ABSTRACT
Cyclosporine A (CsA) is a widely used immunosuppressive drug with a narrow therapeutic index and large individual differences. Its therapeutic and toxic effects are closely related to blood drug concentrations, requiring routine therapeutic drug monitoring (TDM). The current main methods for TDM of CsA are enzyme multiplied immunoassay technique (EMIT) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). However, few study on the method comparison of the EMIT and LC-MS/MS for the measurement of whole blood CsA concentration in children has been reported. In this study, we developed a simple and sensitive LC-MS/MS assay for the determination of CsA, and 657 cases of CsA concentrations were determined from 197 pediatric patients by a routine EMIT assay and by the validated in-house LC-MS/MS method on the same batch of samples, aimed to address the aforementioned concern. Consistency between the two assays was evaluated using linear regression and Bland-Altman analysis. The linear range of LC-MS/MS was 0.500-2000 ng/mL and that of the EMIT was 40-500 ng/mL, respectively. Overall, the correlation between the two methods was significant (r-value ranging from 0.8842 to 0.9441). Unsatisfactory consistency was observed in the concentrations < 40 ng/mL (r = 0.7325) and 200-500 ng/mL (r = 0.6851). Bland-Altman plot showed a mean bias of -18.0 % (±1.96 SD, -73.8 to 37.8 %) between EMIT and LC-MS/MS. For Passing-Bablok regression between EMIT and LC-MS/MS did not differ significantly (p > 0.05). In conclusion, the two methods were closely correlated, but the CsA concentration by LC-MS/MS assay was slightly higher than that by EMIT method. Switching from the EMIT assay to the LC-MS/MS method was acceptable, and the LC-MS/MS method will receive broader application in clinical settings due to its better analytical capabilities, but the results need to be further verified in different laboratories.
Subject(s)
Cyclosporine , Drug Monitoring , Tandem Mass Spectrometry , Humans , Cyclosporine/blood , Tandem Mass Spectrometry/methods , Linear Models , Chromatography, Liquid/methods , Child , Drug Monitoring/methods , Reproducibility of Results , Enzyme Multiplied Immunoassay Technique , Child, Preschool , Male , Limit of Detection , Infant , Immunosuppressive Agents/blood , Immunosuppressive Agents/pharmacokinetics , Female , Adolescent , Liquid Chromatography-Mass SpectrometryABSTRACT
The enzyme multiplied immunoassay technique (EMIT) is a new method for determining the plasma concentration of voriconazole (VRZ). This study aimed to investigate the correlation between EMIT and high-performance liquid chromatography/ultraviolet rays (HPLC/UV) in determining the plasma VRZ trough concentration in children, in China. A total of 419 blood samples were collected, and plasma VRZ concentrations were detected by the EMIT and HPLC methods. The results of 304 samples were analysed after excluding samples that were undetectable or beyond the quantification limit. A test result value of 0 was defined as undetectable, while concentrations outside the detection range (0.2 - 20.0 µg/ml for HPLC and 0.5 - 16.0 µg/ml for EMIT) were defined as beyond the quantification limit. Results from both methods were compared using the Passing Bablok regression, Bland-Altman plot analysis, and paired Wilcoxon test. The plasma VRZ concentrations determined by EMIT and HPLC showed a strong linear correlation through the linear regression equation YEMIT = 1.310 × HPLC +0.149 (R2 = 0.9082). The Bland-Altman plot analysis showed poor level consistency as measured by the two methods. The paired Wilcoxon-test showed a significant difference between the two methods (p < .0001). Compared to EMIT, HPLC accurately detected plasma VRZ concentration, making it suitable for VRZ therapeutic drug monitoring. The numerical values of the EMIT-measured levels were higher than those of HPLC, which may be related to VRZ metabolites interference and co-administrated drugs.
Subject(s)
Enzyme Multiplied Immunoassay Technique , Voriconazole/blood , Adolescent , Child , Child, Preschool , Chromatography, High Pressure Liquid , Female , Humans , Infant , Linear Models , Male , Reference StandardsABSTRACT
Therapeutic drug monitoring (TDM) of immunosuppressive drugs is crucial in organ-transplanted patients to prevent rejection or toxic effects due to inadequate dosage. Mycophenolic acid (MPA) is a commonly used immunosuppressant in this setting. Nowadays, MPA concentrations are monitored by Enzyme Multiplied Immunoassay Technology (EMIT), and Liquid Chromatography (LC)-based techniques, particularly coupled to Tandem Mass Spectrometry (LC-MS/MS). This study evaluates the concordance between TDM results for MPA obtained through CE-IVD EMIT and LC-MS/MS assays in plasma samples. LC-MS/MS quantification was based on a commercial kit and the analytical performance in terms of accuracy was tested through external proficiency tests and inter-laboratory comparison with a home-made HPLC-UV method. Both these evaluations confirmed the reliability of the LC-MS/MS method (1.6 % and 9.0 % of bias, respectively). Conversely, the comparison between EMIT and LC-MS/MS showed overestimation by EMIT of 33.5 %. This bias resulted concentration-dependent, ranging from 46.4 % in the concentration range of 1-2 mg/L, to 21.4 % over 4 mg/L. Considering the theoretical clinical impact of this overestimation, a fraction comprised between 12.4 % and 31.4 % of samples which resulted over three different minimum effective concentration values by EMIT (no indication for dose adjustment) had discordant indications by LC-MS/MS (dose adjustment needed). Concluding, this study highlights a clinically relevant systematic overestimation of MPA concentration by EMIT, supporting the switch to LC-MS/MS techniques for TDM purpose. However, further prospective studies are needed in order to evaluate the clinical impact of switching the TDM activity from EMIT to LC-MS/MS in a larger cohort in a long period.
