Central Institute for Clinical Chemistry and Laboratory Medicine

Stuttgart Mühlhausen, Germany

Central Institute for Clinical Chemistry and Laboratory Medicine

Stuttgart Mühlhausen, Germany
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Shipkova M.,Central Institute for Clinical Chemistry and Laboratory Medicine | Valbuena H.,Nostra Senyora Of Meritxell Hospital
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.


Hulpke-Wette M.,Pediatrician and pediatric cardiology practice | Wieland E.,Central Institute for Clinical Chemistry and Laboratory Medicine
Clinical Research in Cardiology Supplements | Year: 2012

Background: One of the first investigations concerning extracorporeal treatment of hypercholesterolemia was performed in 1967 by plasma exchange in patients with homozygous or severe heterozygous familial hypercholesterolemia (FH). In the following decades, several specific lipid apheresis systems were developed to efficiently eliminate low-density lipoprotein (LDL) cholesterol and Lp(a) cholesterol in hypercholesterolemic patients. In the early 1980s, the main clinical indication has been homozygous FH including mainly children and pregnant women. In consideration of the current development of lipid-lowering regimens and scientific knowledge of preventing progression of cardiovascular diseases, the spectrum of indications to initiate lipid apheresis was extended due to still insufficient lipid-lowering therapy in some clinical cases. However, a generally accepted indication for lipid apheresis treatment is still under discussion. In Germany, the target-oriented distribution of increasingly limited healthcare resources demand data which support the benefit of established treatment procedures such as lipid apheresis. In recent years, the Federal Joint Committee (G-BA), a paramount decision-making body of the German Healthcare System, issued to reassess the approval of chronic lipid apheresis therapy for regular reimbursement. Therefore, in 2005, an interdisciplinary German Apheresis Working Group has been established by members of both the German societies of nephrology. One of the first goals of this working group was a revision of the indications for lipid apheresis corresponding to current guidelines and recommendations for the treatment of lipid disorders. In addition, recently new pathophysiological perceptions of the impact of lipoproteins on atherogenesis and thrombosis were also considered. Methods and Results: Since 2005, the working group met on a regular basis to substantiate the first defined goals. The indications for lipid apheresis were critically revised with respect to actual results from clinical investigations, cardiovascular guidelines, and scientific knowledge and were accepted by the members of the apheresis working group. Conclusions: There is consensus between the medical societies and health insurance funds regarding the need for general accepted guidelines for lipid apheresis. Recommendations for the indications of lipid apheresis were developed, but additionally these results should be confirmed by medical societies to transform them to guidelines. However, due to limited data showing that lipid apheresis has effects on the progression of cardiovascular diseases all members of the apheresis working group support a project for creating a lipid apheresis registry. This apheresis registry has been developed and recently started. The primary goal is to substantiate prospective long-term data on clinical outcome of chronic lipid apheresis treatment and to support additional clinical research activities in this field. In addition, this registry should comply with the actual requests of the Federal Joint Committee (G-BA). © 2012 The Author(s).


PubMed | Central Institute for Transfusion Medicine, Central Institute for Clinical Chemistry and Laboratory Medicine and University Medicine Goettingen
Type: Journal Article | Journal: Clinical biochemistry | Year: 2016

Regulatory T cells (Tregs) which may indicate operational tolerance provide a promising biomarker for individualization of immunosuppression. Naturally thymus-derived Tregs (nTregs) represent the major suppressive phenotype and can be identified by their demethylation status in the Tregs Specific Demethylated Region (TSDR) of the Forkhead-Box-P3 (FOXP3) gene using quantitative PCR (qPCR).The analytical performance of a TSDR demethylation qPCR assay was assessed in whole blood of healthy individuals (HI) and kidney transplant recipients (KTR). The assay was compared to conventional flow cytometry and the agreement of results between two laboratories using a comparable qPCR protocol was assessed. In addition, the effect of gender, age, and medications was investigated.Within and between series imprecision was <20% (n=6). Whole blood samples are suitable for analysis within 3days when stored at room temperature; both whole blood and DNA samples - within 12months when frozen at -80C. A significant correlation between the qPCR results and flow cytometry was lacking both with samples from HI and KTR. qPCR results between laboratories showed a bias of 76% but correlated well (r=0.645; p=0.0002, n=29). nTregs determined by qPCR were significantly (p<0.05) higher in HI (0.73%0.23%, n=60) than in KTR (0.45%0.21%, n=60) and in female HI (1.0%0.27%, n=30) than in male HI (0.45%0.23%, n=27). No effect of drugs or age was observed.The qPCR assay for nTregs provides reproducible results and is of sufficient quality for working with patient samples although inter-laboratory differences can be encountered due to a lack of method standardization. It was confirmed that gender-specific reference ranges are required.


