Plasma/Serum Proteomics based on Mass Spectrometry.

Human blood is a window of physiology and disease. Examination of biomarkers in blood is a common clinical procedure, which can be informative in diagnosis and prognosis of diseases, and in evaluating treatment effectiveness. There is still a huge demand on new blood biomarkers and assays for precision medicine nowadays, therefore plasma/serum proteomics has attracted increasing attention in recent years. How to effectively proceed with the biomarker discovery and clinical diagnostic assay development is a question raised to researchers who are interested in this area. In this review, we comprehensively introduce the background and advancement of technologies for blood proteomics, with a focus on mass spectrometry (MS). Analyzing existing blood biomarkers and newly-built diagnostic assays based on MS can shed light on developing new biomarkers and analytical methods. We summarize various protein analytes in plasma/serum which include total proteome, protein post-translational modifications, and extracellular vesicles, focusing on their corresponding sample preparation methods for MS analysis. We propose screening multiple protein analytes in the same set of blood samples in order to increase success rate for biomarker discovery. We also review the trends of MS techniques for blood tests including sample preparation automation, and further provide our perspectives on their future directions.

Biomedical,clinical and environmental applications of platinum-based nanohybrids: An updated review.

Platinum nanoparticles (Pt NPs) have numerous applications in various sectors, including pharmacology, nanomedicine, cancer therapy, radiotherapy, biotechnology and environment mitigation like removal of toxic metals from wastewater, photocatalytic degradation of toxic compounds, adsorption, and water splitting. The multifaceted applications of Pt NPs because of their ultra-fine structures, large surface area, tuned porosity, coordination-binding, and excellent physiochemical properties. The various types of nanohybrids (NHs) of Pt NPs can be fabricated by doping with different metal/metal oxide/polymer-based materials. There are several methods to synthesize platinum-based NHs, but biological processes are admirable because of green, economical, sustainable, and non-toxic. Due to the robust physicochemical and biological characteristics of platinum NPs, they are widely employed as nanocatalyst, antioxidant, antipathogenic, and anticancer agents. Indeed, Pt-based NHs are the subject of keen interest and substantial research area for biomedical and clinical applications. Hence, this review systematically studies antimicrobial, biological, and environmental applications of platinum and platinum-based NHs, predominantly for treating cancer and photo-thermal therapy. Applications of Pt NPs in nanomedicine and nano-diagnosis are also highlighted. Pt NPs-related nanotoxicity and the potential and opportunity for future nano-therapeutics based on Pt NPs are also discussed.

Prediction of frozen virus stability based on degradation mechanisms, real-time data and modeling.

Aim: Critical virus reagents in regulated bioanalytical assays require stability monitoring. Although stability at ultra-low frozen temperatures is generally assumed, published data are limited and real-time studies are time consuming. Materials & methods: The authors reviewed literature data, typical mechanisms of molecular degradation, glass transition temperatures of commonly used buffers and available real-time storage data to model frozen virus reagent stability. Results: Storage at ultra-low temperatures below the glass transition temperature was critical for virus stability. Modeling of real-time data suggested that virus potency remained within 0.5 log10 of its starting potency at a probability of >99, 90 and 73% after 10, 20 and 30 years, respectively. Conclusion: The study supports the practice of virus storage at -70°C or below for 20-30 years.

A rapid LC-MS/MS assay for detection and monitoring of underivatized branched-chain amino acids in maple syrup urine disease.

Introduction: Quantitation of the isomeric branched-chain amino acids (BCAA; valine, alloisoleucine, isoleucine, leucine) is a challenging task that typically requires derivatization steps or long runtimes if a traditional chromatographic method involving a ninhydrin ion pairing reagent is used. Objectives: To develop and perform clinical validation of a rapid, LC-MS/MS-based targeted metabolomics assay for detection and monitoring of underivatized BCAA in human plasma. Methods: Various columns and modes of chromatography were tested. The final optimized method utilized mixed mode chromatography with an Intrada column under isocratic condition. Sample preparation utilized the 96-well format. Briefly, extraction solvent containing the internal standard is added to 20 uL of sample, followed by shaking and positive pressure filtering, and the resulting extracted sample is analyzed. The assay was validated based on accepted quality standards (e.g., CLIA and CLSI) for clinical assays. Results: The method is linear over a wide range of concentrations, 2.0-1500 µM, with LOD of 0.60 µM and LOQ of 2.0 µM. The precision of the assay was 4-10% across analytes. The method was also validated against reference laboratories via blinded split-sample analysis and demonstrated good agreement with accuracy: 89-95% relative to the external group mean. Conclusion: We have developed a method that is accurate, rapid, and reliable for routine clinical testing of patient sample BCAA, which is used in the diagnosis and management of maple syrup urine disease (MSUD). The assay also has desirable characteristics, such as short run time, small sample volume requirement, simple sample preparation without the need for derivatization, and high throughput.