Subject(s)
Drug Monitoring/methods , Immunosuppressive Agents/pharmacokinetics , Mycophenolic Acid/pharmacokinetics , Chromatography, High Pressure Liquid/methods , Chromatography, Liquid , Enzyme Multiplied Immunoassay Technique , Humans , Immunosuppressive Agents/administration & dosage , Immunosuppressive Agents/analysis , Mycophenolic Acid/administration & dosage , Mycophenolic Acid/analysis , Organ Transplantation/methods , Reproducibility of Results , Tandem Mass SpectrometryABSTRACT
BACKGROUND: Vancomycin remains a mainstay of the treatment of Gram-positive bacterial infections. It is crucial to accurately determine vancomycin serum concentration for adequate dose adjustment. OBJECTIVES: To evaluate the precision and accuracy of commercial assay techniques for vancomycin concentration and to assess the comparability of vancomycin detection methods in Chinese laboratories. METHODS: Human serum samples spiked with known concentrations of vancomycin were provided to laboratories participating in the external quality assessment scheme (EQAS). Assay methods included chemiluminescence, enzyme immunoassay (EIA) and so on. The dispersion of the measurements was analysed and the robust coefficient of variation (rCV), relative percentage difference (RPD) and satisfactory rate for method groups were calculated. Moreover, performance of the Chinese laboratories was assessed. RESULTS: A total of 657 results from 75 laboratories were collected, including 84 samples from 10 Chinese laboratories. The median rCV, median RPD and satisfactory rates classified by methods ranged from 1.85% to 15.87%, -14.75% to 13.34% and 94.59% to 100.00%, respectively. Significant differences were seen in precision, between kinetic interaction of microparticles in solution (KIMS) and other methods, and in accuracy, between enzyme-multiplied immunoassay technique (EMIT), fluorescence polarization immunoassay (FPIA) and other techniques. Vancomycin detection in China mainly depended on the chemiluminescence and EMIT methods, which tended to result in lower measurements. CONCLUSIONS: Although almost all assays in this study achieved an acceptable performance for vancomycin serum concentration monitoring, obvious inconsistencies between methods were still observed. Chinese laboratories were more likely to underestimate vancomycin concentrations. Thus, recognizing inconsistencies between methods and regular participation in vancomycin EQAS are essential.
Subject(s)
Drug Monitoring , Vancomycin , Anti-Bacterial Agents , China , Enzyme Multiplied Immunoassay Technique , Fluorescence Polarization Immunoassay , HumansABSTRACT
This study addresses the clinical and epidemiological aspects of envenoming cases resulting from snakebites treated at a hospital in Cruzeiro do Sul, in the upper Juruá River region, western Brazilian Amazonia. The specific identity of snakes that caused the envenomings was inferred (a) from the diagnosis of patient symptoms and signs upon hospital admission, (b) by enzyme immunoassay for detection of Bothrops atrox and Lachesis muta venom from serum samples taken from patients before antivenom therapy, or (c) by direct identification of the snake, when it was brought along to the hospital or photographed. There were 133 snakebites (76.2 cases per 100,000 inhabitants) registered during one year (July 2017 to June 2018). Most snakebites (88.7%) were caused by Bothrops spp., and the rest by non-venomous snakes or dry bites. Snakebites tended to occur more often during the rainy season, coinciding with the period of greater reproductive activity of the snakes and greater availability of their prey. In addition, the increase in the water level of rivers and lakes during the rainy season tends to concentrate snakes in dry places and, thus, to increase encounters with humans. Information campaigns on prevention and first aid, specially among the most vulnerable groups (indigenous people, farmers, and children and teenagers in rural areas), and the importance of using protective equipment (boots, leggings, leather gloves) in certain high risk activities (e.g. agriculture and extractivism in forests) are fundamental for the reduction of snakebite morbidity. (AU)
Subject(s)
Poisoning , Snakes , Enzyme Multiplied Immunoassay Technique , Bothrops , Animals, PoisonousABSTRACT
Forty-three fatalities involving the potent synthetic cannabinoid, 5-Fluoro-ADB, are summarized. For each case, a description of the terminal event, autopsy findings, cause of death, qualitative identification of 5-Fluoro-ADB and its ester hydrolysis metabolite, 5-Fluoro-ADB metabolite 7, in urine, and the quantitative values obtained in the blood specimens are outlined. Central blood concentrations ranged from 0.010 to 2.2 ng/mL for 5-Fluoro-ADB and 2.0 to 166 ng/mL for 5-Fluoro-ADB metabolite 7. Peripheral blood concentrations ranged from 0.010 to 0.77 ng/mL and 2.0 to 110 ng/mL for 5-Fluoro-ADB and 5-Fluoro-ADB metabolite 7, respectively. The majority of cases resulted in central to peripheral blood concentration ratios greater than 1 for 5-Fluoro-ADB (58%) and 5-Fluoro-ADB metabolite 7 (71%) suggesting that postmortem redistribution occurs to some extent. Combining the increased cardiac weight and/or gastric volume and toxicology data identifying 5-Fluoro-ADB, it is hypothesized that abuse of this substance may precipitate a dysrhythmia and cause sudden death.
