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Coats disease is a rare retinal disease. It is characterised by abnormal dilatation of retinal capillaries (telangiectasia) and increased vascular permeability (intraretinal and subretinal exudation). In advanced stage of disease the patient can have exudative retinal detachment where the retina detaches from its normal attachment. In some cases there will be secondary increase in intraocular pressure if not treated on time. It has a unilateral presentation in most of the cases. This article describes about a four year old boy who presented with retinal detachment due to coats disease. The patient was managed effectively with external subretinal fluid drainage along with laser and cryotherapy. It is essential to rule out aggressive eye cancer (retinoblastoma) as both can have similar presentation.

Purpose: Approximately 15% of women who receive ovarian function suppression (OFS) as adjuvant treatment for high-risk, localized hormone receptor-positive (HR+) breast cancer may have inadequate estradiol suppression which can require therapeutic modification when used in combination with an aromatase inhibitor (AI). We previously reported that abemaciclib may interfere with the estradiol Abbott Alinity chemiluminescent microparticle immunoassay (CMIA) commonly used to monitor estradiol levels and suggested liquid chromatography-mass spectrometry (LC-MS/MS) is preferred in this setting. The aim of this study was to determine discrepancies in estradiol levels using CMIA compared to LC-MS/MS and subsequent treatment changes in a larger patient population., Methods: We conducted a retrospective review of premenopausal women with early-stage HR+ breast cancer at our institution who received adjuvant OFS and abemaciclib with at least 1 CMIA estradiol level drawn during abemaciclib therapy from October 2021 to April 2023., Results: Of the 22 women who met criteria for review, 20 (90.9%) had CMIA estradiol levels in the premenopausal range, of whom 9 also had estradiol measured by LC-MS/MS. All 9 women receiving OFS and abemaciclib with estradiol measurements by both methods had premenopausal range CMIA estradiol levels and postmenopausal range LC-MS/MS estradiol levels. Of the 20 patients with premenopausal estradiol levels by CMIA estradiol, treatment changes included increased OFS dosage or preparation (n = 7), change from AI to tamoxifen (n = 3), and surgical oophorectomy (n = 7)., Conclusion: Our findings suggest the likely interference of abemaciclib with the Abbott Alinity immunoassay which may lead to unnecessary treatment changes. It is recommended that the LC-MS/MS assay be used when monitoring estradiol levels in patients receiving abemaciclib concurrently with OFS., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Amyloidosis is a disease characterized by local and systemic extracellular deposition of amyloid protein fibrils where its excessive accumulation in tissues and resistance to degradation can lead to organ failure. Diagnosis is challenging because of approximately 36 different amyloid protein subtypes. Imaging methods like immunohistochemistry and the use of Congo red staining of amyloid proteins for laser capture microdissection combined with liquid chromatography tandem mass spectrometry (LMD/LC-MS/MS) are two diagnostic methods currently used depending on the expertise of the pathology laboratory. Here, we demonstrate a streamlined in situ amyloid peptide spatial mapping by Matrix Assisted Laser Desorption Ionization-Mass Spectrometry Imaging (MALDI-MSI) combined with Trapped Ion Mobility Spectrometry for potential transthyretin (ATTR) amyloidosis subtyping. While we utilized the standard LMD/LC-MS/MS workflow for amyloid subtyping of 31 specimens from different organs, we also evaluated the potential introduction in the MS workflow variations in data acquisition parameters like dynamic exclusion, or testing Data Dependent Acquisition combined with High-Field Asymmetric Waveform Ion Mobility Spectrometry (DDA FAIMS) versus Data Independent Acquisition (DIA) for enhanced amyloid protein identification at shorter acquisition times. We also demonstrate the use of Mascot's Error Tolerant Search and PEAKS de novo sequencing for the sequence variant analysis of amyloidosis specimens., (© 2024. The Author(s).)

Background: An erroneously high tacrolimus level was reported to a clinician. A root cause analysis investigation failed to determine the cause of the error. It was suspected that the incorrect preanalytical extraction reagent and procedure was used during testing; however, how this would affect the assayed drug concentration was unclear. Here we investigated the effect of the substitution of sirolimus, tacrolimus, and cyclosporine extraction reagents on assayed drug concentration., Methods: Tacrolimus, sirolimus, and cyclosporine concentration were measured on the Abbott Architect i2000 analyzer. Each assay requires a preanalytical extraction step, with a distinct reagent. We investigated the effect of the substitution of the extraction reagents and procedure between the 3 assays on the measured drug concentration. Two experiments were performed, one on samples of known drug concentration and one on samples with no drug present., Results: Substituting cyclosporine and sirolimus extraction procedures increased assayed tacrolimus concentrations from 5.6 to 8.47 (+51.25%) and 8.13 (+45.18%) ng/mL, respectively. Extraction procedure substitutions decreased assayed sirolimus from 13.63 to 4.60 (-66.25%) and 8.07 (-40.79%) ng/mL for cyclosporine and tacrolimus. Cyclosporine concentration increased from 274.60 to 391.30 (+42.50%) ng/mL using sirolimus extraction reagents and to 757.30 (+175.78%) ng/mL using tacrolimus extraction reagents. Cross-reactivity was observed between the tacrolimus assay and sirolimus and cyclosporine extraction reagents., Conclusions: Significant changes, both positive and negative, are observed in assayed drug concentration when incorrect extraction procedures are used in the Abbott i2000 tacrolimus, sirolimus, and cyclosporine assays. Preanalytic extraction procedures should be investigated when performing root cause analysis for erroneous therapeutic drug values., (© Association for Diagnostics & Laboratory Medicine 2024. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Objectives: Soluble B-Cell Maturation Antigen (sBCMA) is a degradation product of plasma cell-bound BCMA found in serum. Serum sBCMA concentrations correlate with bone marrow plasma cellularity, making it an attractive biomarker for monitoring plasma cell disorders, such as multiple myeloma. Here we evaluated the automated BCMA immunoassay for the ProteinSimple ELLA, for the analysis of sBCMA., Design & Methods: Inter and intra-run precision was assessed through replicate sBCMA measurements at 3 different concentration levels. Linearity was determined through serial dilution of a high sBMCA patient sample. Accuracy was assessed through split specimen analysis on two separate lots of reagents. Stability was assessed at 3 temperature levels over 14 days. Cross-reactivity was assessed on BCMA targeting and non-targeting chemotherapeutics. A reference range was established through the analysis of 146 healthy donor samples. The effect of endogenous interferents was assessed through spiking and recovery studies., Results: Inter and intra-run precision studies afforded CVs of <10% at all three concentration levels. Analytical measurement range was confirmed from 0.1 to 7 ng/mL. Accuracy studies afforded a slope of 0.976, intercept of 1.22, R 2 of 0.996. Assayed sBCMA values were unaffected by endogenous interferents and non-BMCA targeting antibodies. BCMA targeting therapeutics negatively affected assayed sBCMA concentrations. The reference range was established at 19-58 ng/mL sBCMA is analytically stable., Conclusions: The ProteinSimple ELLA sBCMA assay shows acceptable performance for the clinical assessment of sBCMA. The assay was highly affected by BCMA targeting therapeutics, thereby patients undergoing this therapy should not have their sBCMA levels assessed by this method., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 Published by Elsevier B.V.)