The impact of RNA extraction method on accurate RNA sequencing from formalin-fixed paraffin-embedded tissues.

BACKGROUND: Utilization of RNA sequencing methods to measure gene expression from archival formalin-fixed paraffin-embedded (FFPE) tumor samples in translational research and clinical trials requires reliable interpretation of the impact of pre-analytical variables on the data obtained, particularly the methods used to preserve samples and to purify RNA. METHODS: Matched tissue samples from 12 breast cancers were fresh frozen (FF) and preserved in RNAlater or fixed in formalin and processed as FFPE tissue. Total RNA was extracted and purified from FF samples using the Qiagen RNeasy kit, and in duplicate from FFPE tissue sections using three different kits (Norgen, Qiagen and Roche). All RNA samples underwent whole transcriptome RNA sequencing (wtRNAseq) and targeted RNA sequencing for 31 transcripts included in a signature of sensitivity to endocrine therapy. We assessed the effect of RNA extraction kit on the reliability of gene expression levels using linear mixed-effects model analysis, concordance correlation coefficient (CCC) and differential analysis. All protein-coding genes in the wtRNAseq and three gene expression signatures for breast cancer were assessed for concordance. RESULTS: Despite variable quality of the RNA extracted from FFPE samples by different kits, all had similar concordance of overall gene expression from wtRNAseq between matched FF and FFPE samples (median CCC 0.63-0.66) and between technical replicates (median expression difference 0.13-0.22). More than half of genes were differentially expressed between FF and FFPE, but with low fold change (median |LFC| 0.31-0.34). Two out of three breast cancer signatures studied were highly robust in all samples using any kit, whereas the third signature was similarly discordant irrespective of the kit used. The targeted RNAseq assay was concordant between FFPE and FF samples using any of the kits (CCC 0.91-0.96). CONCLUSIONS: The selection of kit to purify RNA from FFPE did not influence the overall quality of results from wtRNAseq, thus variable reproducibility of gene signatures probably relates to the reliability of individual gene selected and possibly to the algorithm. Targeted RNAseq showed promising performance for clinical deployment of quantitative assays in breast cancer from FFPE samples, although numerical scores were not identical to those from wtRNAseq and would require calibration.

Development of a CDC-certified total testosterone assay for adult and pediatric samples using LC-MS/MS.

BACKGROUND: Highly accurate and sensitive method to measure testosterone in hypogonadal male, female and children is vital for proper diagnosis of hormone-related conditions and their treatment. OBJECTIVE: To develop an accurate and robust total testosterone ESI-LC-MS/MS quantification method with a simple sample preparation workflow and sufficient sensitivity for serum or plasma samples of all gender and age groups, via ketone functional group derivatization (using Amplifex™ Keto Reagent). METHOD: A simple sample preparation method to accommodate both low and high numbers of samples was developed using simultaneous protein precipitation and derivatization with Amplifex™ Keto reagent, followed by centrifugation and direct injection of supernatant into an LC-MS/MS system (SCIEX Topaz™ IVD LC-MS/MS, in which MS is equivalent to a SCIEX 4500MD Mass Spectrometer). Total testosterone in human serum or plasma samples was quantified using an external calibration curve generated by calibrators spanning a broad concentration range of ∼1-2000 ng/dL (10-20,000 pg/mL), traceable to NIST 971 SRM. 13C3-enriched testosterone was used as an internal standard to correct for both analyte loss during sample preparation and matrix effect during analysis (Supplementary Information: SI Fig. 4C). Two methods, one using a 96-well filter plate and another using Eppendorf tubes, were developed. Both methods were certified by the Centers for Disease Control (CDC) hormone standardization (HoSt) program for total serum testosterone. The feasibility of implementing the method for plasma and serum samples was tested via a small-scale method comparison study between matched pediatric serum and plasma samples derived from the same donor. In addition, plasma samples originating from the same donor collected in two different anticoagulant tube types (Li-heparin and K2EDTA) were compared. RESULTS: Using in-house formulated NIST 971-traceable calibrators, the method was linear (r2 > 0.999) between 1 and 2000 ng/dL (10 and 20,000 pg/mL) with a limit of detection of approximately 1 ng/dL (10 pg/mL). The testosterone concentration bias against 40 reference samples from the HoSt certification program was absolute <3% with an average %CV of ∼3-4%. More than 78% of samples passed the CDC bias criterion of ±6.4%. Comparison between pediatric matched serum and plasma samples resulted in high correlation (r2 = 0.997) and bias of <5%. The calculated % difference between matched adult serum and plasma samples was ∼1%. CONCLUSIONS: Feasibility for an accurate and streamlined method suitable for measuring total testosterone in all human samples was demonstrated with a choice of sample preparation workflow to suit low or high number of samples. The method can potentially be used for plasma matrix from different blood collection tubes (Li-Heparin and K2EDTA).

