RESUMEN
BACKGROUND: The AACC Academy revised the reproductive testing section of the Laboratory Medicine Practice Guidelines: Evidence-Based Practice for Point-of-Care Testing (POCT) published in 2007. METHODS: A panel of Academy members with expertise in POCT and laboratory medicine was formed to develop guidance for the use of POCT in reproductive health, specifically ovulation, pregnancy, premature rupture of membranes (PROM), and high-risk deliveries. The committee was supplemented with clinicians having Emergency Medicine and Obstetrics/Gynecology training. RESULTS: Key recommendations include the following. First, urine luteinizing hormone (LH) tests are accurate and reliable predictors of ovulation. Studies have shown that the use of ovulation predicting kits may improve the likelihood of conception among healthy fertile women seeking pregnancy. Urinary LH point-of-care testing demonstrates a comparable performance among other ovulation monitoring methods for timing intrauterine insemination and confirming sufficient ovulation induction before oocyte retrieval during in vitro fertilization. Second, pregnancy POCT should be considered in clinical situations where rapid diagnosis of pregnancy is needed for treatment decisions, and laboratory analysis cannot meet the required turnaround time. Third, PROM testing using commercial kits alone is not recommended without clinical signs of rupture of membranes, such as leakage of amniotic fluid from the cervical opening. Finally, fetal scalp lactate is used more than fetal scalp pH for fetal acidosis due to higher success rate and low volume of sample required. CONCLUSIONS: This revision of the AACC Academy POCT guidelines provides recommendations for best practice use of POCT in fertility and reproduction.
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Fertilidad , Reproducción , Femenino , Humanos , Pruebas en el Punto de Atención , EmbarazoRESUMEN
Recognizing that race is a social and not a biological construct, healthcare professionals and the public have called for removal of race in clinical algorithms. In response, the National Kidney Foundation and the American Society of Nephrology created the Task Force on Reassessing the Inclusion of Race in Diagnosing Kidney Diseases to examine the issue and provide recommendations. The final report from the Task Force recommends calculating estimated glomerular filtration rate (eGFR) without a race coefficient using the recently published CKD-EPI 2021 creatinine (cr) and creatinine-cystatin C (cr-cys) equations. The Task Force recommends immediately replacing older eGFRcr equations (MDRD Study and CKD-EPI 2009) with the new CKD-EPI 2021 equation. In a 2019 survey by the College of American Pathologists, 23% of 6200 laboratories reporting eGFRcr used an incorrect equation that is not suitable for use with standardized creatinine measurements, 34% used the CKD-EPI 2009 equation and 43% used the MDRD Study 2006 equation re-expressed for standardized creatinine measurement. Rapid transition to using the CKD-EPI 2021 equation is an opportunity for laboratories to standardize to a single equation to eliminate differences in eGFRcr due to different equations used by different laboratories, and to report eGFR without use of race. We provide guidance to laboratories for implementing the CKD-EPI 2021 equations for both eGFRcr and eGFRcr-cys.
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Laboratorios , Insuficiencia Renal Crónica , Creatinina , Tasa de Filtración Glomerular/fisiología , Humanos , Riñón , Laboratorios Clínicos , Insuficiencia Renal Crónica/diagnósticoRESUMEN
BACKGROUND: To further improve workflow efficiency, our laboratory implemented a total laboratory automation (TLA) system that connected our preanalytic processing system with various testing (hematology, coagulation, and chemistry). METHODS: Detailed time and motion studies were performed to create process flow maps before and after TLA. The before maps identified opportunities for workflow improvements. We used postimplementation studies to quantify efficiency gains. RESULTS: The implementation of our TLA system resulted in 86% fewer discrete processing steps in specimen handling, even when starting from a partially automated laboratory. Instrument consolidation reduced the testing footprint by 45% and reduced the number of testing personnel by 2.5 full-time employees (FTEs). An 82% reduction in hands-on time associated with add-on processes was achieved. Combining STAT and outreach work on the testing system did not impact turnaround time. CONCLUSIONS: With careful planning, a TLA system can effectively optimize laboratory processes and efficiency.
