Assay Parameters Research Articles

Adequate Antithrombin III Level Predicts Survival in Severe COVID-19 Pneumonia.

Critically ill patients with COVID-19 are at an increased thrombotic risk, hence thromboprophylaxis with heparin is considered mandatory. Antithrombin III (ATIII) is the most potent endogenous anticoagulant and is required for the clinical efficacy of heparin. Profound hypercoagulable and inflammatory state associated with COVID-19 can result in decreased ATIII levels and ineffective heparin treatment resulting in increased mortality. The present study evaluated ATIII levels in critically ill patients of COVID-19 and correlated them with other coagulation parameters and disease outcomes. A retrospective review of those critically ill COVID-19 patients was performed who were on a therapeutic dose of low molecular weight heparin (LMWH) and had serial measurements of ATIII, anti-factor Xa (antiFXa) assay and other routine coagulation parameters. A total of 27 critically ill COVID-19 patients were identified, out of these, 12 survived and 15 had disease-induced mortality. ATIII levels were found to be significantly lower in non-survivors on the third day of serial measurement along with worsening of other coagulation parameters. AntiFXa levels were found to be higher in non-survivors as compared to survivors. Further studies are required to establish ATIII as a prognostic marker and to determine the utility of monitoring antiFXa levels in COVID-19 patients on LMWH therapy.

APPLICATION OF MICROCOLUMN HPLC-UV FOR RAPID QUANTITATIVE ANALYSIS OF ARBUTIN, BERGEN-IN AND GALLIC ACID IN BERGENIA CRASSIFOLIA

This work is aimed at the development of a microcolumn HPLC-UV assay for the rapid quantitative analysis of arbutin, bergenin, and gallic acid in Bergenia crassifolia. The results obtained indicate appropriate metrological parameters of the developed assay. It was found that the known methods of quantitative analysis of phenolic glycosides using SPE-spectrophotometry cannot be characterized as selective and accurate, due to the fact that the presences of the impurity compounds that do not belong to the group of phenolic glycosides negatively influenced the results. The developed assay was used for quantitative analysis of wild and commercial samples of B. crassifolia raw material. It was found that the content of arbutin, bergenin, and gallic acid in samples of B. crassifolia rhizomes collected in the Republic of Buryatia was 38.58–45.97, 66.74–139.76 and 1.22–1.65 mg/g, respectively, and for commercial batches of raw materials 20.57–41.37, 35.04–83.94 and 0.22–1.28 mg/g, respectively. It was found that the process of gradual enzymatic changes in the color of B. crassifolia leaves (green, red, black) leads to significant changes in the chemical composition. The most pronounced phenomenon is the degradation of bergenin, the presence of which is noted only in green leaves. The concentration of gallic acid is reduced in black leaves. Arbutin is characterized by an increased content in red leaves (102.02 mg/g). Additionally, a quantitative analysis of the peduncles and flowers of B. crassifolia was realized, and it was shown that they are distinguished by a high content of arbutin 48.40 and 42.15 mg/g, respectively, as well as bergenin in flowers (16.89 mg/g). The study demonstrated that the developed technique can be applied for a quick, selective, and accurate quantitative analysis of three compounds in various organs of B. crassifolia.

Characteristics, Yield, Flow Cytometer, DNA Fragmentation, and Comet Assay Parameters of Goat Spermatozoa in Semen of Zaraibi and Baladi Bucks at Young and Old Ages

The aim of this study was to determine goat breed (Zaraibi and Baladi) or buck age (young and old) effect on characteristics, production, flow cytometer, DNA fragmentation and comet assay parameters of spermatozoa. Twelve Baladi (BG) and Zaraibi (ZG) bucks, 3 old (2-4 years) and 3 young (8-12 months) from BG or ZG used for semen collection for 24 weeks. Volume (EV), sperm progressive motility (PM), livability (LS), abnormality (SA), and concentration (SCC) were evaluated, sperm outputs (SO) were calculated. Sperm apoptosis, DNA fragmentation and comet assay were analyzed. Results revealed that PM, LS, SA, SCC, total, motile, live, normal, and functional SO per ejaculate were significantly higher in ZG than BG and in old than young bucks in each breed. Both EV and LS were significantly higher, while SCC was significantly lower in old than young ZG. Each of EV, PM, and SCC was significantly higher, while SA was significantly lower in old than young BG. Viable sperm, haploid and cell cycle percent was significantly higher, while early apoptotic, apoptotic, and necrotic spermatozoa percent was significantly lower in old than young bucks of each breed. Spermatid percent in ZG and diploid percent in BG were significantly higher in old than young bucks. Tailed sperm, tail length, tail DNA percentage, and tail moment were significantly lower in ZG than BG, and in old than young bucks of each breed. It can be stated that Zaraibi bucks has a good potential for semen production than Baladi breed.

