analysis when compared to the tested candidates; however when analyzed in combination, three panels (CA19.9 + REG1B (AUC of 0.88), CA19.9 + SYCN + REG1B (AUC of 0.87) and CA19.9 + AGR2 + REG1B (AUC of 0.87)) showed an AUC that was significantly greater (p < 0.05) than that of CA19.9 alone (AUC of 0.82). In a comparison of early-stage (Stage I-II) PDAC to disease free controls, the combination of SYCN + REG1B + CA19.9 showed the greatest AUC in both sample sets, (AUC of 0.87 and 0.92 in Sets A and B, respectively). CONCLUSIONS: Additional serum biomarkers, particularly SYCN and REG1B, when combined with CA19.9, show promise as improved diagnostic indicators of pancreatic cancer, which therefore warrants further validation.
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Biomarcadores de Tumor/sangre , Antígeno CA-19-9/sangre , Proteínas Portadoras/sangre , Litostatina/sangre , Proteínas de la Membrana/sangre , Neoplasias Pancreáticas/sangre , Neoplasias Pancreáticas/diagnóstico , Adulto , Factores de Edad , Anciano , Aminoácido Oxidorreductasas/sangre , Estudios de Casos y Controles , Femenino , Humanos , Masculino , Persona de Mediana Edad , Mucoproteínas , Estadificación de Neoplasias , Proteínas Oncogénicas , Proteínas/metabolismo , Curva ROC , Reproducibilidad de los Resultados , Estudios Retrospectivos , Factores SexualesRESUMEN
The glomerular filtration barrier in the kidney is formed in part by a specialized intercellular junction known as the slit diaphragm, which connects adjacent actin-based foot processes of kidney epithelial cells (podocytes). Mutations affecting a number of slit diaphragm proteins, including nephrin (encoded by NPHS1), lead to renal disease owing to disruption of the filtration barrier and rearrangement of the actin cytoskeleton, although the molecular basis for this is unclear. Here we show that nephrin selectively binds the Src homology 2 (SH2)/SH3 domain-containing Nck adaptor proteins, which in turn control the podocyte cytoskeleton in vivo. The cytoplasmic tail of nephrin has multiple YDxV sites that form preferred binding motifs for the Nck SH2 domain once phosphorylated by Src-family kinases. We show that this Nck-nephrin interaction is required for nephrin-dependent actin reorganization. Selective deletion of Nck from podocytes of transgenic mice results in defects in the formation of foot processes and in congenital nephrotic syndrome. Together, these findings identify a physiological signalling pathway in which nephrin is linked through phosphotyrosine-based interactions to Nck adaptors, and thus to the underlying actin cytoskeleton in podocytes. Simple and widely expressed SH2/SH3 adaptor proteins can therefore direct the formation of a specialized cellular morphology in vivo.
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Actinas/metabolismo , Citoesqueleto/metabolismo , Riñón/citología , Riñón/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Oncogénicas/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Secuencias de Aminoácidos , Animales , Línea Celular , Citoesqueleto/química , Humanos , Riñón/patología , Proteínas de la Membrana/genética , Ratones , Mutación/genética , Síndrome Nefrótico/congénito , Síndrome Nefrótico/genética , Síndrome Nefrótico/metabolismo , Síndrome Nefrótico/patología , Proteínas Oncogénicas/química , Proteínas Oncogénicas/deficiencia , Proteínas Oncogénicas/genética , Fosforilación , Fosfotirosina/metabolismo , Dominios Homologos srcRESUMEN
Within the glomerulus, the scaffolding protein nephrin bridges the actin-rich foot processes that extend from adjacent podocytes to form the slit diaphragm. Mutations affecting a number of slit diaphragm proteins, including nephrin, cause glomerular disease through rearrangement of the actin cytoskeleton and disruption of the filtration barrier. We recently established that the Nck family of Src homology 2 (SH2)/SH3 cytoskeletal adaptor proteins can mediate nephrin-dependent actin reorganization. Formation of foot processes requires expression of Nck in developing podocytes, but it is unknown whether Nck maintains podocyte structure and function throughout life. Here, we used an inducible transgenic strategy to delete Nck expression in adult mouse podocytes and found that loss of Nck expression rapidly led to proteinuria, glomerulosclerosis, and altered morphology of foot processes. We also found that podocyte injury reduced phosphorylation of nephrin in adult kidneys. These data suggest that Nck is required to maintain adult podocytes and that phosphotyrosine-based interactions with nephrin may occur in foot processes of resting, mature podocytes.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Tasa de Filtración Glomerular/fisiología , Glomérulos Renales/metabolismo , Proteínas Oncogénicas/metabolismo , Podocitos/metabolismo , Uniones Estrechas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Antibacterianos/farmacología , Línea Celular , Modelos Animales de Enfermedad , Doxiciclina/farmacología , Glomerulonefritis/inducido químicamente , Glomerulonefritis/metabolismo , Glomerulonefritis/patología , Glomérulos Renales/patología , Masculino , Proteínas de la Membrana/metabolismo , Ratones , Ratones Transgénicos , Proteínas Oncogénicas/genética , Fosforilación , Podocitos/efectos de los fármacos , Podocitos/ultraestructura , Proteinuria/metabolismo , Proteinuria/patología , Puromicina Aminonucleósido/efectos adversos , Ratas , Ratas Sprague-Dawley , Uniones Estrechas/ultraestructuraRESUMEN