Background: The number of exposures to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and to vaccine antigens affect the magnitude and avidity of the polyclonal response., Methods: We studied binding and avidity of different antibody isotypes to the spike, the receptor-binding domain (RBD), and the nucleoprotein (NP) of wild-type (WT) and BA.1 SARS-CoV-2 in convalescent, mRNA vaccinated and/or boosted, hybrid immune individuals and in individuals with breakthrough cases during the peak of the BA.1 wave., Results: We found an increase in spike-binding antibodies and antibody avidity with increasing number of exposures to infection and/or vaccination. NP antibodies were detectible in convalescent individuals and a proportion of breakthrough cases, but they displayed low avidity. Omicron breakthrough infections elicited high levels of cross-reactive antibodies between WT and BA.1 antigens in vaccinated individuals without prior infection directed against the spike and RBD. The magnitude of the antibody response and avidity correlated with neutralizing activity against WT virus., Conclusions: The magnitude and quality of the antibody response increased with the number of antigenic exposures, including breakthrough infections. However, cross-reactivity of the antibody response after BA.1 breakthroughs, was affected by the number of prior exposures., Competing Interests: Potential conflicts of interest. The Icahn School of Medicine at Mount Sinai has filed patent applications relating to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) serological assays and Newcastle disease virus-based SARS-CoV-2 vaccines, which list F. K. as coinventor. A. F. B., C. C. C., and V. S. are also listed on the SARS-CoV-2 serological assays patent. Mount Sinai has spun out a company, Kantaro Biosciences, to market serological tests for SARS-CoV-2. F. K. has consulted for Merck and Pfizer (before 2020); is currently consulting for Pfizer, Seqirus, Third Rock Ventures, and Avimex; and is a cofounder and scientific advisory board member of CastleVax. The Krammer Laboratory is also collaborating with Pfizer on animal models of SARS-CoV-2. All other authors report no potential conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2023. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

In October 2020, the National Cancer Institute (NCI) Serological Sciences Network (SeroNet) was established to study the immune response to COVID-19, and "to develop, validate, improve, and implement serological testing and associated technologies" (https://www.cancer.gov/research/key-initiatives/covid-19/coronavirus-research-initiatives/serological-sciences-network). SeroNet is comprised of 25 participating research institutions partnering with the Frederick National Laboratory for Cancer Research (FNLCR) and the SeroNet Coordinating Center. Since its inception, SeroNet has supported collaborative development and sharing of COVID-19 serological assay procedures and has set forth plans for assay harmonization. To facilitate collaboration and procedure sharing, a detailed survey was sent to collate comprehensive assay details and performance metrics on COVID-19 serological assays within SeroNet. In addition, FNLCR established a protocol to calibrate SeroNet serological assays to reference standards, such as the U.S. severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) serology standard reference material and first WHO international standard (IS) for anti-SARS-CoV-2 immunoglobulin (20/136), to facilitate harmonization of assay reporting units and cross-comparison of study data. SeroNet institutions reported development of a total of 27 enzyme-linked immunosorbent assay (ELISA) methods, 13 multiplex assays, and 9 neutralization assays and use of 12 different commercial serological methods. FNLCR developed a standardized protocol for SeroNet institutions to calibrate these diverse serological assays to reference standards. In conclusion, SeroNet institutions have established a diverse array of COVID-19 serological assays to study the immune response to SARS-CoV-2 and vaccines. Calibration of SeroNet serological assays to harmonize results reporting will facilitate future pooled data analyses and study cross-comparisons. IMPORTANCE SeroNet institutions have developed or implemented 61 diverse COVID-19 serological assays and are collaboratively working to harmonize these assays using reference materials to establish standardized reporting units. This will facilitate clinical interpretation of serology results and cross-comparison of research data.

Objectives: Novel coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) virus has been declared a pandemic by the World Health Organization as of March 11, 2020. Pregnant women naturally have a reduced immune system due to immunological changes and decreased lung capacity due to respiratory adaptations, making them more susceptible to coronavirus complications. Within the Mount Sinai Health system, more than 15,000 deliveries are performed annually. We began to care for pregnant women with known COVID-19 infections in late March of 2020. In early April 2020, a policy was implemented to perform universal COVID-19 testing for all women planning to deliver within the Mount Sinai Health system. We examined the antibody response of postpartum women who delivered at Mount Sinai Hospital with a SARS-CoV-2 infection between the study intervals during March 15, 2020, through April 30, 2020., Study Design: This was a prospective observational study examining the immune response of pregnant women who delivered at Mount Sinai Hospital with a polymerase chain reaction (PCR)-confirmed SARS-CoV-2 infection. Women with a SARS-CoV-2 infection were contacted via phone to discuss participation in the study. Patients who consented were scheduled for a phlebotomy visit to assess their antibody titer levels to COVID-19. The COVID-19 enzyme-linked immunosorbent assay (ELISA) immunoglobulin (Ig)-G antibody test was used to evaluate the patients' antibody titers. The assay detects IgG antibodies for the detection of IgG seroconversion in patients following a known recent SARS-CoV-2 infection., Results: A total of 120 patients were identified with a documented SARS-CoV-2 infection who delivered within the prespecified time frame. Of those patients, 25 women agreed to participate and were included. Of them, 64.00% were Caucasian with a mean age of 35 years. The mean body mass index (BMI) was 30 kg/m 2 and the majority of patients had commercial insurance (88.00%). The majority of women were asymptomatic for COVID-19 at the time of admission (80.00%) and the average gestational age of delivery and diagnosis of COVID-19 was 39 weeks' gestation. The later the gestational age at the time of diagnosis, the lower the antibody titer response. When examining the interval from diagnosis to antibody titer analysis, patients with the highest titers (2,880) tended to have a shorter interval between their COVID-19 diagnosis and the time at which the titer level was drawn. Patients with symptoms on admission had similar antibody titer levels when compared with women who were asymptomatic., Conclusion: The antibody response among women infected with COVID-19 during pregnancy appears to be greater when the patients are diagnosed at an earlier gestational age., Key Points: · COVID-19 antibody status appears to be greater when diagnosed at an earlier gestational age.. · Asymptomatic and symptomatic pregnant women had similar antibody responses.. · Patients with the highest titers tended to have a shorter interval between their COVID-19 diagnoses.., Competing Interests: None declared., (Thieme. All rights reserved.)