Deep learning from HE slides predicts the clinical benefit from adjuvant chemotherapy in hormone receptor-positive breast cancer patients

We hypothesized that a deep-learning algorithm using HE images might be capable of predicting the benefits of adjuvant chemotherapy in cancer patients. HE slides were retrospectively collected from 1343 de-identified breast cancer patients at the Samsung Medical Center and used to develop the Lunit SCOPE algorithm. Lunit SCOPE was trained to predict the recurrence using the 21-gene assay (Oncotype DX) and histological parameters. The risk prediction model predicted the Oncotype DX score > 25 and the recurrence survival of the prognosis validation cohort and TCGA cohorts. The most important predictive variable was the mitotic cells in the cancer epithelium. Of the 363 patients who did not receive adjuvant therapy, 104 predicted high risk had a significantly lower survival rate. The top-300 genes highly correlated with the predicted risk were enriched for cell cycle, nuclear division, and cell division. From the Oncotype DX genes, the predicted risk was positively correlated with proliferation-associated genes and negatively correlated with prognostic genes from the estrogen category. An integrative analysis using Lunit SCOPE predicted the risk of cancer recurrence and the early-stage hormone receptor-positive breast cancer patients who would benefit from adjuvant chemotherapy.

Optimization and Validation of a New Microbial Inhibition Test for the Detection of Antimicrobial Residues in Living Animals Intended for Human Consumption.

Even though antibiotics are necessary in livestock production, they can be harmful not only due to their toxicity, but also in view of their contribution to the emergence of antimicrobial resistance. Screening tests based on microbial growth inhibition appeared to be useful tools to prevent its entry into the food chain. They have nevertheless been traditionally carried out post mortem, leading to great economical loss and harm to the environment in case a positive sample is found. Hence, the objective was to evaluate the use of a screening test as an ante mortem alternative for the detection of antibiotic residues in meat: thus, Explorer®-Blood test was optimized and validated. After adapting the procedure for matrix preparation, the assay parameters were assessed from 344 antibiotic-free blood serum samples. Limits of Detection (LoDs) were defined by spiking blood serum with several of the most common antimicrobials used in veterinary practice. LoDs were similar to those obtained for meat and were at or below the maximum residue limits set by EU legislation for muscle. Analyses of in vivo injected samples, previously characterized by LC-MS/MS, demonstrated the method’s accuracy and proved that Explorer®-Blood can be considered a suitable alternative to conventional post mortem screening methods.

Farmers exposed to pesticides have almost five times more DNA damage: a meta-analysis study.

We carried out a meta-analytical review of possible DNA damage resulting from occupational exposure to pesticides in farmers in the scientific literature. After the search, screening, and eligibility criteria steps, we included 42 studies to analyze random effect calculation. DNA damage in farmers occupationally exposed to pesticides represents an effect of SMD 4.63 [CI 95% 3.94-5.32; p <0.001]. We observed a high heterogeneity rate between the studies and an asymmetry of the bias analysis results. We performed a meta-regression on the parameters. The Olive Tail Moment (OTM) was the most effective comet assay parameter in the evaluated studies. The Damage Index (DI) was more conservative and highlighted the variability between studies caused by distinct methodologies that showed more significant effects and greater deviations. An analysis of confounding factors demonstrated a slight DNA damage in smokers who were occupationally exposed to pesticides compared to nonsmokers, indicating genotoxicity but smaller than the pesticide effect. The present study shows the greater risk that occupationally exposed rural workers have of developing related diseases due to pesticides' genotoxic effect.