Subject(s)
Illicit Drugs/blood , Illicit Drugs/urine , Indazoles/blood , Indazoles/urine , Marijuana Abuse/mortality , Adult , Chromatography, Gas , Chromatography, Liquid , Enzyme Multiplied Immunoassay Technique , Enzyme-Linked Immunosorbent Assay , Forensic Toxicology , Humans , Illicit Drugs/adverse effects , Indazoles/adverse effects , Male , Mass Spectrometry , Middle Aged , Molecular Structure , Myocardium/pathology , Organ Size , Stomach/pathologyABSTRACT
To find the predictors of High Dose Methotrexate toxicities in childhood Acute Lymphoblastic Leukemia Patients. This study included 198 Childhood Acute Lymphoblastic Leukemia patients (303 infusions) who were treated with High Dose Methotrexate. Methotrexate levels at different time point were measured by modified enzyme multiplied immunoassay technique assay. The correlation between Methotrexate levels and toxicity was evaluated by Receiver Operating Characteristic curve. When the Methotrexate level at 42 h was lower than 0.76 µmol/L, the sensitivity for predicting thorough clearance at 66 h was 90.78%. When the Methotrexate level at 42 h was higher than1.5 µmol/L, the sensitivity for predicting delayed clearance was 82.17%. When the Methotrexate level at 66 h was higher than 0.5 µmol/L, the sensitivity for predicting Methotrexate toxicity was 89.09%. When the Methotrexate level at 66 h was lower than 0.1 µmol/L, the sensitivity for predicting Methotrexate nontoxicity was 92.73%. The Methotrexate level at 42 h could be predictor for delayed clearance. The Methotrexate level at 66 h could be predictor for toxicity.
Subject(s)
Humans , Male , Female , Infant , Child, Preschool , Child , Adolescent , Patients/classification , Methotrexate/administration & dosage , Methotrexate/analysis , Precursor Cell Lymphoblastic Leukemia-Lymphoma/pathology , Forecasting , ROC Curve , Enzyme Multiplied Immunoassay Technique/instrumentation , Dosage/adverse effectsABSTRACT
BACKGROUND: Methotrexate is an important component in many chemotherapy protocols. The blood concentration of Methotrexate is used to determine the regimen of folinic acid. However, the lower limit of Siemens assay kit is 0.30 µmol/L in China. This study extended the limit from 0.3 to 0.05 µmol/L and reduced the test cost by optimizing the parameters of Enzyme Multiplied Immunoassay Technique assay. METHODS: Parameters of Enzyme Multiplied Immunoassay Technique assay were modified to decrease the volume of reagents A and B. Then a standard curve with a new custom set of calibrators was prepared to detect low concentration. Intra-day and inter-day imprecision were assessed by control material and samples. The linearity of the modified assay was verified by analyzing a range of quality controls with known concentration from 0.05 to 1.00 µmol/L. At last, the same samples were tested by modified Enzyme Multiplied Immunoassay Technique assay and Liquid Chromatography-tandem Mass Spectrometry assay respectively. A simple linear regression was performed to verify the validity of the modified Enzyme Multiplied Immunoassay Technique assay. RESULTS: Intra-day and inter-day imprecision show good reproducibility at all levels (0.05, 0.12, 0.43, 0.82 µmol/L). The linearity equation of modified assay was y = 0.9913x + 0.0046, in which y was the mean of measured concentration and x was the target concentration (R2 = 0.9994). In the range of 0.05-10.00 µmol/L, correlation between the Modified assay and Liquid Chromatography-tandem Mass Spectrometry assay was significant (r = 0.9968). In the range of 0.30-10.00 µmol/L, the correlation between modified and commercial assays was significant (r = 0.9987) as well. CONCLUSIONS: The modified assay enhanced the sensitivity of Siemens VIVA-E to 0.05 µmol/L. In addition, the test number of a reagent Kit increased from 140 to 210. This means the cost of detection was reduced about 30%.