Efficacy of an agonist of α-MSH, the palmitoyl tetrapeptide-20, in hair pigmentation.

OBJECTIVE: Hair greying (i.e., canities) is a component of chronological ageing and occurs regardless of gender or ethnicity. Canities is directly linked to the loss of melanin and increase in oxidative stress in the hair follicle and shaft. To promote hair pigmentation and reduce the hair greying process, an agonist of α-melanocyte-stimulating hormone (α-MSH), a biomimetic peptide (palmitoyl tetrapeptide-20; PTP20) was developed. The aim of this study was to describe the effects of the designed peptide on hair greying. METHODS: Effect of the PTP20 on the enzymatic activity of catalase and the production of H2 O2 by Human Follicle Dermal Papilla Cells (HFDPC) was evaluated. Influence of PTP20 on the expression of melanocortin receptor-1 (MC1-R) and the production of melanin were investigated. Enzymatic activity of sirtuin 1 (SIRT1) after treatment with PTP20 was also determined. Ex vivo studies using human micro-dissected hairs allowed to visualize the effect of PTP20 on the expression in hair follicle of catalase, TRP-1, TRP-2, Melan-A, ASIP, and MC1-R. These investigations were completed by a clinical study on 15 human male volunteers suffering from premature canities. RESULTS: The in vitro and ex vivo studies revealed the capacity of the examined PTP20 peptide to enhance the expression of catalase and to decrease (30%) the intracellular level of H2 O2 . Moreover, PTP20 was shown to activate in vitro and ex vivo the melanogenesis process. In fact, an increase in the production of melanin was shown to be correlated with elevated expression of MC1-R, TRP-1, and Melan-A, and with the reduction in ASIP expression. A modulation on TRP-2 was also observed. The pivotal role of MC1-R was confirmed on protein expression analysed on volunteer's plucked hairs after 3 months of the daily application of lotion containing 10 ppm of PTP20 peptide. CONCLUSION: The current findings demonstrate the ability of the biomimetic PTP20 peptide to preserve the function of follicular melanocytes. The present results suggest potential cosmetic application of this newly designed agonist of α-MSH to promote hair pigmentation and thus, reduce the hair greying process.

Truncated aptamers for total and glycated hemoglobin, and their integration into a graphene oxide-based fluorometric method for high-throughput screening for diabetes.

The authors describe the identification of an effective binding region of aptamers against glycated (HbA1c) and total haemoglobin (tHb) by using a fluorometric assay. Truncation of the originally selected aptamers from 60 to 46 and 34 nucleotides for HbA1c and tHb, respectively, enhances the affinity for their targets. Moreover, shortening the aptamer sequences leads to a better conformational change after target binding which enabled the integration of the aptamers in a graphene oxide (GO)-based fluorometric assay. First, fluorescein-labelled truncated aptamers were physically absorbed onto the surface of GO surface via π-stacking interaction. This leads to quenching of fluorescence. Once the truncated aptamers bind the target protein, a conformational change is induced which results (a) )in the release of the aptamers from the surface of GO and (b) in the restoration of green fluorescence that is measured at 515 nm. The assay can be carried out in a microtiter plate format in homogeneous solution, this avoiding the steps of immobilization, incubation, and washing that are often necessary in immunoassays. This also reduces the time and the costs of the overall assay and allows for high throughput screening for diabetes. HbA1c can be detected in the range from 5.4 to 10.6%. The assay is selective for HbA1c over other proteins that commonly exist in blood. The results obtained by using this method compare well with those of a turbidimetric immunoassay that is typically applied in clinical laboratories. Graphical abstract Truncated aptamers for total and glycated hemoglobin were selected and integrated into a graphene oxide-based fluorescence detection assay for high-throughput screening for diabetes.