Towards a functional model of drug sensitivity in advanced-stage ovarian cancer: clinical correlation of drug sensitivity assays in organoid tissue cultures

Objectives: Though many ovarian cancer patients respond to primary treatment, a significant portion will not respond and are considered ‘platinum resistant/refractory.’ Such patients experience a significantly worse prognosis relative to those who are ‘platinum sensitive.’ Pre-treatment prediction of platinum sensitivity has eluded scientists, which forces clinicians to undergo a ‘trial and error’ method of treatment by which resistant patients are forced to try and fail a futile line of therapy. Patient-derived organoid (PDO) cultures represent a potentially powerful functional model which preserves tumor heterogeneity (Hill et al, 2018). Here, we describe the successful culture of 4 PDOs from patients with advanced stage ovarian carcinoma and their correlation with clinical treatment outcome. Methods: Fresh tissue was obtained from the patients’ debulking surgery. It was digested, and a cell pellet was created. The pellet was washed several times, re-suspended in Matrigel, plated, and grown at 37oC over several days. PDOs were treated with standard doses of carboplatin (1 µM) and paclitaxel (10 nM). The percent of viable cells at 72 hours was measured using the CellTiter-Glo 3D platform (Promega, Madison, WI). The patients’ clinical charts were then reviewed 6 months after the completion of primary chemotherapy to determine clinical platinum sensitivity as defined by clinical parameters. Results: PDOs were successfully obtained from each of the four patients with advanced stage ovarian carcinoma. Three patients had primary disease, and 1 patient was being treated for a recurrence. The patient with recurrent disease received chemotherapy immediately prior to her debulking surgery, and the rest underwent primary debulking surgery with chemotherapy to follow. Three patients had high grade serous histology, and 1 patient had low grade serous histology. Three patients ultimately had platinum resistant disease, and their PDOs all demonstrated >50% cell viability in response to carboplatin and paclitaxel with 2 resistant PDOs demonstrating a remarkable 90% and 95% cell viability. The PDO from the patient with platinum sensitive disease by clinical criteria demonstrated 49% cell viability in response to treatment (Table 1). Download : Download high-res image (123KB) Download : Download full-size image Conclusions: We describe the successful creation of PDOs from fresh tissue obtained from debulking surgery in advanced stage ovarian cancer patients. If we use ≥50% cell viability as our initial, preliminary sensitivity threshold, results from the PDO drug sensitivity assay correlate with clinical platinum sensitivity in this pilot feasibility. We propose to confirm the predictability of drug sensitivity thresholds in future, larger studies using PDOs. Currently, there is no standardized drug sensitivity assay or response parameters defined as predictable for PDOs. Such work will be integral in the formation of a functional model of drug sensitivity, which can be used in the clinic to predict platinum sensitivity without requiring a patient to try and fail futile treatments.

Optimization and Validation of an In Vitro Standardized Glycogen Phosphorylase Activity Assay.

Glycogen phosphorylase (GP) is a key enzyme in the glycogenolysis pathway and a potential therapeutic target in the management of type 2 diabetes. It catalyzes a reversible reaction: the release of the terminal glucosyl residue from glycogen as glucose 1-phosphate; or the transfer of glucose from glucose 1-phosphate to glycogen. A colorimetric method to follow in vitro the activity of GP with usefulness in structure-activity relationship studies and high-throughput screening capability is herein described. The obtained results allowed the choice of the optimal concentration of enzyme of 0.38 U/mL, 0.25 mM glucose 1-phosphate, 0.25 mg/mL glycogen, and temperature of 37 °C. Three known GP inhibitors, CP-91149, a synthetic inhibitor, caffeine, an alkaloid, and ellagic acid, a polyphenol, were used to validate the method, CP-91149 being the most active inhibitor. The effect of glucose on the IC50 value of CP-91149 was also investigated, which decreased when the concentration of glucose increased. The assay parameters for a high-throughput screening method for discovery of new potential GP inhibitors were optimized and standardized, which is desirable for the reproducibility and comparison of results in the literature. The optimized method can be applied to the study of a panel of synthetic and/or natural compounds, such as polyphenols.

Large-Scale Data Mining of Rapid Residue Detection Assay Data From HTML and PDF Documents: Improving Data Access and Visualization for Veterinarians.