Subject(s)
Antimetabolites, Antineoplastic/blood , Enzyme Multiplied Immunoassay Technique , Methotrexate/blood , Child , Chromatography, Liquid , Costs and Cost Analysis , Enzyme Multiplied Immunoassay Technique/economics , Humans , Limit of Detection , Precursor Cell Lymphoblastic Leukemia-Lymphoma/blood , Reproducibility of Results , Tandem Mass SpectrometryABSTRACT
INTRODUCTION: Different measured values for tacrolimus were obtained with different automated immunoassays. We aimed to examine the differences in the blood tacrolimus concentrations measured by the major immunoassay systems commercially available in Japan. METHODS: Whole-blood samples from 118 patients were assayed by 3 commercial assays: chemiluminescent enzyme immunoassay (CLIA), affinity column-mediated immunoassay (ACMIA), and enzyme-multiplied immunoassay technique (EMIT). Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used for reference. KEY FINDINGS: The correlation coefficient of immunoassay vs LC-MS/MS was excellent for ACMIA (.83) and CLIA (.81) and good for EMIT (.71). The mean error was negative for ACMIA and positive for CLIA and EMIT. The mean absolute error and root-mean-square error were almost the same for ACMIA and CLIA and lower than those for EMIT. CONCLUSIONS: The ACMIA and CLIA yield considerably better results than the EMIT for monitoring blood tacrolimus concentrations.
Subject(s)
Immunoassay/methods , Tacrolimus/analysis , Tacrolimus/blood , Adolescent , Adult , Aged , Aged, 80 and over , Autoimmune Diseases/blood , Autoimmune Diseases/surgery , Chromatography, Liquid , Enzyme Multiplied Immunoassay Technique , Female , Humans , Immunoassay/classification , Male , Middle Aged , Regression Analysis , Tandem Mass Spectrometry , Young AdultABSTRACT
BACKGROUND: The aim of this study was to characterize the pharmacokinetics of mycophenolic acid (MPA) and MPA glucuronide (MPAG) in Chinese renal transplant patients taking enteric-coated mycophenolate sodium (EC-MPS). Limited sampling strategies (LSSs) were developed to estimate the area under the concentration curve from 0 to 12 hours (AUC0-12h) of total and free MPA. Another objective was to investigate the correlation between high-performance liquid chromatography (HPLC) and enzyme-multiplied immunoassay technology (EMIT) for total MPA determination. METHODS: Serial blood samples were collected over 12 hours from 15 patients who were administered multiple doses of EC-MPS. LSS was developed by multiple stepwise regression analysis. Measurement by HPLC and EMIT was compared using Passing-Bablok regression and Bland-Altman analysis. RESULTS: Normalized to 720 mg twice daily, the AUC0-12h of total MPA and MPAG was 43.0 ± 17.4 and 653 ± 329 mg·h/L, respectively, whereas the free MPA AUC0-12h was 1.368 ± 0.988 mg·h/L. The free fraction of MPA was 3.01% ± 3.15%. The combination of C2h-C4h-C6h and C2h-C4h-C6h-C8h was found to be superior to estimate total and free MPA simultaneously. The EMIT showed an acceptable correlation with HPLC, with an AUC0-12h overestimation of 11.32% ± 15.77%. CONCLUSIONS: The pharmacokinetic profile of total and free MPA and its main metabolite MPAG was examined in Chinese adult renal transplant patients receiving EC-MPS. The use of LSS to estimate individual free and total MPA exposure could be useful in optimizing patient care.