A child with chronic granulomatous disease on vancomycin treatment had V trough levels that became undetectable, as measured in our hospital's clinical laboratory by a commonly employed particle-enhanced turbidometric inhibition assay. An alternative laboratory method yielded appropriate results. Recognizing and resolving erroneously low V trough levels could prevent needless adjustments in dosing that could increase risk for acute kidney injury., Competing Interests: The authors have no funding or conflicts of interest to disclose., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Background: Pandemics are unpredictable and can rapidly spread. Proper planning and preparation for managing the impact of outbreaks is only achievable through continuous and systematic collection and analysis of health-related data. We describe our experience on how to comply with required reporting and develop a robust platform for surveillance data during an outbreak., Materials and Methods: At Mount Sinai Health System, New York City, we applied Visiun, a laboratory analytics dashboard, to support main response activities. Epic System Inc.'s SlicerDicer application was used to develop clinical and research reports. We followed World Health Organization (WHO); federal and state guidelines; departmental policies; and expert consultation to create the framework., Results: The developed dashboard integrated data from scattered sources are used to seamlessly distribute reports to key stakeholders. The main report categories included federal, state, laboratory, clinical, and research. The first two groups were created to meet government and state reporting requirements. The laboratory group was the most comprehensive category and included operational reports such as performance metrics, technician performance assessment, and analyzer metrics. The close monitoring of testing volumes and lab operational efficiency was essential to manage increasing demands and provide timely and accurate results. The clinical data reports were valuable for proper managing of medical surge requirements, such as healthcare workforce and medical supplies. The reports included in the research category were highly variable and depended on healthcare setting, research priorities, and available funding. We share a few examples of queries that were included in the designed framework for research projects., Conclusion: We reviewed here the key components of a conceptual surveillance framework required for a robust response to COVID-19 pandemics. We demonstrated leveraging a lab analytics dashboard, Visiun, combined with Epic reporting tools to function as a surveillance system. The framework could be used as a generic template for possible future outbreak events., Competing Interests: There are no conflicts of interest., (Copyright: © 2022 Journal of Pathology Informatics.)

A novel clinical assay for the detection and quantitation of antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was adapted from an in-house, research-based enzyme-linked immunosorbent assay (ELISA). Development and validation were performed under regulatory guidelines, and the test obtained emergency use authorization (EUA) from the New York State Department of Health (NYSDOH) and the Food and Drug Administration (FDA). The Mount Sinai coronavirus disease 2019 (COVID-19) antibody assay is an orthogonal, quantitative direct ELISA test which detects antibodies reactive to the receptor binding domain (RBD) and the spike protein of the novel SARS-CoV-2. The assay is performed on 96-well plates coated with either SARS-CoV-2 recombinant RBD or spike proteins. The test is divided into two stages, a qualitative screening assay against RBD and a quantitative assay against the full-length spike protein. The test uses pooled high titer serum as a reference standard. Negative pre-COVID-19 and positive post-COVID-19, PCR-confirmed specimens were incorporated in each ELISA test run, and the assays were performed independently at two different locations. The Mount Sinai COVID-19 serology performed with high sensitivity and specificity, 92.5% (95% CI: 0.785-0.980) and 100% (CI: 0.939-1.000) respectively. Between-run precision was assessed with a single run repeated over 22 days; and within-run precision was assessed with 10 replicates per day over 22 days. Both were within reported acceptance criteria (CV ≤ 20%). This population-based study reveals the applicability and reliability of this novel orthogonal COVID-19 serology test for the detection and quantitation of antibodies against SARS-CoV-2, allowing a broad set of clinical applications, including the broad evaluation of SARS-CoV-2 seroprevalence and antibody profiling in different population subsets., Competing Interests: The authors declare the following conflict of interests: Mount Sinai has licensed serological assays to commercial entities and has filed for patent protection for serological assays. The Icahn School of Medicine at Mount Sinai has filed patent applications relating to the COVID-19 serological assay (“Serology Assay”) and NDV-based SARS-CoV-2 vaccines which list Florian Krammer (“Serology Assay”, vaccines), Daniel Stadlbauer (“Serology Assay”), Damodara Rao Mendu (“Serology Assay”), and Carlos Cordon-Cardo (“Serology Assay”) as co-inventors. The foundational “Serology Assay” intellectual property (IP) was licensed by the Icahn School of Medicine at Mount Sinai to commercial entities including Kantaro Biosciences, a company in which Mount Sinai has a financial interest. Florian Krammer consulted for Merck, Curevac, and Pfizer in the past (before 2020) and is currently consulting for Pfizer, Seqirus, and Avimex. The Krammer laboratory is collaborating with Pfizer on animal models of SARS-CoV-2., (© 2021 Published by Elsevier Ltd.)

Current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) serological tests are based on the full-length spike (S), the receptor-binding domain (RBD), or the nucleoprotein (NP) as substrates. Here, we used samples from healthcare workers (HCWs) to perform a longitudinal analysis of the antibody responses using a research-grade RBD and spike-based enzyme-linked immunosorbent assay (ELISA), a commercial RBD and spike-based ELISA, and a commercial NP-based chemiluminescent microparticle immunoassay. Seroprevalence ranged around 28% early during the pandemic and a good correlation was observed between RBD and spike-based ELISAs. Modest correlations were observed between NP and both RBD and spike-based assays. The antibody levels in HCWs declined over time; however, the overall seroprevalence measured by RBD and spike-based assays remained unchanged, while the seroprevalence of NP-reactive antibodies significantly declined. Moreover, RBD and spike-based assays effectively detected seroconversion in vaccinees. Overall, our results consolidate the strength of different serological assays to assess the magnitude and duration of antibodies to SARS-CoV-2., Competing Interests: The Icahn School of Medicine at Mount Sinai has filed patent applications relating to SARS-CoV-2 serological assays (the “Serology Assays”) and NDV-based SARS-CoV-2 vaccines which list Florian Krammer (the “Serology Assays”, vaccines) and Viviana Simon (“Serology Assays”) as co-inventors. The foundational “Serology Assay” intellectual property (IP) was licensed by the Icahn School of Medicine at Mount Sinai to commercial entities including Kantaro Biosciences, a company in which Mount Sinai has a financial interest. Kantaro manufactures and markets serologic tests based on the Mount Sinai IP. It is anticipated that the medical school will receive payments related to commercialization of the “Serology Assay” IP and, as faculty inventors, Drs. Krammer and Simon will be entitled to a portion of these payments. Florian Krammer consulted for Merck, Curevac and Pfizer in the past (before 2020) and is currently consulting for Pfizer, Seqirus and Avimex. The Krammer laboratory is collaborating with Pfizer on animal models of SARS-CoV-2., (© 2021 The Authors.)

Background: Biotin has been reported to interfere with several commonly used laboratory assays resulting in misleading values and possible erroneous diagnosis and treatment. This report describes a prospective study of possible biotin interference in thyroid-related laboratory assays, with a comparison of different commonly used assay platforms. Materials and Methods: Thirteen adult subjects (mean age 45 ± 13 years old) were administered biotin 10 mg/day for eight days. Blood specimens were collected at three time points on day 1 and on day 8 (baseline, two, and five hours after biotin ingestion). Thyrotropin (TSH), free triiodothyronine (fT3), free thyroxine (fT4), total triiodothyronine (TT3), total thyroxine (TT4), thyroxine binding globulin (TBG), and thyroglobulin (Tg) levels were analyzed with four different platforms: Abbott Architect, Roche Cobas 6000, Siemens IMMULITE 2000, and liquid chromatography with tandem mass spectrometry (LC-MS/MS). TSH, fT3, fT4, TT3, and TT4 were measured with Abbott Architect and Roche Cobas 6000. fT3, fT4, TT3, and TT4 were also measured by LC-MS/MS. Tg was measured by Siemens IMMULITE 2000. TBG was assessed with Siemens IMMULITE 2000. Results: Significant changes in TSH, fT4, and TT3 measurements were observed after biotin exposure when the Roche Cobas 6000 platform was used. Biotin intake resulted in a falsely lower Tg level when measurements were performed with Siemens IMMULITE 2000. At the time points examined, maximal biotin interference was observed two hours after biotin exposure both on day 1 and day 8. Conclusions: A daily dose of 10 mg was shown to interfere with specific assays for TSH, fT4, TT3, and Tg. Physicians must be aware of the potential risk of erroneous test results in subjects taking biotin supplements. Altered test results for TSH and Tg can be particularly problematic in patients requiring careful titration of levothyroxine therapy such as those with thyroid cancer.