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 | Schutz E.,University of Gottingen | Besenthal I.,University of Tübingen | Fraunberger P.,Medical Central Laboratory GmbH | Wieland E.,Central Institute for Clinical Chemistry and Laboratory Medicine
Therapeutic Drug Monitoring | Year: 2010

The immunosuppressant mycophenolic acid (MPA) used for solid organ transplantation is predominantly metabolized to a pharmacologically inactive phenolic glucuronide (MPAG) and, to a lesser extent, to the pharmacologically active acyl glucuronide (AcMPAG). The recently introduced CEDIA Mycophenolic Acid Assay from Microgenics has been reported to overestimate MPA in clinical samples and crossreactivity with AcMPAG has been suspected. A detailed investigation of the crossreactivity of AcMPAG and the prodrug mycophenolate mofetil (MMF) in the CEDIA assay is presented using pure substances. In addition, MPA concentrations in plasma were compared with a validated high-performance liquid chromatography-ultraviolet method. Plasma samples from kidney (KTx, n = 50), heart (HTx, n = 50), and liver (LTx, n = 50) transplant recipients were analyzed by the CEDIA (MPA) and a high-performance liquid chromatography-ultraviolet method (MMF, MPA, MPAG, AcMPAG). Crossreactivity of MMF (0.93-46.3 mg/L), MPAG (50-1000 mg/L), and AcMPAG (0.5-10 mg/L) was investigated using spiked drug-free plasma. Method comparison was performed using Bland & Altman and Passing & Bablok analysis. The method bias was correlated to AcMPAG concentrations using Spearman's rank correlation. Crossreactivity with AcMPAG and MMF was concentration-dependent and reached 215% and 143%, respectively. There was no crossreactivity with MPAG. The CEDIA assay showed a mean positive bias of 36.3% in patient samples. The mean bias was lowest with HTx samples (15%), 41.7% with KTx samples, and highest with LTx samples (52.3%). There was a positive correlation between the method bias and AcMPAG concentrations (r = 0.829; P < 0.001). No MMF was detected in patient samples. The CEDIA overestimates MPA concentrations on average by 36%. This bias is mainly the result of AcMPAG as previously observed with the EMIT MPA assay. It should be considered that the putative therapeutic range for MPA with the CEDIA assay will be higher than the range using high-performance liquid chromatography. Copyright © 2010 by Lippincott Williams & Wilkins.


Wieland E.,Central Institute for Clinical Chemistry and Laboratory Medicine | Shipkova M.,Central Institute for Clinical Chemistry and Laboratory Medicine
Clinical Biochemistry | Year: 2015

Immunosuppression is mandatory after solid organ transplantation between HLA mismatched individuals. It is a lifelong therapy that needs to be closely monitored to avoid under- and over-immunosuppression. For many drugs, pharmacokinetic monitoring has been proven to be beneficial. However, the therapeutic ranges are statistically derived surrogate markers for the effects that cannot predict the individual response of single patients. Better tailored immunosuppression biomarkers are needed that indicate immune activation. T cells are critically involved in organ rejection, and the means to assess their activation state may be promising to individualize immunosuppressive therapies. Activated T cells can be monitored with flow cytometry based on surface molecules that are typically up regulated or with molecules that are cleaved off the cell surface. Among these molecules are the interleukin-2 receptor (CD25); transferrin receptor (CD71); the T cell co-stimulatory molecules CD28, CD69, and CD154 and sCD30, which is a member of the TNF-alpha family. The effect of immunosuppressive drugs on T cell activation can be recorded with indirect cell function assays or by directly monitoring activated T cells in whole blood. Soluble proteins can be measured with immunoassays. This review provides a summary of the experimental and clinical studies investigating the potential of surface molecules as a tool for immune monitoring. It critically discusses the obstacles and shortcomings from an analytical and diagnostic perspective that are currently preventing their use in multicenter trials and clinical routine monitoring of transplant patients. © 2015.