Subject(s)
Glucuronides/pharmacokinetics , Kidney Transplantation , Mycophenolic Acid/analogs & derivatives , Mycophenolic Acid/pharmacokinetics , Adolescent , Adult , Area Under Curve , Asian People , Chromatography, High Pressure Liquid , Enzyme Inhibitors/blood , Enzyme Inhibitors/pharmacokinetics , Enzyme Multiplied Immunoassay Technique , Female , Glucuronides/blood , Humans , Male , Middle Aged , Mycophenolic Acid/blood , Tablets, Enteric-Coated/pharmacokinetics , Young AdultABSTRACT
PURPOSE: To assess the pharmacokinetic properties of mycophenolate mofetil (MMF) dispersible tablets and capsules by the enzyme multiplied immunoassay technique (EMIT) in Chinese kidney transplant recipients in the early post-transplantation phase and to develop the equations to predict mycophenolic acid (MPA) area under the 12-hour concentration-time curve (AUC0-12h) using a limited sampling strategy (LSS). METHODS: Forty patients who underwent renal transplantation from brain-dead donors were randomly divided into dispersible tablets (Sai KE Ping; Hangzhou Zhongmei Huadong Pharma) and capsules (Cellcept; Roche Pharma, Why, NSW, Australia) groups, and treated with MMF combined with combination tacrolimus and prednisone as a basic immunosuppressive regimen. Blood samples were collected before treatment (0) and at 0.5,1, 1.5, 2, 4, 6, 8, 10, and 12 hours post-treatment and 7 days after renal transplantation. Plasma MPA concentrations were measured using EMIT. LSS equations were identified using multiple stepwise linear regression analysis. RESULTS: The peak concentration (Cmax) in the MMF dispersible tablets (MMFdt) group (7.0 ± 2.8) mg/L was reduced compared with that in the MMF capsules (MMFc) group (10.8 ± 6.2 mg/L; P = .012); time to peak concentration in the MMFdt group was 3.2 ± 2.3 hours, which was nonsignificantly elevated compared with that of the MMFc group (2.2 ± 1.7 hours). Three-point estimation formulas were generated by multiple linear regression for both groups: MPA-AUCMMFdt = 3.542 + 3.332C0.5h + 1.117C1.5h + 3.946C4h (adjusted r2 = 0.90, P < .001); MPA-AUCMMFc = 8.149 + 1.442C2h + 1.056C4h + 7.133C6h (adjusted r2 = 0.88, P < .001). Both predicted and measured AUCs showed good consistency. CONCLUSIONS: After treatment with MMF dispersible tables or MMF capsules, the Cmax of MPA for the MMFdt group was significantly lower than that of the MMFc group; there was no significant difference in other pharmacokinetic parameters. Three-time point equations can be used as a predictable measure of the AUC0-12h of MPA.
Subject(s)
Drug Monitoring/methods , Immunosuppressive Agents/pharmacokinetics , Kidney Transplantation , Mycophenolic Acid/pharmacokinetics , Adult , Area Under Curve , China , Enzyme Multiplied Immunoassay Technique , Female , Humans , Immunosuppressive Agents/blood , Immunosuppressive Agents/chemistry , Linear Models , Male , Middle Aged , Mycophenolic Acid/blood , Mycophenolic Acid/chemistry , Postoperative Period , Prednisone/therapeutic use , Tacrolimus/therapeutic useABSTRACT
Tacrolimus is the most commonly used immunosuppressant. Because of its narrow therapeutic range, it is necessary to frequently monitor its concentration. We report the case of a 25-year-old man who underwent kidney transplantation whose tacrolimus concentrations, as measured by an affinity column-mediated immunoassay, were falsely elevated. As we reduced the dose of tacrolimus, the recipient developed T cell-mediated rejection. Using the same blood samples, an enzyme-multiplied immunoassay technique showed that the patient's levels of tacrolimus were extremely low. A further examination indicated that the false increase in the tacrolimus concentration was likely due to an unknown interfering substance. We administered methylprednisolone and antithymocyte-globulin. The patient's serum creatinine level decreased and remained stable after these treatments.
Subject(s)
Antilymphocyte Serum/therapeutic use , Graft Rejection/chemically induced , Graft Rejection/drug therapy , Immunosuppressive Agents/blood , Immunosuppressive Agents/therapeutic use , Methylprednisolone/therapeutic use , Tacrolimus/blood , Tacrolimus/therapeutic use , Adult , Chromatography, Affinity , Enzyme Multiplied Immunoassay Technique , Humans , Kidney Transplantation/adverse effects , Male , T-Lymphocytes/drug effects , Treatment OutcomeABSTRACT
OBJECTIVE: Evaluate whether saliva could be a useful alternative to serum for routine therapeutic drug monitoring of caffeine in preterm infants using the enzyme multiplied immunoassay technique (EMIT) assay. METHODS: We conducted a prospective study including preterm infants (less than 34 weeks' amenorrhea) admitted to the intensive care and neonatal medicine department. All infants received 5, 10, 15, 20 and 25mg/kg/day of citrate caffeine intravenously from the first to the fifth day of birth, respectively. For each patient, two concomitant blood and saliva samples corresponding to the trough concentrations were collected 24hours after each caffeine dose. The caffeine concentrations were determined using the EMIT®2000 caffeine assay. RESULTS: Thirteen preterm infants were included. The saliva and the serum caffeine concentration increased proportionally to the administered dose. Saliva and serum kinetics were comparable and the saliva caffeine concentrations were correlated to the serum ones (r2=0.76). CONCLUSION: Saliva caffeine monitoring by EMIT is a valid, useful and safe alternative to serum in preterm infants.