Abstract: Elsberg syndrome is a rare cause of lumbosacral radiculitis with concomitant thoracic and lumbosacral myelitis that can be seen after an acute or reactivated viral infection. After the initial coronavirus surge in New York City, a 68-year-old man developed progressive lower extremity weakness and a defined sensory level at the lower abdomen. He had highly elevated SARS-CoV-2 IgG antibodies despite an absence of preceding COVID-19 symptoms. Serial electrodiagnostic testing revealed absent lower extremity late responses, with otherwise normal distal sensorimotor conductions. Electromyography revealed active neurogenic changes and reduced motor unit recruitment in the L3-L4 myotomes. Treatment with methylprednisolone and intravenous immunoglobulin was followed by minimal clinical improvement but re-emergence of the lower extremity late responses on electrodiagnostic testing. We report here, to the best of our knowledge, the first case of suspected COVID-19-associated Elsberg syndrome, which expands the spectrum of neuromuscular manifestations associated with SARS-CoV-2 infection and sheds light on ways to approach diagnostic and treatment options for these patients., Competing Interests: The authors report no conflicts of interest., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

The COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2, created an unprecedented need for comprehensive laboratory testing of populations, in order to meet the needs of medical practice and to guide the management and functioning of our society. With the greater New York metropolitan area as an epicenter of this pandemic beginning in March 2020, a consortium of laboratory leaders from the assembled New York academic medical institutions was formed to help identify and solve the challenges of deploying testing. This report brings forward the experience of this consortium, based on the real-world challenges which we encountered in testing patients and in supporting the recovery effort to reestablish the health care workplace. In coordination with the Greater New York Hospital Association and with the public health laboratory of New York State, this consortium communicated with state leadership to help inform public decision-making addressing the crisis. Through the length of the pandemic, the consortium has been a critical mechanism for sharing experience and best practices in dealing with issues including the following: instrument platforms, sample sources, test performance, pre- and post-analytical issues, supply chain, institutional testing capacity, pooled testing, biospecimen science, and research. The consortium also has been a mechanism for staying abreast of state and municipal policies and initiatives, and their impact on institutional and laboratory operations. The experience of this consortium may be of value to current and future laboratory professionals and policy-makers alike, in dealing with major events that impact regional laboratory services., Competing Interests: Declaration of Conflicting Interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (© The Author(s) 2021.)

Competing Interests: All authors have completed and submitted the International Committee of Medical Journal Editors form for disclosure of potential conflicts of interest. Mount Sinai has licensed serological assays to commercial entities and has filed for patent protection for serological assays. Daniel Stadlbauer and Florian Krammer are listed as inventors on the pending patent application. No other potential conflicts of interest were disclosed.

Passive transfer of antibodies from COVID-19 convalescent patients is being used as an experimental treatment for eligible patients with SARS-CoV-2 infections. The United States Food and Drug Administration's (FDA) guidelines for convalescent plasma initially recommended target antibody titers of 160. We evaluated SARS-CoV-2 neutralizing antibodies in sera from recovered COVID-19 patients using plaque reduction neutralization tests (PRNT) at moderate (PRNT50) and high (PRNT90) stringency thresholds. We found that neutralizing activity significantly increased with time post symptom onset (PSO), reaching a peak at 31-35 days PSO. At this point, the number of sera having neutralizing titers of at least 160 was approximately 93% (PRNT50) and approximately 54% (PRNT90). Sera with high SARS-CoV-2 antibody levels (>960 enzyme-linked immunosorbent assay titers) showed maximal activity, but not all high-titer sera contained neutralizing antibody at FDA recommended levels, particularly at high stringency. These results underscore the value of serum characterization for neutralization activity., (© The Author(s) 2020. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic with millions infected and more than 1 million fatalities. Questions regarding the robustness, functionality, and longevity of the antibody response to the virus remain unanswered. Here, on the basis of a dataset of 30,082 individuals screened at Mount Sinai Health System in New York City, we report that the vast majority of infected individuals with mild-to-moderate COVID-19 experience robust immunoglobulin G antibody responses against the viral spike protein. We also show that titers are relatively stable for at least a period of about 5 months and that anti-spike binding titers significantly correlate with neutralization of authentic SARS-CoV-2. Our data suggest that more than 90% of seroconverters make detectable neutralizing antibody responses. These titers remain relatively stable for several months after infection., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a new human disease with few effective treatments 1 . Convalescent plasma, donated by persons who have recovered from COVID-19, is the acellular component of blood that contains antibodies, including those that specifically recognize SARS-CoV-2. These antibodies, when transfused into patients infected with SARS-CoV-2, are thought to exert an antiviral effect, suppressing virus replication before patients have mounted their own humoral immune responses 2,3 . Virus-specific antibodies from recovered persons are often the first available therapy for an emerging infectious disease, a stopgap treatment while new antivirals and vaccines are being developed 1,2 . This retrospective, propensity score-matched case-control study assessed the effectiveness of convalescent plasma therapy in 39 patients with severe or life-threatening COVID-19 at The Mount Sinai Hospital in New York City. Oxygen requirements on day 14 after transfusion worsened in 17.9% of plasma recipients versus 28.2% of propensity score-matched controls who were hospitalized with COVID-19 (adjusted odds ratio (OR), 0.86; 95% confidence interval (CI), 0.75-0.98; chi-square test P value = 0.025). Survival also improved in plasma recipients (adjusted hazard ratio (HR), 0.34; 95% CI, 0.13-0.89; chi-square test P = 0.027). Convalescent plasma is potentially effective against COVID-19, but adequately powered, randomized controlled trials are needed.

Several studies have revealed that the hyper-inflammatory response induced by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a major cause of disease severity and death. However, predictive biomarkers of pathogenic inflammation to help guide targetable immune pathways are critically lacking. We implemented a rapid multiplex cytokine assay to measure serum interleukin (IL)-6, IL-8, tumor necrosis factor (TNF)-α and IL-1β in hospitalized patients with coronavirus disease 2019 (COVID-19) upon admission to the Mount Sinai Health System in New York. Patients (n = 1,484) were followed up to 41 d after admission (median, 8 d), and clinical information, laboratory test results and patient outcomes were collected. We found that high serum IL-6, IL-8 and TNF-α levels at the time of hospitalization were strong and independent predictors of patient survival (P < 0.0001, P = 0.0205 and P = 0.0140, respectively). Notably, when adjusting for disease severity, common laboratory inflammation markers, hypoxia and other vitals, demographics, and a range of comorbidities, IL-6 and TNF-α serum levels remained independent and significant predictors of disease severity and death. These findings were validated in a second cohort of patients (n = 231). We propose that serum IL-6 and TNF-α levels should be considered in the management and treatment of patients with COVID-19 to stratify prospective clinical trials, guide resource allocation and inform therapeutic options.