Extra-label drug use in food animal medicine is authorized by the US Animal Medicinal Drug Use Clarification Act (AMDUCA), and estimated withdrawal intervals are based on published scientific pharmacokinetic data. Occasionally there is a paucity of scientific data on which to base a withdrawal interval or a large number of animals being treated, driving the need to test for drug residues. Rapid assay commercial farm-side tests are essential for monitoring drug residues in animal products to protect human health. Active ingredients, sensitivity, matrices, and species that have been evaluated for commercial rapid assay tests are typically reported on manufacturers' websites or in PDF documents that are available to consumers but may require a special access request. Additionally, this information is not always correlated with FDA-approved tolerances. Furthermore, parameter changes for these tests can be very challenging to regularly identify, especially those listed on websites or in documents that are not publicly available. Therefore, artificial intelligence plays a critical role in efficiently extracting the data and ensure current information. Extracting tables from PDF and HTML documents has been investigated both by academia and commercial tool builders. Research in text mining of such documents has become a widespread yet challenging arena in implementing natural language programming. However, techniques of extracting tables are still in their infancy and being investigated and improved by researchers. In this study, we developed and evaluated a data-mining method for automatically extracting rapid assay data from electronic documents. Our automatic electronic data extraction method includes a software package module, a developed pattern recognition tool, and a data mining engine. Assay details were provided by several commercial entities that produce these rapid drug residue assay tests. During this study, we developed a real-time conversion system and method for reflowing contents in these files for accessibility practice and research data mining. Embedded information was extracted using an AI technology for text extraction and text mining to convert to structured formats. These data were then made available to veterinarians and producers via an online interface, allowing interactive searching and also presenting the commercial test assay parameters in reference to FDA-approved tolerances.

Rosmarinic acid relieves cisplatin-induced ovary toxicity in female mice via suppression of oxidative stress and inflammation.

Rosmarinic acid having potential anti-inflammatory and free radical scavenging activity. We examined the chemotherapeutic effect of rosmarinic against cisplatin (CIS)-induced ovarian toxicity via modulation of oxidative stress and inflammation. Swiss BALB mice used in experimental protocol and mice were divided into different groups. Intraperitoneal injection of CIS (7 mg/kg) was used for ovarian cancer induction. The rats were received rosmarinic acid (2.5, 5, and 10 mg/kg, body weight) treatment for 22 weeks. Body weight, ovary weight food, and water intake were estimated at regular time intervals. Hormonal and antioxidant parameters were estimated in the ovary tissue and serum at the end of the study. Cytokines, inflammatory, and apoptosis parameters were determined at the end of the study. Finally, the ovary tissue histopathology was performed at end of the experimental study. Rosmarinic acid significantly (p < 0.001) improved the body weight and reduced the ovary weight. Rosmarinic acid considerably reduced the hormonal assay parameters, such as antimullerian hormone,estradiol, luteinizing hormone,and follicle-stimulating hormonecompared to model control mice. Rosmarinic treatment significantly (p < 0.001) reduced the level of nitric oxide, myeloperoxidase, and boosted the level of antioxidant parameters, such as glutathione, superoxide dismutase, catalase,and glutathione peroxidaseinserum and ovary tissue. Rosmarinic acid downregulated the cytokines like interleukin-6, tumor necrosis factor-α, interleukin-1β; inflammatory parameter includes prostaglandin E2 , cyclooxygenase-2,and inducible nitric oxide synthaseat a dose-dependently. Ovary tissue histopathology showed improvement after rosmarinic acid treatment. The result suggests that rosmarinic acid is a protective effect in ameliorating CIS-induced ovary toxicity via alteration of inflammatory and apoptosis parameters.

Optimization of the feeding rate of Anopheles farauti s.s. colony mosquitoes in direct membrane feeding assays