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Automatización de Laboratorios/métodos , Laboratorios/organización & administración , Automatización de Laboratorios/normas , Hematología , Laboratorios/normas , Flujo de TrabajoRESUMEN
OBJECTIVE: To concretely outline a process for selecting a total laboratory automation system that connects clinical chemistry, hematology, and coagulation analyzers and to serve as a reference for other laboratories. METHODS: In Phase I, a committee including the laboratory's directors and technologists conducted a review of 5 systems based on formal request for information process, site visits, and vendor presentations. We developed evaluation criteria and selected the 2 highest performing systems. In Phase II, we executed a detailed comparison of the 2 vendors based on cost, instrument layout, workflow design, and future potential. RESULTS: In addition to selecting a laboratory automation system, we used the process to ensure employee engagement in preparation for implementation. CONCLUSION: Selecting a total laboratory automation system is a complicated process. This paper provides practical guide in how a thorough selection process can be done with participation of key stakeholders.
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Automatización de Laboratorios , Laboratorios , Compromiso Laboral , Humanos , Laboratorios/organización & administración , Laboratorios/normasRESUMEN
After damage, cells reseal their plasma membrane and repair the underlying cortical cytoskeleton. Although many different proteins have been implicated in cell repair, the potential role of specific lipids has not been explored. Here we report that cell damage elicits rapid formation of spatially organized lipid domains around the damage site, with different lipids concentrated in different domains as a result of both de novo synthesis and transport. One of these lipids-diacylglycerol (DAG)-rapidly accumulates in a broad domain that overlaps the zones of active Rho and Cdc42, GTPases that regulate repair of the cortical cytoskeleton. Formation of the DAG domain is required for Cdc42 and Rho activation and healing. Two DAG targets, protein kinase C (PKC) ß and η, are recruited to cell wounds and play mutually antagonistic roles in the healing process: PKCß participates in Rho and Cdc42 activation, whereas PKCη inhibits Rho and Cdc42 activation. The results reveal an unexpected diversity in subcellular lipid domains and the importance of such domains for a basic cellular process.
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Estructuras de la Membrana Celular/fisiología , Diglicéridos/fisiología , Animales , Diacilglicerol Quinasa/metabolismo , Proteínas de Unión al GTP Monoméricas/metabolismo , Oocitos/metabolismo , Fosfatidilcolinas/metabolismo , Fosfolipasa D/metabolismo , Transporte de Proteínas , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Análisis de la Célula Individual , Transferasas (Grupos de Otros Fosfatos Sustitutos)/metabolismo , Fosfolipasas de Tipo C/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevisRESUMEN
Asymmetric cell growth and division rely on polarized actin cytoskeleton remodeling events, the regulation of which is poorly understood. In budding yeast, formins stimulate the assembly of an organized network of actin cables that direct polarized secretion. Here we show that the Fer/Cip4 homology-Bin amphiphysin Rvs protein Hof1, which has known roles in cytokinesis, also functions during polarized growth by directly controlling the activities of the formin Bnr1. A mutant lacking the C-terminal half of Hof1 displays misoriented and architecturally altered cables, along with impaired secretory vesicle traffic. In vitro, Hof1 inhibits the actin nucleation and elongation activities of Bnr1 without displacing the formin from filament ends. These effects depend on the Src homology 3 domain of Hof1, the formin homology 1 (FH1) domain of Bnr1, and Hof1 dimerization, suggesting a mechanism by which Hof1 "restrains" the otherwise flexible FH1-FH2 apparatus. In vivo, loss of inhibition does not alter actin levels in cables but, instead, cable shape and functionality. Thus Hof1 tunes formins to sculpt the actin cable network.
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Actinas/metabolismo , Polaridad Celular , Proteínas del Citoesqueleto/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/crecimiento & desarrollo , Citoesqueleto de Actina/metabolismo , Proliferación Celular , Tamaño de la Célula , Microscopía Fluorescente , Proteínas Asociadas a Microtúbulos/química , Mutación/genética , Fenotipo , Profilinas/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/químicaRESUMEN
To test if proteolysis is involved in tumor cell extravasation, we developed an in vitro model where tumor cells cross an endothelial monolayer cultured on a basement membrane. Using this model we classified the ability of the cells to transmigrate through the endothelial cell barrier onto the underlying matrix, and scored this invasion according to the stage of passage through the endothelium. Metalloproteinase inhibitors reduced tumor cell extravasation by at least 35%. Visualization of protease and cell adhesion molecules by confocal microscopy demonstrated the cell surface localization of MMP-2, MMP-9, MT1-MMP, furin, CD44 and αvß3, during the process of transendothelial migration. By the addition of inhibitors and bio-modulators we assessed the functional requirement of the aforementioned molecules for efficient migration. Proteolytic digestion occurred at the cell-matrix interface and was most evident during the migratory stage. All of the inhibitors and biomodulators affected the transition of the tumor cells into the migratory stage, highlighting the most prevalent use of proteolysis at this particular step of tumor cell extravasation. These data suggest that a proteolytic interface operates at the tumor cell surface within the tumor-endothelial cell microenvironment.