The COVID-19 pandemic caused by infection with Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has led to more than 100,000 deaths in the United States. Several studies have revealed that the hyper-inflammatory response induced by SARS-CoV-2 is a major cause of disease severity and death in infected patients. However, predictive biomarkers of pathogenic inflammation to help guide targetable immune pathways are critically lacking. We implemented a rapid multiplex cytokine assay to measure serum IL-6, IL-8, TNF-α, and IL-1β in hospitalized COVID-19 patients upon admission to the Mount Sinai Health System in New York. Patients (n=1484) were followed up to 41 days (median 8 days) and clinical information, laboratory test results and patient outcomes were collected. In 244 patients, cytokine measurements were repeated over time, and effect of drugs could be assessed. Kaplan-Meier methods were used to compare survival by cytokine strata, followed by Cox regression models to evaluate the independent predictive value of baseline cytokines. We found that high serum IL-6, IL-8, and TNF-α levels at the time of hospitalization were strong and independent predictors of patient survival. Importantly, when adjusting for disease severity score, common laboratory inflammation markers, hypoxia and other vitals, demographics, and a range of comorbidities, IL-6 and TNF-α serum levels remained independent and significant predictors of disease severity and death. We propose that serum IL-6 and TNF-α levels should be considered in the management and treatment of COVID-19 patients to stratify prospective clinical trials, guide resource allocation and inform therapeutic options. We also propose that patients with high IL-6 and TNF-α levels should be assessed for combinatorial blockade of pathogenic inflammation in this disease.

Objectives: Chyluria is a medical condition with presence of chyle in urine. The disease is most prevalent in South East Asian countries mostly caused by parasitic (Wuchereria bancrofti) infections. Our objective was to investigate the spontaneous remission of non-parasitic chyluria., Design and Methods: The spontaneous remission of non-parasitic chyluria cases were worked up with diagnostic investigations, clinical assessment and studied in detail with respect to their natural evolution., Results: We present two patients who were evaluated in the nephrology clinic with symptoms of milky urine and painless hematuria. Midnight blood smear was negative for filarial parasites. Urine culture was without mycobacteria. Urine cytology and IgG western blot for cysticercus were negative. Imaging for a lymphatic leak by lymphoscintigraphy was unrevealing. Chyluria resolved spontaneously in both patients., Conclusions: In our cases, radiologic visualization via lymphoscintigraphy was unrevealing. The patients were managed conservatively and fortunately underwent spontaneous remission marked by the disappearance of chyluria within several months of her initial diagnosis. In our opinion this spontaneous remission could be due to unrevealed lymphatico-renal fistula collapse or sclerosis of lymphatics caused by contrast media., (Copyright © 2017 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.)

Background: Very sensitive measurements of serum estrogens and testosterone are important in adult and pediatric endocrinology and immunoassays are known to lack the required performance at very low levels. Our aim was to develop a sensitive HPLC-MS/MS assay for both estradiol (E 2 ) and testosterone (Te) in serum without the need for chemical derivatization and using commercially available calibrators., Methods: Serum samples were prepared by the addition of internal standards followed by extraction using hexane:ethyl acetate. Chromatographic separation was achieved using a C 18 column and mass spectrometry was performed in both positive and negative ion modes., Results: The lower limits of quantitation (LLOQs) of E 2 and Te were 5pg/mL and 1ng/dL, respectively. The analytical measurement range (AMR) for E 2 was 5-600pg/mL and 1-1,170ng/dL for Te. Assay accuracy was determined both by comparison with a LC-MS/MS method performed at a national laboratory and the CDC HoSt program. Comparison with samples analyzed by both methods showed excellent correlation. Within-day (N=10) and between-day (N=20) CVs at concentrations spanning the AMR were less than 7% for both analytes., Conclusion: We have developed an accurate and highly sensitive assay to measure E 2 and Te levels in serum by HPLC-MS/MS without chemical derivatization and using commercially available calibrators., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Oral fluid (OF) offers a simple, non-invasive, directly observable sample collection for clinical and forensic drug testing. Given that chronic cannabis smokers often engage in drug administration multiple times daily, evaluating OF cannabinoid pharmacokinetics during ad libitum smoking is important for practical development of analytical methods and informed interpretation of test results. Eleven cannabis smokers resided in a closed research unit for 51 days, and underwent four, 5-day oral delta-9-tetrahydrocannabinol (THC) treatments. Each medication period was separated by 9 days of ad libitum cannabis smoking from 12:00 to 23:00 h daily. Ten OF samples were collected from 9:00-22:00 h on each of the last ad libitum smoking days (Study Days 4, 18, 32, and 46). As the number of cannabis cigarettes smoked increased over the study days, OF THC, cannabinol (CBN), and 11-nor-9-carboxy-THC (THCCOOH) also increased with a significant effect of time since last smoking (Δtime; range, 0.0-17.4 h) and ≥88% detection rates; concentrations on Day 4 were significantly lower than those on Days 32 and 46 but not Day 18. Within 30 min of smoking, median THC, CBN, and THCCOOH concentrations were 689 µg/L, 116 µg/L, and 147 ng/L, respectively, decreasing to 19.4 µg/L, 2.4 µg/L, and 87.6 ng/L after 10 h. Cannabidiol and 11-hydroxy-THC showed overall lower detection rates of 29 and 8.6%, respectively. Cannabinoid disposition in OF was highly influenced by Δtime and composition of smoked cannabis. Furthermore, cannabinoid OF concentrations increased over ad libitum smoking days, in parallel with increased cannabis self-administration, possibly reflecting development of increased cannabis tolerance., (Copyright © 2014 John Wiley & Sons, Ltd.)

Background: D-lactic acidosis, also referred as D-lactate encephalopathy, has been reported in patients with short bowl syndrome (SBS)., Case Report: The neurologic symptoms include altered mental status, slurred speech, and ataxia. Onset of neurological symptoms is accompanied by metabolic acidosis and high anion gap. We present here a case of D-lactic acidosis in a patient with acute lymphoblastic leukemia (ALL) who developed severe neurological symptoms and metabolic acidosis due to vancomycin-resistant enterococci (VRE) infection, and elevated D-lactic acid., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Oral fluid (OF) is an increasingly popular alternative matrix for drug testing, with cannabinoids being the most commonly identified illicit drug. Quantification of multiple OF cannabinoids and understanding differences in OF cannabinoid pharmacokinetics between frequent and occasional smokers improve test interpretation. The new Oral-Eze® OF collection device has an elution buffer that stabilizes analytes and improves drug recovery from the collection pad; however, its performance has not been independently evaluated. After controlled smoking of a 6.8% Δ(9) -tetrahydrocannabinol (THC) cannabis cigarette by frequent and occasional smokers, OF was collected with the Oral-Eze device for up to 30 h. Samples were analyzed for multiple cannabinoids by a validated 2D-GC-MS method. Frequent smokers had significantly greater OF THCCOOH concentrations than occasional smokers at all times, and showed positive results for a significantly longer time. We evaluated multiple cannabinoid cut-offs; the shortest last detection times were observed when THC ≥ 1 μg/L was combined with CBD or CBN ≥ 1 μg/L. With these cut-offs, last detection times(1-13.5 h) were not significantly different between groups, demonstrating suitability for short-term cannabinoid detection independent of smoking history. Cut-offs utilizing THC alone or combined with THCCOOH showed significantly different last detection times between groups. The widest detection windows were observed with THC ≥ 1 or 2 μg/L or THCCOOH ≥ 20 ng/L. Our data illustrate the effectiveness of the Oral-Eze® device for OF collection, the impact of self-administered smoked cannabis history on OF cannabinoid results, and the ability to improve interpretation and tailor OF cannabinoid cut-offs to fulfill the detection window needs of a given program., (Published 2014. This article is a U.S. Government work and is in the public domain in the USA.)