BackgroundDirect membrane feeding assays (DMFA) are an important tool to study parasite transmission to mosquitoes. Mosquito feeding rates in these artificial systems require optimization, as there are a number of factors that potentially influence the feeding rates and there are no standardized methods that apply to all anopheline species.MethodsA range of parameters prior to and during direct membrane feeding (DMF) were evaluated for their impact on Anopheles farauti sensu stricto feeding rates, including the starving conditions and duration of starving prior to feeding, membrane type, DMF exposure time, mosquito age, feeding in the light versus the dark, blood volume, mosquito density and temperature of water bath.ResultsThe average successful DMFA feeding rate for An. farauti s.s. colony mosquitoes increased from 50 to 85% when assay parameters were varied. Overnight starvation and Baudruche membrane yielded the highest feeding rates but rates were also affected by blood volume in the feeder and the mosquito density in the feeding cups. Availability of water during the pre-feed starvation period did not significantly impact feeding rates, nor did the exposure duration to blood in membrane feeders, the age of mosquitoes (3, 5 and 7 days post-emergence), feeding in the light versus the dark, or the temperature (34 °C, 38 °C, 42 °C and 46 °C) of the water bath.ConclusionOptimal feeding conditions in An. farauti s.s. DMFA were to offer 50 female mosquitoes in a cup (with a total surface area of ~ 340 cm2 with 1 mosquito/6.8 cm2) that were starved overnight 350–500 µL of blood (collected in heparin-coated Vacutainer tubes) per feeder in feeders with a surface area ~ 5 cm2 (with a maximum capacity of 1.5 mL of blood) via a Baudruche membrane, for at least 10–20 min.Graphical

Feasibility of singlicate-based analysis in bridging ADA assay on Meso-Scale Discovery platform: comparison with duplicate analysis.

Aim: To investigate the feasibility of singlicate analysis in anti-drug antibody (ADA) assay by comparing performance characteristics for assays qualified in duplicate and singlicate formats. Materials & methods: We employed modeling to assess and quantify the impact of singlicate to cut point factor (CPF) in scenarios with the duplicate precision from 1-20% and the proportion of well-to-well variance to overall assay variance from 0.01-0.90. The impact to CPF by singlicate is marginal if the well-to-well coefficient of variation is <10% and represents <25% of the overall variability. Results & conclusion: The assay parameters including sensitivity, precision, selectivity, drug and target tolerance were comparable between singlicate and duplicate based assays. Our results suggested the minimal impact of singlicate analysis on ADA assay with good duplicate precision. The study provided additional supportive evidence that the singlicate-based analysis is feasible in ADA ligand bindingassays.

Impact of Dietary or Drinking Water Ruminococcus sp. Supplementation and/or Heat Stress on Growth, Histopathology, and Bursal Gene Expression of Broilers.

This research was conducted to evaluate the impact of dietary or drinking water Ruminococcus sp. supplementation and/or heat stress (HS) on the growth, serum biochemistry, tissue antioxidant, phagocytic assay, histopathology, and bursa gene expression of broilers. Day-old broiler chicks were allotted into six groups according to HS and/or Ruminococcus with or without enzyme supplementation. The first group was the control one, with a formulated diet and normal environmental temperature but without any supplement. The second group fed on Ruminococcus-supplemented diet (1 kg/kg diet). The third group fed on a formulated diet without supplement, and Ruminococcus and digestive enzymes were given in drinking water (0.1 ml/L). The fourth one was the heat stress group, with a normal formulated diet. The fifth and the sixth groups served as second and third groups, respectively, but with heat stress. The results of this experiment indicated that thermal temperature negatively affected the parameters of growth performance, serum biochemical, tissue antioxidants, and phagocytic assay. Moreover, heat stress led to pathological lesions in the internal organs and affected the expression of some genes related to heat stress, including proapoptotic genes such as caspase8 and bax, inflammatory genes such as NF-κβ1, and heat shock protein such as HSP 70 in the bursal tissue. These bad effects and abnormalities were mitigated by Ruminococcus alone or with enzyme supplementation, which improved all the above-mentioned parameters.

Effect of extrusion technology on hempseed (Cannabis sativa L.) oil cake: Polyphenol profile and biological activities.

Effects of extrusion with varying barrel temperature, moisture content, and screw speed on hempseed oil cake were studied for the first time. Extrusion at lower moisture (30%) and higher screw speed (300rpm) significantly increased the proportion of free polyphenols, flavonoids, and phenylpropionamide content, and -glucosidase and acetylcholinesterase inhibition activities. Full factorial design confirmed the three-way interactions among all extrusion parameters for all chemical assays with the bound phenolic fraction, total flavonoid content, and DPPH inhibition activity of the free phenolic fraction. HPLC-DAD-ESI-QTOF-MS/MS analysis tentatively identified 26 phenylpropionamides, and the contents of N-trans-caffeoyltyramine (66.26µg/g) and total phenylpropionamides (85.77µg/g) were significantly increased after extrusion at the lower moisture and higher screw speed extrusion conditions. The higher -glucosidase inhibition activity at higher screw speed could be due to the N-trans-caffeoyltyramine (r=0.99, p<0.01), while the AChE inhibition activity appeared to be influenced more by the cannabisins A-C, M (r>0.8, p<0.01). PRACTICAL APPLICATION: Hempseed oil cake is a byproduct of oil extraction, with high protein and high fiber contents. The results of this research could be used directly in food industry to improve the nutritional and commercial value of hempseed oil cake by extrusion technology.