Rapid and Reliable Detection of Alkaline Phosphatase by a Hot Spots Amplification Strategy Based on Well-Controlled Assembly on Single Nanoparticle.

The first appeal of clinical assay is always accurate and rapid. For alkaline phosphatase (ALP) monitoring in medical treatment, a rapid, reliable surface-enhanced Raman scattering (SERS) test kit is designed based on a "hot spots" amplification strategy. Consisting of alkyne-tagged Au nanoparticles (NPs), Ag+, and enzyme substrate, the packaged test kit can achieve one-step clinical assay of ALP in human serum within several minutes, while the operation is simple as it directly inputs the sample into the test kit. Here, Ag+ ions are adsorbed onto the surface of Au core due to electrostatic interaction between Ag+ and the negatively charged donor surface, then enzymatic biocatalysis of ALP triggers the reduction of Ag+ and subsequently silver growth occurs on every Au core surface in a controllable manner, forming "hot spots" between the Au core and Ag shell, in which the SERS signal of alkyne Raman reporters would be highly amplified. Meanwhile, ALP mediates a redox reaction of Ag+ as well as the dynamic silver coating process so the increase of SERS intensity is well-controlled and can be recognized with increasing amounts of the targets. Instead of conventional NP aggregation, this leads to a more reproducible result. In particular, the distinct Raman emission from our self-synthesized alkyne reporter is narrow and stable with zero background in the Raman silent region, suffering no optical fluctuation from biosystem inputs and the detection results are therefore reliable with a limit of detection of 0.01 U/L (2.3 pg/mL). Along with ultrahigh stability, this SERS test kit therefore is an important point-of-care candidate for a reliable, efficacious, and highly sensitive detection method for ALP, which potentially decreases the need for time-consuming clinical trials.

Prognostic Validation of SKY92 and Its Combination With ISS in an Independent Cohort of Patients With Multiple Myeloma.

BACKGROUND: High risk and low risk multiple myeloma patients follow a very different clinical course as reflected in their PFS and OS. To be clinically useful, methodologies used to identify high and low risk disease must be validated in representative independent clinical data and available so that patients can be managed appropriately. A recent analysis has indicated that SKY92 combined with the International Staging System (ISS) identifies patients with different risk disease with high sensitivity. PATIENTS AND METHODS: Here we computed the performance of eight gene expression based classifiers SKY92, UAMS70, UAMS80, IFM15, Proliferation Index, Centrosome Index, Cancer Testis Antigen and HM19 as well as the combination of SKY92/ISS in an independent cohort of 91 newly diagnosed MM patients. RESULTS: The classifiers identified between 9%-21% of patients as high risk, with hazard ratios (HRs) between 1.9 and 8.2. CONCLUSION: Among the eight signatures, SKY92 identified the largest proportion of patients (21%) also with the highest HR (8.2). Our analysis also validated the combination SKY92/ISS for identification of three classes; low risk (42%), intermediate risk (37%) and high risk (21%). Between low risk and high risk classes the HR is >10.

A novel sandwich electrochemiluminescence immunosensor for ultrasensitive detection of carbohydrate antigen 19-9 based on immobilizing luminol on Ag@BSA core/shell microspheres.