Oral fluid (OF) enables non-invasive sample collection for on-site drug testing, but performance of on-site tests with occasional and frequent smokers' OF to identify cannabinoid intake requires further evaluation. Furthermore, as far as we are aware, no studies have evaluated differences between cannabinoid disposition among OF collection devices with authentic OF samples after controlled cannabis administration. Fourteen frequent (≥4 times per week) and 10 occasional (less than twice a week) adult cannabis smokers smoked one 6.8% ∆(9)-tetrahydrocannabinol (THC) cigarette ad libitum over 10 min. OF was collected with the StatSure Saliva Sampler, Oral-Eze, and Draeger DrugTest 5000 test cassette before and up to 30 h after cannabis smoking. Test cassettes were analyzed within 15 min and gas chromatography-mass spectrometry cannabinoid results were obtained within 24 h. Cannabinoid concentrations with the StatSure and Oral-Eze devices were compared and times of last cannabinoid detection (t(last)) and DrugTest 5000 test performance were assessed for different cannabinoid cutoffs. 11-nor-9-Carboxy-THC (THCCOOH) and cannabinol concentrations were significantly higher in Oral-Eze samples than in Stat-Sure samples. DrugTest 5000 t(last) for a positive cannabinoid test were median (range) 12 h (4-24 h) and 21 h (1- ≥ 30 h) for occasional and frequent smokers, respectively. Detection windows in screening and confirmatory tests were usually shorter for occasional than for frequent smokers, especially when including THCCOOH ≥20 ng L(-1) in confirmation criteria. No differences in t(last) were observed between collection devices, except for THC ≥2 μg L(-1). We thus report significantly different THCCOOH and cannabinol, but not THC, concentrations between OF collection devices, which may affect OF data interpretation. The DrugTest 5000 on-site device had high diagnostic sensitivity, specificity, and efficiency for cannabinoids.

Background: Recently, high-dose oral synthetic delta-9-tetrahydrocannabinol (THC) was shown to alleviate cannabis withdrawal symptoms. The present data describe cannabinoid pharmacokinetics in chronic, daily cannabis smokers who received high-dose oral THC pharmacotherapy and later a smoked cannabis challenge., Methods: Eleven daily cannabis smokers received 0, 30, 60, or 120 mg/d THC for four 5-day medication sessions, each separated by 9 days of ad libitum cannabis smoking. On the fifth day, participants were challenged with smoking one 5.9% THC cigarette. Plasma collected on the first and fifth days was quantified by two-dimensional gas chromatography mass spectrometer for THC, 11-hydroxy-THC (11-OH-THC), and 11-nor-9-carboxy-THC (THCCOOH). Linear ranges (ng/mL) were 0.5-100 for THC, 1-50 for 11-OH-THC, and 0.5-200 for THCCOOH., Results: During placebo dosing, THC, 11-OH-THC, and THCCOOH concentrations consistently decreased, whereas all cannabinoids increased dose dependently during active dronabinol administration. THC increase over time was not significant after any dose, 11-OH-THC increased significantly during the 60- and 120-mg/d doses, and THCCOOH increased significantly only during the 120-mg/d dose. THC, 11-OH-THC, and THCCOOH concentrations peaked within 0.25 hours after cannabis smoking, except after 120 mg/d THC when THCCOOH peaked 0.5 hours before smoking., Conclusions: The significant withdrawal effects noted during placebo dronabinol administration were supported by significant plasma THC and 11-OH-THC concentration decreases. During active dronabinol dosing, significant dose-dependent increases in THC and 11-OH-THC concentrations support withdrawal symptom suppression. THC concentrations after cannabis smoking were only distinguishable from oral THC doses for 1 hour, too short a period to feasibly identify cannabis relapse. THCCOOH/THC ratios were higher 14 hours after overnight oral dronabinol abstinence but cannot distinguish oral THC dosing from the smoked cannabis intake.

Background: Oral Δ(9)-tetrahydrocannabinol (THC) is effective for attenuating cannabis withdrawal and may benefit treatment of cannabis use disorders. Oral fluid (OF) cannabinoid testing, increasing in forensic and workplace settings, could be valuable for monitoring during cannabis treatment., Methods: Eleven cannabis smokers resided on a closed research unit for 51 days and received daily 0, 30, 60, and 120 mg of oral THC in divided doses for 5 days. There was a 5-puff smoked cannabis challenge on the fifth day. Each medication session was separated by 9 days of ad libitum cannabis smoking. OF was collected the evening before and throughout oral THC sessions and analyzed by 2-dimensional GC-MS for THC, cannabidiol (CBD), cannabinol (CBN), 11-hydroxy-THC (11-OH-THC), and 11-nor-9-carboxy-THC (THCCOOH)., Results: During all oral THC administrations, THC OF concentrations decreased to ≤ 78.2, 33.2, and 1.4 μg/L by 24, 48, and 72 h, respectively. CBN also decreased over time, with concentrations 10-fold lower than THC, with none detected beyond 69 h. CBD and 11-OH-THC were rarely detected, only within 19 and 1.6 h after smoking, respectively. THCCOOH OF concentrations were dose dependent and increased over time during 120-mg THC dosing. After cannabis smoking, THC, CBN, and THCCOOH concentrations showed a significant dose effect and decreased significantly over time., Conclusions: Oral THC dosing significantly affected OF THCCOOH but minimally contributed to THC OF concentrations; prior ad libitum smoking was the primary source of THC, CBD, and CBN. Higher cannabinoid concentrations following active oral THC administrations vs placebo suggest a compensatory effect of THC tolerance on smoking topography.

Oral fluid (OF) is a valuable biological alternative for clinical and forensic drug testing. Evaluating OF to plasma (OF/P) cannabinoid ratios provides important pharmacokinetic data on the disposition of drug and factors influencing partition between matrices. Eleven chronic cannabis smokers resided on a closed research unit for 51 days. There were four 5-day sessions of 0, 30, 60, and 120 mg oral ∆(9)-tetrahydrocannabinol (THC)/day followed by a five-puff smoked cannabis challenge on Day 5. Each session was separated by 9 days ad libitum cannabis smoking. OF and plasma specimens were analyzed for THC and metabolites. During ad libitum smoking, OF/P THC ratios were high (median, 6.1; range, 0.2-348.5) within 1 h after last smoking, decreasing to 0.1-20.7 (median, 2.1) by 13.0-17.1 h. OF/P THC ratios also decreased during 5-days oral THC dosing, and after the smoked cannabis challenge, median OF/P THC ratios decreased from 1.4 to 5.5 (0.04-245.6) at 0.25 h to 0.12 to 0.17 (0.04-5.1) at 10.5 h post-smoking. In other studies, longer exposure to more potent cannabis smoke and oromucosal cannabis spray was associated with increased OF/P THC peak ratios. Median OF/P 11-nor-9-carboxy-THC (THCCOOH) ratios were 0.3-2.5 (range, 0.1-14.7) ng/μg, much more consistent in various dosing conditions over time. OF/P THC, but not THCCOOH, ratios were significantly influenced by oral cavity contamination after smoking or oromucosal spray of cannabinoid products, followed by time-dependent decreases. Establishing relationships between OF and plasma cannabinoid concentrations is essential for making inferences of impairment or other clinical outcomes from OF concentrations.