OBJECTIVES: Point-of-care testing (POCT) is testing performed outside the traditional laboratory, often at the patient bedside. In hospital settings, blood glucose is the most common POCT. Staff performing POCT are not usually laboratory trained; they are clinical staff with a primary focus on treating patients. Clinical staff find POCT quality assurance (QA) practices burdensome and are often non-compliant. In hospitals within EORLA (Eastern Ontario Regional Laboratories Association), all critically high POCT glucose results must be repeated prior to acting, according to policy. Compliance with this policy is audited regularly. DESIGN: and methods: All POCT glucose tests performed in participating sites between January and June 2018 and June and December 2019 were audited for compliance with the critical repeat policy. The discordant repeat rate was also determined for each audit period. Between January and May 2019, there were interventions aimed at improving compliance with the repeat policy. RESULTS: Compliance with the critical repeat policy increased from 30 to 57% in 2019 compared to 2018, following nursing education and implementation of notifications on the glucose meters themselves. The rate of discordant repeat results (>20% different from initial) also improved at most sites in 2019 compared to 2018. Nurses cited insufficient cleaning of patient hands prior to initial testing as the primary reason for discordant repeats. CONCLUSIONS: Operator compliance with POCT QA policies is an ongoing challenge requiring continual audit, feedback and education. A strong POCT multi-disciplinary committee with supports from senior and clinical leadership in an organization are key to improving compliance.
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Clinical laboratories across the world are working to validate and perform testing for SARS-CoV-2 antibodies. Herein, we present interim consensus guidance for Canadian clinical laboratories testing and reporting SARS-CoV-2 serology, with emphasis on the capabilities and limitations of these tests and recommendations for interpretative comments in an effort to achieve harmonized laboratory practices. The consensus document provides a broad overview of topics including sample type and contamination risk; kinetics of antibody response to COVID-19 and the impact on serology testing; clinical utility of SARS-CoV-2 serology testing; clinical performance of commercial laboratory-based assays commonly deployed in North America; recommendations for interim reporting; utility of SARS-CoV-2 antibody testing for pediatric patients; and utility of point-of-care testing. The information is based on the current literature and is subject to change as additional information becomes available.
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Prueba Serológica para COVID-19 , COVID-19 , Química Farmacéutica , Pandemias , SARS-CoV-2 , Sociedades Médicas , COVID-19/sangre , COVID-19/epidemiología , Canadá , Consenso , HumanosRESUMEN
OBJECTIVES: To evaluate the Siemens N-latex kappa free light chain (κFLC) and lambda FLC (λFLC) assays on the BNII nephelometer and assess agreement with The Binding Site Freelite FLC assays on the SPAPlus. DESIGN AND METHODS: Over 180 patient serum samples from routine analysis of κFLC and λFLC measured by the Freelite assay were collected for the study and measured with the N-latex κFLC and λFLC assays to assess precision, linearity, method comparison and dilutional effects. RESULTS: Complex precision showed coefficients of variation of 4.8-7.2% for the κFLC assay and 3.6-6.0% for the λFLC assay. Linearity assessment showed both assays were linear (κFLC, y=1.00x-0.09 and λFLC, y=1.050x-1.252). Qualitative method comparison showed 87.9% (116/132) agreement and Cohen's kappa of 80.4% between the κFLC assays and 72.6% (98/135) agreement and Cohen's kappa of 55.4% for the λFLC assays. Quantitative method comparison for κFLC<150mg/L was y=0.92x+2.21, R=0.661 and for λFLC<150mg/L was y=7.90x-137.96, R=0.526. Dilutional effects including antigen excess and non-linearity were also examined. CONCLUSIONS: The N-latex assay showed good precision and linearity with reasonable agreement to the Freelite assay. However, the assays should not be used interchangeably to monitor patients.