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Metaloproteasas/metabolismo , Proteolisis , Migración Transendotelial y Transepitelial/fisiología , Células Tumorales Cultivadas/fisiología , Microambiente Tumoral/fisiología , Western Blotting , Moléculas de Adhesión Celular/metabolismo , Técnica del Anticuerpo Fluorescente , Humanos , Técnicas In Vitro , Metaloproteasas/antagonistas & inhibidores , Microscopía ConfocalRESUMEN
In the United States, the nonmedical use of prescription drugs is the second most common illicit drug use, behind only marijuana. This article discusses the abuse issues with three of the most widely abused prescription drugs: opioids, central nervous system (CNS) depressants (eg, benzodiazepines), and stimulants (eg, amphetamine-dextroamphetamine and methylphenideate) in the United States. Efforts to deal with the problem are described as well.
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Epidemias , Medicamentos bajo Prescripción/administración & dosificación , Trastornos Relacionados con Sustancias/epidemiología , Humanos , Estados Unidos/epidemiologíaRESUMEN
BACKGROUND: Diagnosis and management of diabetic ketoacidosis (DKA) often rely on the measurement of urine ketones along with blood glucose, anion gap, and pH. These values, however, do not reliably reflect the severity of ketoacidosis. The Abbott Precision Xceed Pro® meter is an FDA-approved device that quantitatively measures ß-hydroxybutyrate (BOH) in whole blood. This study was undertaken to determine whether the ketone meter meets the analytical criteria to aid DKA diagnosis and management in the hospital. METHODS: 54 heparinized venous whole blood BOH concentrations from 27 diabetic patients were measured by the Abbott meter, and compared with the plasma BOH concentrations measured with Stanbio reagent (reference method). Measurements were done in the hospital central laboratory. RESULTS: Of the 54 pairs of specimens analyzed, 17 pairs displayed a difference of >15% between the two methods. Nearly all discrepant points occurred when BOH >5 mmol/L (reference method). Linearity evaluation revealed that the meter is not linear from 0.0 to 8.0 mmol/L, contrary to the claim by the manufacturer. Further, we identified acetoacetate, a metabolite commonly present in DKA patients, as a potential interfering substance for the meter BOH measurement. CONCLUSIONS: BOH measurements by the Abbott meter up to 3 mmol/L correlate well with the reference method, but become discrepant above that point. While this characteristic may be useful in the diagnosis of DKA, it may not allow clinicians to serially follow the response to therapy in hospitalized DKA patients with BOH values greater than 5 mmol/L (reference method).
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Ácido 3-Hidroxibutírico/sangre , Análisis Químico de la Sangre/instrumentación , Cetoacidosis Diabética/diagnóstico , Cetoacidosis Diabética/sangre , HumanosRESUMEN
BACKGROUND: The Rho GTPases-Rho, Rac, and Cdc42-regulate the dynamics of F-actin (filamentous actin) and myosin-2 with considerable subcellular precision. Consistent with this ability, active Rho and Cdc42 occupy mutually exclusive zones during single-cell wound repair and asymmetric cytokinesis, suggesting the existence of mechanisms for local crosstalk, but how local Rho GTPase crosstalk is controlled is unknown. RESULTS: Using a candidate screen approach for Rho GTPase activators (guanine nucleotide exchange factors; GEFs) and Rho GTPase inactivators (GTPase-activating proteins; GAPs), we find that Abr, a protein with both GEF and GAP activity, regulates Rho and Cdc42 during single-cell wound repair. Abr is targeted to the Rho activity zone via active Rho. Within the Rho zone, Abr promotes local Rho activation via its GEF domain and controls local crosstalk via its GAP domain, which limits Cdc42 activity within the Rho zone. Depletion of Abr attenuates Rho activity and wound repair. CONCLUSIONS: Abr is the first identified Rho GTPase regulator of single-cell wound healing. Its novel mode of targeting by interaction with active Rho allows Abr to rapidly amplify local increases in Rho activity using its GEF domain while its ability to inactivate Cdc42 using its GAP domain results in sharp segregation of the Rho and Cdc42 zones. Similar mechanisms of local Rho GTPase activation and segregation enforcement may be employed in other processes that exhibit local Rho GTPase crosstalk.