A novel sandwich-type electrochemiluminescence immunosensor based on immobilizing luminol on Ag@BSA core/shell microspheres (Ag@BSA-luminol) for ultrasensitive detection of tumor marker carbohydrate antigen 19-9 (CA19-9) has been developed. Herein, magnetic carbon nanotubes (MAGCNTs) decorated with polyethylenimine (PEI) was used to construct the base of the immunosensor. MAGCNTs with prominent electrical conductivity and high surface area could be beneficial for promoting the electron transfer and loading plenty of primary antibodies (Ab1) via glutaraldehyde (GA). Meanwhile, the magnetic property of MAGCNTs makes it easy to be attached to the surface of magnetic glass carbon electrode (MGCE) through magnetism interaction, which provides an outstanding platform for this immunosensor. Moreover, Ag@BSA microspheres with large surface area, good stability, and excellent biocompatibility were desirable candidates for effective cross-link of CA19-9 detection antibodies (Ab2). A more interesting thing was that ELISA color reaction was used as an ultrasensitive strategy for identifying Ab2 was successfully coated on Ag@BSA with the naked eye. Additionally, we immobilized the luminol on the surface of Ag@BSA to prepare the target immunosensor. Immobilization of luminol on the surface of Ag@BSA could decrease the distance between luminophores and the electrode surface, leading to great enhancement of the ECL intensity of luminol in the present of hydrogen peroxide (H2O2). Under the optimal conditions, the intensity of the ECL immunosensor increased linearly with the logarithm of CA19-9 concentration in a wide linear range from 0.0005 to 150UmL(-1) with a detection limit of 0.0002UmL(-1) (S/N=3). All the results suggested the prepared CA19-9 immunosensor displayed high sensitivity, excellent stability and good specificity. The developed method opened a new avenue to clinical bioassay.

Cancer Biomarkers Discovery and Validation: State of the Art, Problems and Future Perspectives.

Cancer is one of the major public health problems worldwide representing the leading cause of morbidity and mortality in industrialized countries. To reduce cancer morbidity and mortality as well as to facilitate the evolution from the traditional "one size fits all" strategy to a new "personalized" cancer therapy (i.e., the right drug to the right patient at the right time, using the right dose and schedule), there is an urgent need of reliable, robust, accurate and validated cancer biomarker tests.Unfortunately, despite the impressive advances in tumor biology research as well as in high-powerful "omics" technologies, the translation of candidate cancer biomarkers from bench to bedside is lengthy and challenging and only a few tumor marker tests have been adopted successfully into routine clinical care of oncologic patients.This chapter provides an updated background on biomarkers research in oncology, including biomarkers clinical uses, and discusses the problems of discovery pipeline, biomarkers failures and future perspectives.

Development and validation of CALR mutation testing for clinical diagnosis.

OBJECTIVES: To validate a diagnostic assay for detecting CALR mutations in the clinical setting. METHODS: Traditional polymerase chain reaction (PCR) was performed on DNA previously extracted from 60 specimens (30 bone marrow aspirates [BMAs] and 30 peripheral blood [PB] samples) from 55 patients. Nearly all reported CALR mutations are insertions or deletions in exon 9. Therefore, we performed amplicon sizing by capillary electrophoresis and fragment length analysis (FLA) to determine mutation status. Mutations were confirmed by Sanger sequencing. RESULTS: Fourteen samples from 10 patients with JAK2 and MPL wild-type myeloproliferative neoplasms were positive for CALR mutation. Detected mutations included a 52-base pair (bp) deletion (n = 6), a 5-bp insertion (n = 2), a 31-bp deletion (n = 1), and a 61-bp deletion (n = 1). Sanger sequencing of 15 samples showed 100% concordance. Matched patient PB and BMA samples (n = 5) harbored identical mutations, and samples run multiple times (n = 8) showed 100% reproducibility. CONCLUSIONS: We conclude that CALR mutations may be quickly and accurately detected by FLA of PCR amplicons by capillary electrophoresis. These methods are routine procedures for most molecular laboratories and should allow for straightforward incorporation of the CALR assay into the clinical diagnostic testing menu.