A Bispecific Antibody Lacks Measurability in Routine Coagulation Assays and Comparability to Factor VIII

Background : Novel therapeutic approaches for hemophilia A include the bispecific antibody emicizumab (ACE910), for which clinical phase III results were recently reported (Oldenburg et al. NEJM 2017). During preclinical and clinical development, ACE910's activity was tracked using thrombin generation assay (TGA), chromogenic assay, and activated partial thromboplastin time assay (aPTT) (Shima et al. NEJM 2016, Kitazawa et al. Nat Med. 2012). The most suitable method to test the efficacy of non-factor therapy in a routine setting is not yet known.Aim: To examine the effect of assay type on hemostatic activity monitoring of a sequence identical analogue (SIA) to ACE910, and to assess FVIII-activity equivalence in chromogenic and aPTT assays, and in TGA.Methods : SIA ACE910 was expressed in HEK293 cells, purified as previously described (Sampei et al. PLoS One. 2013), and analyzed using the following five commercial FVIII chromogenic assay kits: Coatest factor VIII, Coamatic factor VIII, Siemens FVIII:C, Technochrom FVIII:C, and Biophen FVIII:C. aPTT or clot waveform was analyzed using five differently composed trigger reagents (Dapttin, Actin FSL, Pathromtin SL, Triniclot, and APTT-SP). In thrombin generation assays, SIA was tested in comparison with recombinant FVIII (ADVATE) using extrinsic (TF) and intrinsic (FXIa) trigger.Depending on method, SIA concentrations ranged from 2-1200 nM.Results: SIA ACE910's lack of cross-reactivity to bovine factors, which are commonly used in chromogenic assay kits, rendered only the Biophen FVIII:C test, which uses human coagulation factors, suitable for analysis. However, as the concentration response curve of SIA ACE910 maxed out and deviated from the FVIII reference curve, the chromogenic assay was not suitable to assess FVIII-equivalence. The aPTT test was highly sensitive to SIA ACE910, resulting in a substantial reduction in clotting time at low and potentially non-therapeutic concentrations. Different aPTT reagents yielded a FVIII-equivalence of 18-69% for 20 nM SIA and >200% at ≥200 nM SIA ACE910 - the plasma concentrations observed in patients in the phase III trial. SIA ACE910 (600 nM) only partially restored thrombin generation (TG) in hemophilia A patient plasma, resulting in a FVIII-equivalence of 4-8% (intrinsic) and 16-36% (extrinsic) based on peak thrombin. Assessment of other TG parameters demonstrated in FVIII-equivalence of <1 to >100% . Assay results for 60, 200, and 600 nM SIA are summarized in Table 1.Conclusions : Analysis of SIA and its equivalence to FVIII is challenged by its strongly differing mechanism, rendering standard FVIII protocols unsuitable.Results are highly influenced by assay type, analytical conditions, and parameters used for calculation. Chromogenic assays, aPTT, and TGA give largely discrepant values. Thus, new methods and likely a product specific standard are required to better predict the hemostatic effect of non-factor therapies. [Display omitted] DisclosuresHartmann:Shire: Employment. Feenstra:Shire: Employment. Knappe:Shire: Employment. Schrenk:Shire: Employment. Scheiflinger:Shire: Employment, Equity Ownership; Baxter: Equity Ownership. Dockal:Baxter: Equity Ownership, Patents & Royalties; Shire: Employment, Equity Ownership; Baxalta: Patents & Royalties.