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Proteínas Activadoras de GTPasa/metabolismo , Regulación de la Expresión Génica/fisiología , Proteínas de Unión al GTP rho/metabolismo , Animales , Membrana Celular , Oocitos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Xenopus laevis , Proteína de Unión al GTP cdc42/genética , Proteína de Unión al GTP cdc42/metabolismoAsunto(s)
Trasplante de Médula Ósea , Calcio/sangre , Hipocalcemia/tratamiento farmacológico , Leucemia/terapia , Albúmina Sérica/metabolismo , Transfusión de Componentes Sanguíneos , Calcio/orina , Cationes Bivalentes , Hematócrito , Humanos , Hipocalcemia/complicaciones , Hipocalcemia/metabolismo , Leucemia/complicaciones , Leucemia/metabolismo , Recuento de Plaquetas , Unión ProteicaRESUMEN
INTRODUCTION: Bronze drinking vessels famous for their intricate carvings and used by the aristocracy in the Chinese Shang dynasty (1555-1145 BCE) are known to have been fabricated with alloys containing soft metallic lead. The contribution of lead leaching from such vessels into the fermented grain wines drunk by the Chinese nobility in ancient times has not been previously estimated. METHODS: Three bronze vessels containing 8% lead by weight were fabricated to resemble the late Shang bronze goblets. Shaoxing drinking rice wine was purchased locally and placed in the vessels, using a white grape wine and water as comparisons. Sampling was performed at baseline, 2 min, and then at days 1, 2, 4, and 7. Lead concentrations in the liquid matrix were measured using atomic absorption spectroscopy. RESULTS: Significant amounts of lead leached into the liquid within one day: 13,900 µg/L in water, 45,900 µg/L in rice wine, and 116,000 µg/L in white wine. Lead continued to leach into both the grape and rice wines with the passage of time. DISCUSSION: Significant lead contamination of Shaoxing rice wine was detected when it was left in bronze goblets fabricated to resemble the Shang dynasty vessels. If a liter of contaminated wine was drunk daily, the daily intake of lead could have been as high as 85 mg. Such a high degree of contamination could cause chronic lead poisoning, affecting the health of the Shang nobility who used bronze beverage containers, before lead was excluded from the manufacture of bronze.
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Contaminación de Alimentos/análisis , Intoxicación por Plomo/etiología , Plomo/análisis , Vino/análisis , Consumo de Bebidas Alcohólicas , HumanosRESUMEN
Single cells and multicellular tissues rapidly heal wounds. These processes are considered distinct, but one mode of healing--Rho GTPase-dependent formation and closure of a purse string of actin filaments (F-actin) and myosin-2 around wounds--occurs in single cells and in epithelia. Here, we show that wounding of one cell in Xenopus embryos elicits Rho GTPase activation around the wound and at the nearest cell-cell junctions in the neighbor cells. F-actin and myosin-2 accumulate at the junctions and around the wound itself, and as the resultant actomyosin array closes over the wound site, junctional F-actin and myosin-2 become mechanically integrated with the actin and myosin-2 around the wound, forming a hybrid purse string. When cells are ablated rather than wounded, Rho GTPase activation and F-actin accumulation occur at cell-cell junctions surrounding the ablated cell, and the purse string closes the hole in the epithelium. Elevation of intracellular free calcium, an essential upstream signal for the single-cell wound response, also occurs at the cell-cell contacts and in neighbor cells. Thus, the single and multicellular purse string wound responses represent points on a signaling and mechanical continuum that are integrated by cell-cell junctions.
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Actinas/fisiología , Uniones Intercelulares/fisiología , Miosina Tipo II/fisiología , Cicatrización de Heridas/fisiología , Proteínas de Unión al GTP rho/fisiología , Animales , Blastómeros , Señalización del Calcio , Embrión no Mamífero/lesiones , Embrión no Mamífero/metabolismo , Activación Enzimática , Cicatrización de Heridas/genética , Xenopus laevis/embriologíaRESUMEN
Actin is involved in endocytosis in organisms ranging from yeast to mammals. In activated Xenopus eggs, exocytosing cortical granules (CGs) are surrounded by actin "coats," which compress the exocytosing compartments, resulting in compensatory endocytosis. Here, we examined the roles of two myosins in actin coat compression. Myosin-2 is recruited to exocytosing CGs late in coat compression. Inhibition of myosin-2 slows coat compression without affecting actin assembly. This differs from phenotype induced by inhibition of actin assembly, where exocytosing CGs are trapped at the plasma membrane (PM) completely. Thus, coat compression is likely driven in part by actin assembly itself, but it requires myosin-2 for efficient completion. In contrast to myosin-2, the long-tailed myosin-1e is recruited to exocytosing CGs immediately after egg activation. Perturbation of myosin-1e results in partial actin coat assembly and induces CG collapse into the PM. Intriguingly, simultaneous inhibition of actin assembly and myosin-1e prevents CG collapse. Together, the results show that myosin-1e and myosin-2 are part of an intricate machinery that coordinates coat compression at exocytosing CGs.