Experiencia en enfermería, ensayo clínico y atención a pacientes con cáncer de pulmón / Experience in nursing, clinical assay and care of patients with lung cancer

INTRODUCCIÓN: los ensayos clínicos con nuevas sustancias, como anticuerpos y vacunas han tenido un incremento debido a la necesidad de encontrar nuevas terapéuticas para el tratamiento del cáncer. En la aplicación del tratamiento el éxito depende del buen desempeño profesional, el nivel de preparación y capacitación que tenga el personal de enfermería, como parte del equipo de investigación al ejecutar un rol importante. OBJETIVO: evaluar la experiencia del personal de enfermería en este ensayo relacionado con la atención a pacientes con cáncer de pulmón. MÉTODOS: se realizó un estudio descriptivo con una revisión bibliográfica sobre el actuar, y la experiencia de enfermería en el ensayo clínico. Se evaluó la seguridad de CIMAvax®- EGF en pacientes avanzados con tumores del pulmón, de células no pequeñas y tratados en la asistencia primaria de salud. Para ello se realizó un análisis documental de la buena práctica clínica, y los protocolos de estudios sobre el tema. RESULTADOS: fueron incluidos 11 pacientes. El promedio de diagnóstico y tratamiento de pacientes, con cáncer de pulmón estuvo en 23 y 14 meses al respecto. CONCLUSIONES: enfermero (a) en su desempeño debe tener dominio del protocolo en su totalidad sobre el producto, propiedades farmacéuticas y dosis a administrar. Además, identificar las reacciones adversas que pueden presentarse y las medidas a tomar para de esta forma lograr evaluar la efectividad del nuevo producto en investigación y mitigar cualquier riesgo que influya en la calidad y excelencia de nuestros servicios de salud.

INTRODUCTION: an increase in clinical assays of new substances, such as antibodies and vaccines, has taken place in response to the need to find new cancer therapies. Success in the application of treatment depends on the good professional performance, background and training of nursing personnel, for they play an important role as research team members. OBJECTIVE: evaluate the experience of nursing personnel in an assay about the care of patients with lung cancer. METHODS: a descriptive study was conducted based on a bibliographic review of the role and experience of nurses in the clinical assay performed. The safety of CIMAvax®- EGF was evaluated in patients with advanced non-small-cell lung tumors treated at primary health care services. For this purpose a document analysis was performed about good clinical practice and study protocols about the subject. RESULTS: eleven patients were included. Average diagnosis and treatment of patients with lung cancer was 23 and 14 months, respectively. CONCLUSIONS: nurses should be thoroughly aware of the entire protocol, particularly the product, its pharmaceutical properties and the dose to administer. They should also be able to identify the potential adverse reactions and the measures to take to assess the effectiveness of the new product under research, as well as mitigate any risk affecting the quality and excellence of our health services.

Validation and clinical use of a sensitive HIV-2 viral load assay that uses a whole virus internal control.

BACKGROUND: Human immunodeficiency virus type 2 (HIV-2) is distantly related to the more widespread HIV-1. Although HIV-2 infection is rare in the U.S., cases are concentrated in the Northeast. No FDA-approved HIV-2 viral load assays exist. A clinically validated laboratory-developed assay is currently available in the U.S., however it is not currently approved for use on New York State patients. OBJECTIVE: To develop a sensitive viral load assay to quantify HIV-2 RNA in plasma and to validate it for clinical use. METHODS: The real-time RT-PCR assay simultaneously amplifies HIV-2 and a whole virus internal control, added during the lysis step. Two extraction volumes can be used. Results are reported in HIV-2 RNA International Units (IU). RESULTS: The assay has a limit of detection of 7 IU/mL and a lower limit of quantification of 29 IU/mL. The assay detects multiple strains of HIV-2 group A and B and generates reproducible results. Samples exchanged with a comparator laboratory produced similar viral load results, with 74% of positives differing by <0.5 log10 IU/mL. To date, we have tested 52 clinical specimens from 25 individuals. Twenty-eight (54%) specimens had measurable HIV-2 viral loads (range: 1.63-5.14 log10 IU/mL), 10 (19%) were positive but not quantifiable, and 14 were negative. HIV-2 RNA was detected in at least one specimen from 19 of 25 (76%) individuals tested. CONCLUSIONS: We developed a sensitive and accurate HIV-2 viral load assay. Validation data indicate the assay is suitable for clinical use and its availability in New York State will improve clinical monitoring of HIV-2 infected patients.

High-throughput asparaginase activity assay in serum of children with leukemia.