Ewing sarcoma family of tumors (ESFT) is a group of aggressive pediatric malignancies driven by the EWS-FLI1 fusion protein, an aberrant transcription factor up-regulating specific target genes, such as neuropeptide Y (NPY) and its Y1 and Y5 receptors (Y5Rs). Previously, we have shown that both exogenous NPY and endogenous NPY stimulate ESFT cell death via its Y1 and Y5Rs. Here, we demonstrate that this effect is prevented by dipeptidyl peptidases (DPPs), which cleave NPY to its shorter form, NPY(3-36), not active at Y1Rs. We have shown that NPY-induced cell death can be abolished by overexpression of DPPs and enhanced by their down-regulation. Both NPY treatment and DPP blockade activated the same cell death pathway mediated by poly(ADP-ribose) polymerase (PARP-1) and apoptosis-inducing factor (AIF). Moreover, the decrease in cell survival induced by DPP inhibition was blocked by Y1 and Y5R antagonists, confirming its dependence on endogenous NPY. Interestingly, similar levels of NPY-driven cell death were achieved by blocking membrane DPPIV and cytosolic DPP8 and DPP9. Thus, this is the first evidence of these intracellular DPPs cleaving releasable peptides, such as NPY, in live cells. In contrast, another membrane DPP, fibroblast activation protein (FAP), did not affect NPY actions. In conclusion, DPPs act as survival factors for ESFT cells and protect them from cell death induced by endogenous NPY. This is the first demonstration that intracellular DPPs are involved in regulation of ESFT growth and may become potential therapeutic targets for these tumors.

Background: Accurate measurement of free thyroxine (FT(4)) is important for diagnosing and managing thyroid disorders. Most laboratories measure FT(4) by direct analogue immunoassay methods. The validity of these methods have recently been questioned. The inverse log-linear relationship between FT(4) and thyroid-stimulating hormone (TSH) is well described and provides a physiological rationale on which to base an evaluation of FT(4) assays., Methods: The study included 109 participants for whom FT(4) measurement was requested by their clinician. Samples were selected for inclusion to reflect a wide spectrum of TSH and albumin results. FT(4) and TSH were measured by use of the Siemens Immulite immunoassay (IA). FT(4) was also measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS) (MS-FT(4))., Results: The inverse log-linear correlation coefficient between TSH and FT(4) was significantly better (P < 0.0001) for MS-FT(4) (0.84, 95% CI, 0.77-0.88) than for IA-FT(4) (0.45, 95% CI, 0.29-0.59). IA-FT(4) showed a significant correlation with albumin (Spearman correlation coefficient 0.45, 95% CI, 0.29-0.5, P < 0.0001) and thyroxine-binding globulin (TBG) (Spearman correlation coefficient 0.23, 95% CI, 0.05-0.41, P = 0.02). In contrast, FT(4) measurement by LC-MS/MS did not show a significant correlation with albumin or TBG., Conclusions: The inverse log-linear relationship between FT(4) and TSH was significantly better for FT(4) measured by LC-MS/MS than by IA. The MS-FT(4) method therefore provides FT(4) results that agree clinically with those obtained for TSH. Additionally, the significant correlation between IA-FT(4) with albumin and TBG suggests that this FT(4) method depends on binding protein concentrations and consequently does not accurately reflect FT(4).

Background: Cyclosporine (CsA) monitoring is essential for transplant success. We report a performance study of the recently released, fully automated Siemens ADVIA Centaur CsA assay., Methods: Whole blood samples from 248 transplant patients were prepared using a new 1-step extraction method. Performance evaluations vs. HPLC-tandem MS (LC-MS/MS), Abbott TDx and AxSYM assays were conducted according to CLSI EP5-A2 and EP9-A2 guidelines., Results: The correlation coefficient for LC-MS/MS and ADVIA Centaur was > or = 0.97 at each site, and for each transplant type. Regression analysis yielded y=0.94x+19 for all sites: 95% CI=0.91-0.96 (slope) and 10-28 (intercept). Absolute and relative bias was minimal for C0 and C2 sampling. Centaur vs. Abbott TDx and AxSYM assays: y = 0.72x+6, r = 0.98, 95% CI = 0.70-0.73 (slope), 3-9 (intercept); and y = 0.69x+18, r = 0.97, 95% CI = 0.67-0.71 (slope), 8-27(intercept). Within run CVs were 4.5%-7.1%, total CVs were 5.3%-7.7%., Conclusions: The ADVIA Centaur assay compared favorably with LC-MS/MS and Abbott assays, displaying good correlation for all transplant types and methods., (Copyright 2010 Elsevier B.V. All rights reserved.)

Objective: Levetiracetam and its acid metabolite have almost identical MRMs. They therefore need to be separated chromatographically prior to quantitation., Research Design and Methods: The sample is deproteinized with acetonitrile containing Ritonavir as internal standard, centrifuged and the supernatant diluted with water (1:2 v/v). Sixty microliters of the supernatant is injected into the LC-MS/MS and Levetiracetam (LEV) and LEV metabolite separated chromatographically at room temperature employing a Supelco C(18) column and a 0.1% formic acid methanol gradient at pH of 2.5., Results: The retention times for LEV metabolite, LEV and Ritonavir were 4.50, 5.38 and 9.18 min, respectively. Calibration curves in spiked plasma were linear over the concentration range of 0-50 microg/mL for LEV and 0.0-5.0 microg/mL for LEV metabolite. Intra- and inter-run imprecision (n=10) gave CVs of 2.3-4.7%, 3.4-8.9% for LEV and 2.9-3.9%, 3.3-7.4% for LEV metabolite. Recoveries of both LEV and LEV metabolite were close to 100%. Results for LEV were compared with those obtained by a commercial reference laboratory (r=0.974)., Conclusion: The procedure is reliable, quick, and inexpensive. LEV and LEV metabolite co-elute using C-18 columns at pHs >3.0 and previously published methods employing these conditions could therefore be subject to metabolite interference. In this method LEV and LEV metabolite are separated at pH 2.5. The total run time including the washing step is 10 min/sample, making this method suitable when moderate throughput is needed such as in clinical or commercial reference laboratories., (Copyright 2009 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.)