Shipkova M.,Central Institute for Clinical Chemistry and Laboratory Medicine | Wieland E.,Central Institute for Clinical Chemistry and Laboratory Medicine
Clinical Biochemistry | Year: 2016

Solid organ transplantation is inevitably associated with the activation of the immune system of the graft recipient. An advanced knowledge of the immunological mechanisms leading to acute and chronic rejection, the advent of powerful immunosuppressive drugs, and refined surgical techniques have made solid organ transplantation a standard therapy to replace irretrievable loss of vital functions.The immune system is a complex network involving immune cells, cytokines, chemokines, antibodies, and the complement system. Monitoring and ideally influencing the allo-response of the organ recipient against the donor antigens may help to personalize the immunosuppressive therapy including the disclosure of those patients who are suitable for weaning or even discontinuation of immunosuppression. Immune monitoring comprises as plethora of candidate biomarkers capable of reflecting the donor specific and non-donor specific net activation state of the immune system in transplant recipients both before and after initiation of the immunosuppressive therapy.This special issue of Clinical Biochemistry on Immune Monitoring addresses the basic effects of immune activation in solid organ transplantation and critically reviews candidate biomarkers for immune monitoring and their analytical as well as clinical performance. © 2016 The Canadian Society of Clinical Chemists.


Shipkova M.,Central Institute for Clinical Chemistry and Laboratory Medicine | Franz J.,Gastroenterology | Abe M.,Central Institute for Clinical Chemistry and Laboratory Medicine | Klett C.,Central Institute for Clinical Chemistry and Laboratory Medicine | And 2 more authors.
Therapeutic Drug Monitoring | Year: 2011

Background: Inosine triphosphate pyrophosphatase (ITPA) catalyzes the pyrophosphohydrolysis of inosine triphosphate to inosine monophosphate. Recently, single-nucleotide polymorphisms in the ITPA gene, associated with decreased enzyme activity, have been reported. Some clinical studies have demonstrated that the 94C>A mutation is linked to flu-like symptoms, rash, and pancreatitis during azathioprine (AZA) therapy and to early AZA discontinuation. In this study, we investigated whether the enzyme phenotype is also related to adverse effects (AEs). Methods: Patients suffering from inflammatory bowel disease who were treated with AZA (N = 160; age 43 ± 12 years) were included. Data were categorized into quartiles according to the ITPA activity. Information about the therapeutic regimen, AEs [leucopenia, increased hepatic enzymes (alanine aminotransferase, aspartate aminotrasnferase, gamma-glutamyl transferase), flu-like symptoms, and pancreatitis], cotherapy, and comorbidity was obtained from the responsible clinicians and patients by using a standardized questionnaire. ITPA activity was measured by a validated high-performance liquid chromatography procedure. In patients with decreased ITPA activity, the 94C>A and IVS2 + 21A>C genotypes were determined. Results: AEs were reported significantly more often for patients with low ITPA activity than for patients with high ITPA activity; the highest odds ratio for occurrence of AEs was found to be below a threshold of 59.9 μmol/ (gHb·h) [hemoglobin (Hb)]. Decreased ITPA activities [particularly <89.2 μmol/(gHb·h)] were frequently accompanied by leucopenias, whereas very low enzyme activities [<37.3 μmol/(gHb·h)] were associated with a higher incidence of increased liver enzymes. Conclusions: The results demonstrate a relationship between low ITPA activity and AEs and support the idea that the determination of ITPA phenotype might be an appropriate alternative to genotyping. Copyright © 2011 by Lippincott Williams & Wilkins.


PubMed | Central Institute for Clinical Chemistry and Laboratory Medicine
Type: Editorial | Journal: Clinical biochemistry | Year: 2016

Solid organ transplantation is inevitably associated with the activation of the immune system of the graft recipient. An advanced knowledge of the immunological mechanisms leading to acute and chronic rejection, the advent of powerful immunosuppressive drugs, and refined surgical techniques have made solid organ transplantation a standard therapy to replace irretrievable loss of vital functions. The immune system is a complex network involving immune cells, cytokines, chemokines, antibodies, and the complement system. Monitoring and ideally influencing the allo-response of the organ recipient against the donor antigens may help to personalize the immunosuppressive therapy including the disclosure of those patients who are suitable for weaning or even discontinuation of immunosuppression. Immune monitoring comprises as plethora of candidate biomarkers capable of reflecting the donor specific and non-donor specific net activation state of the immune system in transplant recipients both before and after initiation of the immunosuppressive therapy. This special issue of Clinical Biochemistry on Immune Monitoring addresses the basic effects of immune activation in solid organ transplantation and critically reviews candidate biomarkers for immune monitoring and their analytical as well as clinical performance.

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