Utility of Global Coagulation Assays in Myeloproliferative Neoplasm: Thromboelastography, Thrombin Generation and Overall Haemostatic Potential

IntroductionMyeloproliferative neoplasm (MPN), such as polycythaemia vera (PV) and essential thrombocythaemia (ET), are independent risk factors for arterial and venous thrombotic events, with the estimated risks of thrombotic complications at diagnosis to be approximately 7-26% in ET patients and 19-32% in PV patients. As such, one of the management goals for MPN is directed towards modifying thrombosis risk via cytoreduction and anti-platelet therapies. However, there are no routinely available laboratory tests to evaluate the patient's thrombotic risk beyond the assessment of blood counts. Global coagulation assays such as thromboelastography, thrombin and fibrin generation may be better surrogate measures of thrombosis. This pilot study evaluates a cohort of MPN patients on current treatment, to further elucidate the impact of their prothrombotic milieu on global coagulation assays.MethodsParticipants with MPN (proven on bone marrow biopsy and/or molecular diagnosis) were recruited. Thromboelastography (using TEG® 5000) was performed on citrated whole blood while additional citrated blood samples were double spun at -2500G to obtain platelet-poor plasma (PPP) and stored in at -80°C within 2 hours of sample collection. The stored PPP were then used for thrombin generation assessment using the calibrated automated thrombogram (CAT), fibrinolysis evaluation using overall haemostatic potential assay (OHP) as well as p-selectin testing. The results were compared to our previously collected age-matched healthy normal controls (n=41).ResultsThirty-eight MPN patients (20 females, 18 males) with median age 65 years (range: 29-84) were recruited. There were 26 patients with ET (68.4%) including a patient with post-ET myelofibrosis, 8 patients with PV (20.5%), 3 patients with primary myelofibrosis and one with MPN, unclassifiable. The majority of the patients are JAK2 V617F positive (30/38, 79%). The haemoglobin level was comparable between both groups (p=0.73) although the MPN group had significantly increased platelet count (526 vs 237 x 109/L, p<0.01).When compared to normal controls, the thromboelastography parameters were mostly comparable including no significant difference in maximum amplitude or clot strength (59.6 vs 60.1 mm, p=0.42) although the clot lysis time (LY30) was significantly higher (5.1% vs 0.9%, p<0.01) in the MPN group, independent of the use of aspirin. Higher LY30 (>5%) was associated with higher haemoglobin (150.3 vs 138.4 g/L, p=0.03) but not platelet count. CAT parameters, however, showed higher thrombin peak (260.8 vs 227.3 nM; p=0.01) and velocity index (93.4 vs 70.1 nM.min; p<0.01) compared to the normal controls, but interestingly no difference in endogenous thrombin potential (1397.5 vs 1363.0 nM.min, p=0.52).Fibrin generation parameters, namely the overall coagulation potential (OCP) and OHP were significantly reduced in the MPN cohort (OCP - 56.5 vs 66.7, p<0.001; OHP - 27.4 vs 33.0, p<0.001) with preserved overall fibrinolytic potential (p=0.66). P-selectin was markedly increased in MPN patients compared to the normal controls (116.3 vs 54.6 ng/mL, p<0.001), with higher P-selectin levels correlating with a higher platelet counts (p<0.01).ConclusionThis study demonstrates that global coagulation assays can provide additional information regarding the thrombotic risks in MPN patients. The MPN patients in our study had significantly higher lysis time and a reduction in fibrin generation parameters, which is contradictory to the prothrombotic nature of MPN. It may be a protective prognostic marker in the setting of treatment and could represent an underlying compensatory effect. Despite being a treated MPN cohort, the P-selectin remained higher in these patients and correlated with higher platelet count, suggesting that the higher P-selectin seen is likely of platelet subtype, though this did not correlate with changes in other global coagulation assay parameters despite thrombin being a known trigger of P-selectin exocytosis. Further prospective clinical studies including assessment of fibrinolysis markers and markers of platelet and endothelial function to evaluate and confirm these findings are proposed. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Development and Validation of a LC-MS/MS Method for the Profiling of Impurities Formed during Stress Study of Antifungal Agent-Efinaconazole.

