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Abe M.,Central Institute for Clinical Chemistry and Laboratory Medicine
Folia medica | Year: 2010

Clinically, both viral load and genotypes have been found to be major predictors of antiviral therapy outcome regarding chronic hepatitis C and they are, under normal circumstances, performed as separate assays. In order to improve the diagnostic strategy and subsequently reduce the reagent costs we have developed and established the simultaneous quantification and genotyping of hepatitis C virus RNA by a two-step real-time PCR on the LightCycler Instrument (Roche Diagnostics). The quantification assay was calibrated against WHO Standard 96/790. The detection limit was 30 IU/ml, the dynamic range up to 500,000,000 IU/ml. Intra- and interassay imprecisions were 1.2% and 1.9% (n = 10), respectively. The HCV RNA values obtained by real-time PCR assay were highly correlated with those obtained by the Cobas Amplicor HCV monitor test (r = 0.992; p < 0.001). The genotyping was performed by means of the melting temperature analysis. The concordance between our new genotyping method and the Trugene HCV 5'NC Kit was at the level of genotypes 100%. This rapid (3 h) and convenient assay is suitable for HCV genotyping, HCV detection and disease monitoring.


Shipkova M.,Central Institute for Clinical Chemistry and Laboratory Medicine | Svinarov D.,Medical University-Sofia
Clinical Biochemistry | Year: 2016

Therapeutic Drug monitoring (TDM) is a multidisciplinary endeavor encompassing skills of the laboratory, clinical pharmacologists and physicians aimed at providing the best possible patient care via the individualization of drug therapy. The expanding role of liquid chromatography - tandem mass spectrometry (LC-MS/MS) in TDM is based on dramatic improvements in analytical instrumentation, thereby enabling unique specificity, extreme sensitivity, high throughput, the simultaneous analysis of multiple drugs and metabolites in a drop of blood within several minutes and the use of multiplexed platforms for the rapid determination of single analytes. However, analysis by LC-MS/MS does not automatically ensure reliable results and superiority over other assays. The aim of this article is to provide an overview of the current use, advantages, and challenges of LC-MS/MS methods in TDM services, to summarize ongoing issues, and to provide an outlook on new perspectives. © 2016 The Canadian Society of Clinical Chemists.


Shipkova M.,Central Institute for Clinical Chemistry and Laboratory Medicine | Valbuena H.,Clinical Laboratory
TrAC - Trends in Analytical Chemistry | Year: 2016

Currently therapeutic drug monitoring (TDM) of immunosuppressive drugs is one of the best established fields of application of TDM as a tool dedicated to therapy guidance and patient care improvement. LC-MS/MS techniques were introduced to this field about 20 years ago, and enabled a huge progress towards providing more adequate overall quality and turnaround time for TDM services. However, numerous LC-MS/MS difficulties have also been recognized and approaches to deal with them have been developed. Moreover, some further issues must be resolved to realize the full potential of this technique. This article aims to provide an update of the LC-MS/MS literature with a focus on new developments and applications that have been reported during the last five years. In addition, it summarizes important lessons and defines current needs in the context of TDM services, which must be addressed in the near future. © 2016 Elsevier B.V.


Wieland E.,Central Institute for Clinical Chemistry and Laboratory Medicine
Therapeutic Apheresis and Dialysis | Year: 2011

Lipid apheresis treatment has been suggested to cause oxidative stress. Cells respond to oxidative stress in many ways, including, among others, altered gene expressions. In the present investigation we investigated whether the gene expression of known stress genes was affected in the WBCs of patients undergoing lipid apheresis. For this purpose cellular early-growth-response gene-1 (Egr-1), c-Jun, c-Fos, and heat shock protein 70 (Hsp70) mRNA expression was followed before and immediately after lipid apheresis treatments (N=24). Gene expression was determined by quantitative reverse transcription-polymerase chain reaction. With the exception of c-Fos, the expression of Egr-1, c-Jun, and Hsp70 mRNA was not affected in WBCs by a single lipid apheresis treatment (median [16th percentile; 84th percentile]): Egr-1, before 0.30 (0.13; 0.53), after 0.31 (0.14; 1.33); c-Jun, before 0.03 (0.03; 0.16), after 0.05 (0.03; 0.18); Hsp70, before 0.49 (0.23; 1.07), after 0.53 (0.20; 1.61)). Expression of c-Fos was significantly decreased (P<0.01) after lipid apheresis treatment (before 2.18 [1.06; 5.27], after 1.65 [0.74; 4.12]). Hsp70 and c-Fos expression in lipid apheresis patients was not different from that in 35 healthy blood donors, whereas Egr-1 and c-Jun were significantly decreased (P<0.05) in lipid apheresis patients when compared to controls (Egr-1 0.96 [0.42; 1.83], c-Jun 0.64 [0.40; 0.98], c-Fos 2.77 [1.32; 4,02], Hsp70 0.43 [0.28; 0.61]). These results show that lipid apheresis procedures do not induce stress gene expression in WBCs. Therefore, all the lipid apheresis systems used seem to be safe with respect to oxidative stress and other injuries induced in WBCs due to contact with extracorporeal tubing and membranes. © 2010 The Authors. Therapeutic Apheresis and Dialysis © 2010 International Society for Apheresis.


Valbuena H.,University of Barcelona | Shipkova M.,Central Institute for Clinical Chemistry and Laboratory Medicine | Kliesch S.-M.,Central Institute for Clinical Chemistry and Laboratory Medicine | Muller S.,Muon Statistics | Wieland E.,Central Institute for Clinical Chemistry and Laboratory Medicine
Clinical Chemistry and Laboratory Medicine | Year: 2016

Background: Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is routinely used for analysis of immunosuppressive drugs. This study investigated whether replacing analog internal standards (ANISs) with isotopically labeled internal standards (ILISs) has an impact on the performance of a LC-MS/MS method for the quantification of tacrolimus (TAC), sirolimus (SIR), ciclosporin A (CsA) and everolimus (EVE) in whole blood. Methods: Following hemolysis, protein precipitation, and extraction with either ANISs (ascomycin, desmethoxy-rapamycin, CsD), or ILISs (TAC-13C,D2; SIR-13C,D3; CsA-D12; EVE-D4), samples were centrifuged and injected into a LC-MS/MS device equipped with a C18 reversed phase column. The effect of the two ISs on the linearity, precision, accuracy, trueness, matrix effects, and carryover was investigated by using the same patient-, proficiency testing-, and quality control samples. Statistical analysis of agreement between results includes a standard random effects model and Passing-Bablok regression. Results: Within-day imprecision was <10%, between-day <8%, and trueness 91%-110% for all the analytes with both ISs. No carryover or matrix effects were observed. The median accuracy was -2.1% for CsA, 9.1% for EVE, 12.2% for SIR, and -1.2% for TAC with the ILISs; and -2% for CsA, 9.8% for EVE, 11.4% for SIR, and 0.2% for TAC with the ANISs. Results of patient and proficiency testing samples were not statistically different. Conclusions: Although ILISs are generally considered superior to ANISs, they may not be always essential. When optimizing a LC-MS/MS method other factors must be also considered. © 2016 by De Gruyter 2016.

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