Subject(s)
Caffeine/pharmacokinetics , Central Nervous System Stimulants/pharmacokinetics , Citrates/pharmacokinetics , Drug Monitoring/methods , Enzyme Multiplied Immunoassay Technique , Caffeine/administration & dosage , Central Nervous System Stimulants/administration & dosage , Citrates/administration & dosage , Dose-Response Relationship, Drug , Female , Humans , Infant, Newborn , Infant, Premature , Male , Prospective Studies , Reproducibility of Results , Saliva/chemistryABSTRACT
Propósito: Tres capas celulares componen el urotelio: basal, intermedio y luminal ("celulas en paraguas") y diferentes enfermedades pueden surgir de diferentes poblaciones celulares. El objetivo de este estudio es analizar la capacidad de cuantificación de dichas poblaciones celulares utilizando 4 protocolos diferentes. Métodos: Se aleatorizaron 20 ratas macho (Wistar) en 4 grupos de 5 animales: raspado, enzimático 30, 45 y 60 minutos. Las células se aislaron, se analizaron mediante citómetro de flujo y se procesaron datos mediante el software BD FACSDIVA (TM). Resultados: El urotelio se separó en 2 poblaciones celulares que son diferentes en tamaño y complejidad. El grupo que mostró más eficiencia en la disociación de células y la separación celular fue el protocolo enzimático de 45 minutos. Conclusiones: El protocolo enzimático de 45 minutos fue capaz de aislar las poblaciones de células uroteliales, y podría ser explorado como herramienta pronóstica potencial, selección de pacientes y objetivo terapéutico en las enfermedades uroteliales. Estudios futuros deberían validar la aplicación clínica potencial al racional propuesto del paradigma luminal basal en el cáncer urotelial como esperanza para un abordaje individualizado
Purpose: Three cell layers compose the urothelium: basal, intermediate and luminal ("umbrella cells") and different diseases might arise from different cell populations. The aim of this study is to analyze the quantification ability of such cell populations by using four different protocols. Methods: Twenty male rats (Wistar) were randomized in four groups of five animals: scraping, enzymatic 30, 45 and 60 minutes. The cells were isolated, analyzed by flow cytometer and data processed by BD FACSDIVA (TM) software. Results: The urothelium was separated in two cell populations that are different in size and complexity. The group that showed more efficiency in cells dissociation and cells separation was enzymatic protocol 45 minutes. Conclusions: Enzymatic protocol 45 minutes was able to isolate urothelial cell populations and might be explored as potential prognostic tool, patient selection and therapeutic target in urothelial diseases. Future studies should validate the potential clinical application to the proposed rational of luminal-basal paradigm in the urothelial cancer as hope for individualized approach
Subject(s)
Humans , Urothelium/cytology , Carcinoma, Transitional Cell/pathology , Models, Animal , Urinary Bladder/ultrastructure , Enzyme Multiplied Immunoassay TechniqueABSTRACT
Cyclosporin A (CyA) is an immunosuppressive agent widely used in clinical therapy. In the therapeutic process, the blood concentration of CyA should be monitored to avoid or prevent rejection and toxicity. The objectives of this study were to compare the correlation of liquid chromatography-tandem mass spectrometry (LC-MS/MS) and enzyme-multiplied immunoassay technique (EMIT) for the determination of the CyA concentration in human blood and to provide evidence for the rational usage of EMIT in clinical practice. Blood samples collected from 132 patients undergoing a liver or kidney transplant or patients with aplastic anemia at Qilu Hospital of Shandong University were tested using the two methods. The calibration curve was linear from 25-500 ng·mL-1 for LC-MS/MS and from 50-450 ng·mL-1 for EMIT. The inter- and intra-day RSDs were less than 15%. The CyA blood concentration according to EMIT was 3.5 ng·mL-1 more than that according to LC-MS/MS. The 95% confidence interval was -10.0~16.9 ng·mL-1. The CyA blood concentration according to the two methods did not differ significantly (p > 0.05). LC-MS/MS and EMIT were suitable methods for determining the CyA blood concentration. The two methods were closely correlated (r2 = 0.969), but the CyA blood concentration according to EMIT was slightly higher than that according to LC-MS/MS. The clinical significance of this finding needs to be further evaluated.
Subject(s)
Asian People , Chromatography, Liquid/methods , Cyclosporine/blood , Enzyme Multiplied Immunoassay Technique , Tandem Mass Spectrometry/methods , Calibration , Humans , Limit of Detection , Reference Standards , Regression AnalysisABSTRACT
OBJECTIVE: Ovarian clear cell carcinoma (OCCC) and high grade serous ovarian cancer (HGSOC) are associated with the highest risk of VTE among patients with epithelial ovarian cancer (EOC). Tissue factor (TF) is a transmembrane glycoprotein which can trigger thrombosis. We sought to evaluate if there is an association between VTE and tumor expression of tissue factor (TF), plasma TF, and microvesicle TF (MV TF) activity in this high-risk population. METHODS: We performed a case-control study of OCCC and HGSOC patients with and without VTE. 105 patients who underwent surgery at a tertiary care center between January 1995 and October 2013 were included. Plasma TF was measured with an enzyme-linked immunosorbent assay. A TF-dependent Factor Xa generation assay was used to measure MV TF activity. Immunohistochemical (IHC) analysis was performed to evaluate tumor expression of TF. RESULTS: 35 women with OCCC or HGSOC diagnosed with VTE within 9months of surgery were included in the case group. Those with VTE had a worse OS, p<0.0001, with a greater than three-fold increase in risk of death, HR 3.33 (CI 1.75-6.35). There was no significant difference in median plasma TF level or MV TF activity level between patients with and without VTE. OCCC patients had greater expression of TF in their tumors than patients with HGSOC, p<0.0001. CONCLUSIONS: TFMV activity and plasma TF level were not predictive of VTE in this patient population. Given the extensive expression of TF in OCCC tumors, it is unlikely IHC expression will be useful in risk stratification for VTE in this population.