IbeA of Escherichia coli K1 was cloned, expressed and purified as a His(6)-tag fusion protein. The purified fusion protein inhibited E. coli K1 invasion of human brain microvascular endothelial cells and was heat-modifiable. The structural and functional aspects, along with equilibrium unfolding of IbeA, were studied in solution. The far-UV CD spectrum of IbeA at pH 7.0 has a strong negative peak at 215 nm, indicating the existence of beta-sheet-like structure. The acidic unfolding curve of IbeA at pH 2.0 shows the existence of a partially unfolded molecule (molten globule-like structure) with beta-sheet-like structure and displays strong 8-anilino-2-naphthyl sulfonic acid (ANS) binding. The pH dependent intrinsic fluorescence of IbeA was biphasic. At pH 2.0, IbeA exists in a partially unfolded state with characteristics of a molten globule-like state, and the protein is in extended beta-sheet conformation and exhibits strong ANS binding. Guanidine hydrochloride denaturation of IbeA in the molten globule-like state is noncooperative, contrary to the cooperativity seen with the native protein, suggesting the presence of two domains (possibly) in the molecular structure of IbeA, with differential unfolding stabilities. Furthermore, tryptophan quenching studies suggested the exposure of aromatic residues to solvent in this state. Acid denatured unfolding of IbeA monitored by far-UV CD is non-cooperative with two transitions at pH 3.0-1.5 and 1.5-0.5. At lower pH, IbeA unfolds to the acid-unfolded state, and a further decrease in pH to 2.0 drives the protein to the A state. The presence of 0.5 m KCl in the solvent composition directs the transition to the A state by bypassing the acid-unfolded state. Additional guanidine hydrochloride induced conformational changes in IbeA from the native to the A-state, as monitored by near- and far-UV CD and ANS-fluorescence.

Application of statistical experimental designs for optimization of fermentation parameters to enhance ethanol production, which is an economical and renewable energy source using Saccharomyces cerevisiae NCIM 3090 from palmyra jaggery, was studied in a batch fermentor. Using Plackett-Burman design, impeller speed, concentrations of CoCl2 and KH2PO4 were identified as significant variables, which highly influenced ethanol production, and these variables were further optimized using a central composite design (CCD). The ethanol production was adequately approximated with a full quadratic equation obtained from three factors and five levels of CCD. Maximum ethanol concentration of 132.56 g/l (16.8% [v/v]) was obtained for an impeller speed of 247.179 ( approximately 250) rev/min, CoCl2 of 0.263 g/l and KH2PO4 of 2.39 g/l. A second-order polynomial regression model was fitted and was found adequate with R 2 of 0.8952. This combined statistical approach enables rapid identification and investigation of significant parameters for improving the ethanol production and could be very useful in optimizing processes.

Human immunodeficiency virus type-1 (HIV-1) envelope glycoprotein gp140 interacts with its specific receptors on the surface of the target cells leading to cellular activation through various signaling pathways. The effect of blocking the chemokine repertoire in human brain microvascular endothelial cells in HIV dementia (HAD) disease has not been reported. Characterizing the nature of HIV-1 envelope protein gp140 (T-tropic, HXBc2) receptor binding conditions to HBMEC is critical to gain insight into the HIV dementia, and eventually to rationally design the agents to block envelope protein receptor interactions. HIV-1 gp140 oligomers were purified and separated to monomers, dimers, and trimers. The binding conditions of gp140 to HBMEC chemokine receptor, CXCR4, were optimized with an aim of understanding the structural interactions in HAD. Analysis of the interaction between HIV-1 gp140 and CXCR4 of HBMEC by saturation binding, cross-competition analysis with radiolabeled SDF and gp140, revealed a strong interaction, specificity between HIV-1 gp140 and CXCR4. Our binding data demonstrate that HIV-1 envelope protein gp140 enters cells by protein receptor mediated interactions that are regulated by the conformational state of the gp140 at physiological environment (pH and temperature). The CXCR4 antibody 12G5 inhibited SDF-1 binding to HBMEC indicating the specificity of gp140 binding to HBMEC. Scatchard analysis revealed the presence of approximately 70250 gp140 binding sites per cell with a K(d) of 4.5 nM. Cross-competition experiments using labeled SDF-1 and gp140 revealed that both unlabeled SDF-1 and gp140 are capable of displacing their radiolabeled counterparts. The binding assay conditions and radioligand binding assay are highly valuable to identify and design better HIV inhibitors for HAD.

Caffeine is widely used in children's hospitals to treat neonatal apnea. Amikacin is used for treating hospital-acquired infections caused by Gram-negative bacteria resistant to other aminoglycosides. The blood levels, however, have to be monitored carefully because of its ototoxicity and nephrotoxicity. Methotrexate (MTX) is used as a chemotherapeutic agent in the treatment of leukemia and lymphoma as well as of certain solid tumors. Current enzyme multiplied immunoassay technique (EMIT) assays for caffeine, amikacin, and MTX lack low-end precision. In addition, the EMIT assays for MTX lack the sensitivity of reliable quantification to 0.05 micromol/L needed because of today's more rigorous requirements. The goal of the present study was to optimize the EMIT method parameters on the Dimension RxL Max, thereby providing applications with improved precision for all the three analytes and enhancing the sensitivity of the EMIT methotrexate assay. Serum samples were measured for caffeine, amikacin, and MTX by EMIT on the Dimension RxL Max and by EMIT (on the Olympus AU 600 for caffeine) and fluorescence polarization immunoassay [for MTX and amikacin (FPIA; TDx FLx)] at Quest Diagnostics. The new instrument method parameters that use larger sample volumes and longer observation of optical density changes (caffeine, MTX) provide improved sensitivity for MTX permitting reliable measurement at 0.05 micromol/L and improved precision for all three analytes. Within- and between-day imprecision were less than 6% for low to high concentrations of caffeine and amikacin controls and are less than 7.5% for MTX concentrations greater than 0.3 micromol/L and 12.3% at 0.06 micromol/L. The correlation coefficients for caffeine, amikacin, and MTX plotted for the Dimension RxL Max versus the methods used at Quest Diagnostics were 0.973, 0.986, and 0.992, respectively. These EMIT method applications now compare well with other established assays. The new Dimension RxL Max method parameters provide greatly improved precision and also meet today's clinical sensitivity guidelines (0.05 micromol/L) for MTX.

Background: Levetiracetam (Keppra) is a novel antiepileptic drug recently approved by the U.S. Food and Drug Administration as an add-on therapy in the treatment of partial onset seizures in patients. We developed and describe a simple and rapid high performance liquid chromatography-electrospray tandem mass spectrometry (HPLC-ESI-MS/MS) assay for the determination of levetiracetam in human matrix (plasma, serum, or saliva)., Methods: An API-3000 or API-4000 triple-quadrupole mass spectrometer (Sciex, Concord, Canada) coupled with the IonSpray source and Shimadzu HPLC system (Shimadzu Scientific Instruments, Columbia, MD) was used employing ritonavir as internal standard (IS) for levetiracetam. One hundred microliters of serum (or plasma, saliva) was deproteinized by adding 150 microl of acetonitrile containing internal standard. After centrifugation, 100 microl of supernatant was diluted with 300 microl of water and 10 microl aliquot was injected onto a C-18 column. After a 2.5 min wash the valve was activated to initiate the isocratic elution program which eluted the levetiracetam and internal standard into the MS/MS system. Quantitation by MRM analysis was performed in the positive ion mode. Within-day and between-day imprecision were evaluated for levetiracetam using three levels of in-house controls. Reliability and accuracy of this method were assessed by comparison of targets with external QC material (ChromSystems), between laboratory comparisons and by recovery studies., Results: Within-day coefficients of variation (CVs) were <6.1% and between-day CVs were <8.2%. The average spiked recoveries of levetiracetam added to the drug-free human plasma samples were 108% at low concentration level and 103% at high concentration level., Conclusions: The method was found both specific and sensitive for the rapid and accurate measurement of levetiracetam in human matrices and correlated well with the Quest/Chantilly tandem mass spectrometric method (r=0.983).