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Cadenas kappa de Inmunoglobulina/sangre , Cadenas lambda de Inmunoglobulina/sangre , Sitios de Unión , Humanos , Técnicas de Dilución del Indicador , Látex , Paraproteinemias/sangre , Reproducibilidad de los ResultadosRESUMEN
Protein electrophoresis is commonly used as an aid in the diagnosis of monoclonal gammopathies and is performed in many laboratories in Canada and throughout the world. However, unlike many other diagnostic tests, there is limited guidance for standardization and neither guidance nor specific recommendations for clinical reporting of serum (SPE) or urine (UPE) protein electrophoresis and immunotyping available in the literature. Therefore, a Canadian effort was undertaken to recommend standards that cover all aspects of clinical reporting with an ultimate goal towards reporting standardization. The Canadian Society of Clinical Chemists (CSCC) Monoclonal Gammopathy Interest Group (MGIG), which is composed of CSCC members with an interest in protein electrophoresis, has formed a Monoclonal Gammopathy Working Group (MGWG) to take initial steps towards standardization of SPE, UPE and immunotyping. Candidate standardization recommendations were developed, discussed and voted upon by the MGWG. Candidate recommendations that achieved 90% agreement are presented as consensus recommendations. Recommendations that did not achieve 90% consensus remain candidate recommendations and are presented with accompanying MGWG discussion. Eleven consensus recommendations along with candidate recommendations for nomenclature, protein fraction reporting, test utilization, interference handling and interpretive reporting options are presented.
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Electroforesis de las Proteínas Sanguíneas/métodos , Guías como Asunto , Paraproteinemias/sangre , Sociedades Médicas , Canadá , HumanosRESUMEN
The family of polo like kinases (Plks) regulate cell cycle progression through key functional roles in mitosis. While the four mammalian family members, Plk1-4, share overlapping functions, each member possesses unique functions that may be dictated in part by their ability to phosphorylate different substrates. Numerous cellular substrates for Plk1, 2, and 3 have been characterized, but the protein targets for Plk4/Sak remain unknown. We have purified the kinase domain of Sak and demonstrated that it has robust kinase activity in vitro. Using in vitro kinase assays on peptide spots arrays, we determined the consensus phosphorylation motif for Sak to be yen-[Ile/Leu/Val]-Ser/Thr-phi-phi-X- yen/Pro (where phi denotes a large hydrophobic residue, yen is a charged residue dependent on the context of the surrounding sequence, and residues in brackets are unfavoured). This consensus phosphorylation motif differs from that of Plk1, and provides a basis for future studies to identify in vivo substrates of Sak.
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Péptidos/química , Procesamiento Proteico-Postraduccional , Proteínas Serina-Treonina Quinasas/química , Secuencias de Aminoácidos , Animales , Ratones , Péptidos/metabolismo , Fosforilación , Análisis por Matrices de Proteínas , Procesamiento Proteico-Postraduccional/fisiología , Proteínas Serina-Treonina Quinasas/aislamiento & purificación , Proteínas Serina-Treonina Quinasas/metabolismo , Estructura Terciaria de Proteína , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
BACKGROUND: CUB and zona pellucida-like domain-containing protein 1 (CUZD1) was identified as a pancreas-specific protein and was proposed as a candidate biomarker for pancreatic related disorders. CUZD1 protein levels in tissues and biological fluids have not been extensively examined. The purpose of the present study was to generate specific antibodies targeting CUZD1 to assess CUZD1 expression within tissues and biological fluids. METHODS: Mouse monoclonal antibodies against CUZD1 were generated and used to perform immunohistochemical analyses and to develop a sensitive and specific enzyme-linked immunosorbent assay (ELISA). CUZD1 protein expression was assessed in various human tissue extracts and biological fluids and in gel filtration chromatography-derived fractions of pancreatic tissue extract, pancreatic juice and recombinant protein. RESULTS: Immunohistochemical staining of CUZD1 in pancreatic tissue showed that the protein is localized to the acinar cells and the lumen of the acini. Western blot analysis detected the protein in pancreatic tissue extract and pancreatic juice. The newly developed ELISA measured CUZD1 in high levels in pancreas and in much lower but detectable levels in several other tissues. In the biological fluids tested, CUZD1 expression was detected exclusively in pancreatic juice. The analysis of gel filtration chromatography-derived fractions of pancreatic tissue extract, pancreatic juice and recombinant CUZD1 suggested that the protein exists in high molecular weight protein complexes. CONCLUSION: This study describes the development of tools targeting CUZD1 protein, its tissue expression pattern and levels in several biological fluids. These new tools will facilitate future investigations aiming to delineate the role of CUZD1 in physiology and pathobiology.