Asparaginase is an antineoplastic agent used in combination therapy for acute lymphoblastic leukemia (ALL). The asparaginase activity measured in serum reflects the effectiveness of the drug. However, the wide inter-individual variability in the pharmacokinetics of asparaginase suggests that the serum activity should be closely monitored in patients during therapy. In order to identify patients with low asparaginase exposure during treatment, a fast, sensitive, and high-throughput assay is required for measuring asparaginase activity in patient sera. In this study, asparaginase activity was determined by monitoring the enzymatically-coupled oxidation of reduced nicotinamide adenine dinucleotide (NADH) to NAD(+) in a 96-well format. The rate of disappearance of NADH (ΔmOD/minute) was directly proportional to the activity of asparaginase, and the linear range of the assay was established from 0.025 to 2.2 IU/mL (R(2) = 0.998) with a reportable range that was extended to 4.0 IU/mL by dilution with serum albumin. Inter-assay precision was established (low control CV% = 8.8, high control CV% = 9.0), as was intra-assay precision (low control CV% = 3.3, high control CV% = 2.7). The method is high-throughput and provides a broader linear range of detection compared to previously described assays. The speed, ease, and accuracy of the assay make it suitable for assessing serum asparaginase activity after standard doses of native E. coli, Erwinia, and PEGylated E. coli asparaginase given to children during the treatment of leukemia.

Estudo randomizado, duplamente mascarado, placebo controlado do uso do misoprostol versus placebo para histeroscopia diagnostica em mulheres na pos-menopausa / A randomized, double-masked, placebo-controlled study of the use of misoprostol versus placebo for diagnostic hysteroscopy in postmenopausal women

A histeroscopia é um dos procedimentos mais realizados em ginecologia. Muitos ensaios controlados sugerem vantagens no uso prévio de misoprostol para diminuir a resistência da cérvix uterina; entretanto este tipo de estudo ainda é escasso quando se compara o uso do misoprostol prévio à histeroscopia diagnóstica em mulheres na pós-menopausa. avaliar os efeitos intra e pós-operatórios em mulheres na pós-menopausa, submetidas à histeroscopia diagnóstica, sem anestesia, com uso prévio de misoprostol para amadurecimento do colo uterino. Sujeitos e métodos: foi realizado um estudo tipo ensaio clínico, duplamente mascarado, randomizado, em pós-menopausadas que se submeteram à histeroscopia diagnóstica sem anestesia no IMIP e no hospital Barão de Lucena, em Recife, Pernambuco. Foram incluídas 120 pacientes alocadas aleatoriamente em dois grupos, sendo 60 com uso de 200µg de misoprostol via vaginal e 60 com uso de 200µg de placebo. Foram estudadas as seguintes variáveis: tempo do procedimento, freqüência e intensidade da dor durante o exame, necessidade de dilatação cervical adicional, efeitos colaterais (sangramento genital, náuseas, vômitos, diarréia, hipertermia) e complicações (perfuração uterina, falso pertuito, laceração cervical, infecções, dor no pós-operatório imediato). Para análise estatística, utilizaram-se os testes de qui-quadrado de associação, testes exato de Fisher e Mann-Whitney para comparação dos grupos, considerando-se significativo um erro alfa menor que 5 por cento. Resultados: os grupos foram semelhantes em relação...

Hysteroscopy is one of the most useful procedure to study uterine cavity and widely used in gynecologic clinic. Although many controlled clinical trials have shown advantages in the use of misoprostol in realeasing cérvix uterine resistanse, there are few evidence regarding its use before office hysteroscopy in postmenopausal women. Objective: to compare the trans and postoperative results of postmenopausal women underwent office hysteroscopy without anesthesia that used misoprostol or placebo previouslly to the procedure to mature the uterine cérvix in two teaching hospitals in Recife. Patients and methods: a randomized, double masked, clinical trial was conducted enrolling 120 postmenopausal women who had been submitted to office hysteroscopy without anesthesia at IMIP and Barão de Lucena teaching hospital. Among those patients 60 were randomized allocated in the study group using 200µg of vaginal misoprostol and 60 took part in the placebo group using 200µg of a vaginal placebo.The following variable were studied: duration of the procedure, frequency and intensity of pain during the exame, necessity of additional dilatation, side effect as (genital bleeding, nauseas, vomits, diarrhea, and hyperthermia) and complications (uterine perforation, false way, cervical laceration, infections and pain in the imediate postoperatory period). For analysis statistics we used the tests of association qui-square, accurate test of Fisher and Mann-Whitney for comparison of the groups, considering itself significant with lesser alpha error that 5 por cento. Results: We found no significant statistic difference between the...