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Actinas/metabolismo , Endocitosis , Miosinas/metabolismo , Animales , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Citocalasina D/farmacología , Gránulos Citoplasmáticos/efectos de los fármacos , Gránulos Citoplasmáticos/metabolismo , Endocitosis/efectos de los fármacos , Exocitosis/efectos de los fármacos , Femenino , Óvulo/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , XenopusRESUMEN
Xenopus oocytes undergo dynamic structural changes during maturation and fertilization. Among these, cortical granule exocytosis and compensatory endocytosis provide effective models to study membrane trafficking. This study documents an important role for myosin 1e in cortical granule exocytosis. Myosin 1e is expressed at the earliest stage that cortical granule exocytosis can be detected in oocytes. Prior to exocytosis, myosin 1e relocates to the surface of cortical granules. Overexpression of myosin 1e augments the kinetics of cortical granule exocytosis, whereas tail-derived fragments of myosin 1e inhibit this secretory event (but not constitutive exocytosis). Finally, intracellular injection of myosin 1e antibody inhibits cortical granule exocytosis. Further experiments identified cysteine string proteins as interacting partners for myosin 1e. As constituents of the membrane of cortical granules, cysteine string proteins are also essential for cortical granule exocytosis. Future investigation of the link between myosin 1e and cysteine string proteins should help to clarify basic mechanisms of regulated exocytosis.
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Gránulos Citoplasmáticos/metabolismo , Miosinas/fisiología , Oocitos/metabolismo , Xenopus/metabolismo , Fosfatasa Alcalina/metabolismo , Amilosa/química , Animales , Cisteína/metabolismo , Exocitosis , Proteínas del Choque Térmico HSP40/metabolismo , Cinética , Proteínas de la Membrana/metabolismo , Microscopía Confocal , Microscopía Fluorescente , Miosinas/metabolismo , Fracciones Subcelulares/metabolismoRESUMEN
Local actin assembly is associated with sites of exocytosis in processes ranging from phagocytosis to compensatory endocytosis. Here, we examine whether the trigger for actin-coat assembly around exocytosing Xenopus egg cortical granules is 'compartment mixing'--the union of the contents of the plasma membrane with that of the secretory granule membrane. Consistent with this model, compartment mixing occurs on cortical granule-plasma membrane fusion and is required for actin assembly. Compartment mixing triggers actin assembly, at least in part, through diacylglycerol (DAG), which incorporates into the cortical granule membranes from the plasma membrane after cortical granule-plasma membrane fusion. DAG, in turn, directs long-term recruitment of protein kinase Cbeta (PKCbeta) to exocytosing cortical granules, where it is required for activation of Cdc42 localized on the cortical granules. The results demonstrate that mixing of two membrane compartments can direct local actin assembly and indicate that this process is harnessed during Xenopus egg cortical granule exocytosis to drive compensatory endocytosis.
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Actinas/metabolismo , Compartimento Celular/fisiología , Membrana Celular/metabolismo , Gránulos Citoplasmáticos/metabolismo , Fusión de Membrana , Animales , Diglicéridos/metabolismo , Exocitosis/fisiología , Femenino , Oocitos/metabolismo , Proteína Quinasa C/metabolismo , Proteína Quinasa C beta , Xenopus/metabolismoRESUMEN
Cellular damage triggers rapid resealing of the plasma membrane and repair of the cortical cytoskeleton. Plasma membrane resealing results from calcium-dependent fusion of membranous organelles and the plasma membrane at the site of the damage. Cortical cytoskeletal repair results from local assembly of actin filaments (F-actin), myosin-2 and microtubules into an array that closes around the original wound site. Control of the cytoskeletal response is exerted by local activation of the small GTPases, Rho and Cdc42. Recent work has given insight into both the membrane fusion and cytoskeletal responses to plasma membrane damage and we propose that Rho GTPase activation results at least in part from the events that drive membrane repair.