Subject(s)
Neoplasms, Glandular and Epithelial/blood , Neoplasms, Glandular and Epithelial/pathology , Ovarian Neoplasms/blood , Ovarian Neoplasms/pathology , Thromboplastin/metabolism , Venous Thrombosis/blood , Venous Thrombosis/pathology , Adult , Aged , Aged, 80 and over , Carcinoma, Ovarian Epithelial , Case-Control Studies , Enzyme Multiplied Immunoassay Technique , Female , Humans , Immunohistochemistry , Middle Aged , Neoplasm Staging , Survival Analysis , Thromboplastin/biosynthesisABSTRACT
BACKGROUND: The enteric-coated mycophenolate sodium (EC-MPS), whose active constituent is mycophenolic acid (MPA), has been widely clinically used for organ transplant recipients. However, its absorption is delayed due to its special designed dosage form, which results in difficulty to monitor the exposure of the MPA in patients receiving the EC-MPS. This study was aimed at developing a relatively practical and precise model with limited sampling strategy to estimate the 12-hour area under the concentration-time curve (AUC0-12 h) of MPA for Chinese renal transplant recipients receiving EC-MPS. METHODS: A total of 36 Chinese renal transplant recipients receiving the EC-MPS and tacrolimus were recruited in this study. The time point was 2 weeks after the transplantation for all the patients. The MPA concentrations were measured with enzyme-multiplied immunoassay technique for 11 blood specimens collected predose and at 0.5, 1, 1.5, 2, 2.5, 3, 4, 6, 8, and 12 hours after the morning dose of EC-MPS. The measured AUC was calculated with these 11 points of MPA concentrations with the linear trapezoidal rule. Limited sampling strategy was used to develop models for estimated AUC in the model group (n = 18). The bias and precision of different models were evaluated in the validation group (n = 18). RESULTS: C4 showed the strongest correlation with the measured AUC. The best 3 time point equation was 6.629 + 8.029 × C0 + 0.592 × C3 + 1.786 × C4 (R = 0.910; P < 0.001), whereas the best 4 time point equation was 3.132 + 5.337 × C0 + 0.735 × C3 + 1.783 × C4 + 3.065 × C8 (R = 0.959; P < 0.001). When evaluated in the validation group, the 4 time point model had a much better performance than the 3 time point model: for the 4 time point model: R = 0.873, bias = 0.505 [95% confidence interval (CI), -10.159 to 11.170], precision = 13.370 (95% CI, 5.186-21.555), and 77.8% of estimated AUCs was within 85%-115% of the measured AUCs; for the 3 time point model: R = 0.573, bias = 6.196 (95% CI, -10.627 to 23.018), precision = 21.286 (95% CI, 8.079-34.492), and 50.0% of estimated AUCs was within 85%-115% of the measured AUCs. CONCLUSIONS: It demanded at least 4 time points to develop a relatively reliable model to estimate the exposure of MPA in renal transplant recipients receiving the EC-MPS. The long time span needed restricted its application, especially for the outpatients, but it could be a useful tool to guide the personalized prescription for the inpatients.
Subject(s)
Immunosuppressive Agents/administration & dosage , Kidney Transplantation/methods , Models, Biological , Mycophenolic Acid/administration & dosage , Adult , Area Under Curve , Asian People , Drug Monitoring/methods , Drug Therapy, Combination , Enzyme Multiplied Immunoassay Technique , Female , Humans , Immunosuppressive Agents/pharmacokinetics , Male , Middle Aged , Mycophenolic Acid/pharmacokinetics , Reproducibility of Results , Tablets, Enteric-Coated , Tacrolimus/administration & dosage , Time FactorsABSTRACT
AIMS: To investigate the area under the curve (AUC) of mycophenolic acid (MPA) in adult Chinese renal allograft recipients receiving concomitant enteric-coated mycophenolate sodium (EC-MPS) and cyclosporine (CsA) during the early post-transplant phase and to develop optimal model equations for estimation of the MPA area under the plasma concentration-time curve from 0 to 12 hours (AUC0-12h) using a limited-sampling strategy (LSS). METHODS: The present study enrolled 24 Chinese renal recipients treated with EC-MPS, CsA, and corticosteroid, from whom 24 serial blood samples were collected over 12 hours. MPA concentration was evaluated with enzyme multiplied immunoassay technique (EMIT). LSS was developed by multiple stepwise regression analysis using a two-group method (test group, n = 12; and validation group, n = 12). RESULTS: The MPA predose concentration had a poor correlation with MPA AUC0-12h, and the best equations obtained from the test group were the following: 25.73 + 0.59 × C1.5 + 0.79 × C2 + 2.03 × C4 (for three time points, r2 = 0.761) and 22.13 + 1.7 × C0.5 + 0.61 × C1.5 + 0.78 × C2 + 1.83 × C4 (for four time points, r2 = 0.853). When these equations were tested in the validation group, there were no signiï¬cant differences in prediction errors. CONCLUSION: An LSS using time points at 1.5, 2, and 4 hours or 0.5, 1.5, 2, and 4 hours provides the most accurate and reliable estimation of the MPA AUC0-12h in Chinese adult renal recipients treated concomitantly with EC-MPS and CsA during the early post-transplant phase.â©.