Liquid chromatography interfaced with mass spectroscopy (LC-MS) is a sensitive and effective analytical method that combines high-resolution separation as well as specific mass detection of impurities in formulated products and as well as in active pharmaceutical ingredient (API). In this study, LC-MS/MS was used to determine impurities formed during the stability period of Efinaconazole (EFZ, a triazole antifungal agent). The method was validated for its assay parameters such as specificity, linearity, accuracy, precision and also for solution stability. The calibration curve was found to be linear for the concentration range from 0.5μg/mL to 30μg/mL. A thorough investigation was performed by exposing Efinaconazole (1mg/mL) to various stress conditions such as hydrolysis (acid and alkaline), oxidation, photolysis and temperature at various time intervals to achieve 10% degradation. The impurities formed during forced degradation were separated using high-performance liquid chromatography later it was identified using LC-MS/MS technique. The obtained results showed that EFZ is prone to oxidation when exposed to a 3% aqueous medium of hydrogen peroxide solution and quite stable in all the remaining stress conditions for a longer period.

Development and validation of a reporter gene assay for bioactivity determination of Anti-CGRP monoclonal antibodies

Calcitonin gene-related peptide (CGRP) is critical for the pathophysiology of migraine, and four therapeutic antibodies targeting CGRP and its corresponding receptors have been approved by the Food and Drug Administration (FDA), while many others are in the different stages of clinical trials. Bioactivity determination is essential for the quality control and clinical application of therapeutic monoclonal antibodies (mAbs). However, no bioassay has been reported to date. In this study, we developed a reporter gene assay (RGA) based on SK-N-MC cells stably expressing firefly luciferase driven by cAMP response element (CRE). The key assay parameters were optimized according to signal-to-noise (SNR), the response value, and the fitted dose-response curve. Validation of the RGA in accordance with ICH-Q2 guidelines showed that the method had good specificity, accuracy, linearity, and precision. The established RGA can be utilized as a reference method for release testing and stability studies of relevant antibodies.

Determining the approximate factor VIII level of patients with severe haemophilia A on emicizumab using in vivo global haemostasis assays.

Emicizumab is a recombinant, humanized bispecific monoclonal antibody that mimics the function of factor VIII (FVIII) which results in a significant reduction in the annualized bleeding rate in patients with haemophilia A (HA), however, the degree with which emicizumab corrects the coagulation defect remains unclear. The objective of this study was to predict the approximate FVIII level in severe haemophilia A patients with inhibitors on emicizumab using global haemostasis assays. Patients with moderate and mild HA in the non-bleeding state and healthy controls had FVIII levels and thrombin generation assessed. Linear regression was utilized to model the FVIII levels as a function of the thrombin generation assay parameters and to make a calibration curve of FVIII levels versus peak thrombin and endogenous thrombin potential. Patients with severe haemophilia A with inhibitors on emicizumab had thrombin generation performed in the same manner and their peak thrombin and endogenous thrombin potential results were placed on the calibration curve to calculate their FVIII Equivalency of Emicizumab by Thrombin Generation (F8EmT). All patients with severe HA with inhibitors on emicizumab had F8EmT>10%, suggesting they had been converted to a mild haemophilia phenotype. The patient's weight was inversely correlated to their F8EmT. The results from this study suggest that the F8EmT in patients with severe HA on emicizumab falls within the range of mild haemophilia which is consistent with the data noted in the emicizumab clinical trials and in vivo studies in animals.

Natural occurrence of Azospirillum brasilense in petunia with capacity to improve plant growth and flowering.

To evaluate the natural occurrence of the plant growth-promoting bacterium Azospirillum brasilense and petunia plants, local strains were isolated and characterized by biochemical and molecular methods. Three strains were assessed in greenhouse conditions using Petunia × hybrida Ultra™. Treatments: Plants without bacterial inoculation or chemical fertilization; fertilized with NPK and KNO3 ; and independently inoculated with the strains 2A1, 2A2, and 2E1 by submerging their roots in a bacterial suspension (~106 CFU·ml-1 ). Root length, dry weight of roots and shoots, leaf area, leaf greenness, and nutrient content were evaluated. The number of days from transplanting to the opening of the first flower and the number of flowers per plant were also determined. As a result, five isolates were characterized as A. brasilense, showing the capacity to produce indoles and siderophores, to solubilize phosphate, nitrogenase activity, and nifH-PCR amplification. In general, all the parameters of the plant assay were improved in plants inoculated with A. brasilense, with variations among the strains, as well as the onset of flowering and the number of flowers per plant, compared with uninoculated or fertilized plants. This is the first report on the natural occurrence of A. brasilense in petunia with the capacity to improve plant growth and flowering.