Subject(s)
Drug Monitoring/methods , Immunosuppressive Agents/administration & dosage , Kidney Transplantation , Models, Biological , Mycophenolic Acid/administration & dosage , Administration, Oral , Adult , Area Under Curve , Asian People , China , Cyclosporine/administration & dosage , Drug Compounding , Drug Therapy, Combination , Enzyme Multiplied Immunoassay Technique , Female , Humans , Immunosuppressive Agents/blood , Immunosuppressive Agents/pharmacokinetics , Kidney Transplantation/adverse effects , Male , Mycophenolic Acid/blood , Mycophenolic Acid/pharmacokinetics , Predictive Value of Tests , Reproducibility of Results , Tablets, Enteric-Coated , Treatment Outcome , Young AdultABSTRACT
Introduction: The progress of civilization and the development of pharmaceuticals industry have caused an increase in psychoactive substances abuse. That is why there are often cases of overdose and poisoning with these substances. Benzodiazepines are among the most commonly used drugs. They are a group of medications producing sedative, anxiolytic, hypnotic, myorelaxant and anticonvulsant effects. Benzodiazepines are a large and diversified group of compounds (over 50 different benzodiazepines are used in clinical treatment), and their metabolites are biologically active. The progress of technology allows the use of more advanced and accurate diagnostic methods. The enzyme multiplied immunoassay technique (EMIT) is a routinely used method in toxicology laboratories, and it is often employed to determine the concentration of benzodiazepines in tested material. This technique is quite easy and quick to perform. However, these advantages have different kinds of consequences, e.g. false-positive results. Therefore, it is important to confirm the results with reference methods. The aim of this study was to evaluate the reactivity of chosen benzodiazepines using the EMITRtoxTMSerum Benzodiazepines Assay. Materials and methods: The precision and accuracy of the results were calculated. Nine benzodiazepines were analyzed (chlordiazepoxide, estazolam, flurazepam, medazepam, nitrazepam, nordazepam, oxazepam, prazepam, and temazepam) using the V-Twin System with EMIT technology from Siemens. Every drug was tested 3 times using different concentrations: 300 ng/mL, 1000 ng/mL, and 2000 ng/mL. Results and conclusions: The EMIT test showed the highest precision for the quantitative determination of prazepam, and the lowest precision for the determination of nitrazepam and medazepam, whereas the test's accuracy was highest for the determination of prazepam, and lowest in the case of nitrazepam.
Subject(s)
Benzodiazepines/blood , Enzyme Multiplied Immunoassay Technique , Benzodiazepines/chemistry , Data Accuracy , False Positive Reactions , HumansABSTRACT
BACKGROUND: A general non-specific marker of disease activity that could alert the clinician and prompt further investigation would be of value in patients with HIV/AIDS, especially in resource limited environments. OBJECTIVE: To investigate the potential of neopterin as non-specific biomarker in patients with advanced HIV/AIDS. METHODS: Cross-sectional study in 105 HIV positive patients (75 on highly active antiretroviral treatment (HAART). Neopterin was assessed by enzyme linked immune-absorbent assay and cytokines by flow cytometry. RESULTS: Neopterin levels were significantly higher (p<0.001) for the total patient than for the control group. Significant correlations between neopterin and plasma indicators of inflammation showed neopterin to be a good indicator of active inflammatory status and of the effect of HAART on the immune system. Neopterin was superior to C-reactive protein and to individual cytokines as indicator of immune deficiency. Increased neopterin levels were associated with a decline in albumin, haemoglobin and the albumin/globulin ratio, and with increases in red cell distribution width. CONCLUSIONS: Plasma neopterin is a good non-specific biomarker of disease activity in HIV/AIDS patients. It is a good indicator of inflammatory activity, perpetuation of inflammation-associated co-morbidities, degree of immune deficiency and has predictive value for underlying disease, and for monitoring the HAART response.