To recreate the in vivo hematopoietic cell microenvironment or niche and to study the impact of extracellular matrix (ECM) biophysical properties on hematopoietic progenitor cell (HPC) proliferation and function, mouse bone-marrow derived HPC (Lin-Sca1+cKit+/(LSK) were cultured within three-dimensional (3D) type I collagen oligomer matrices. To generate a more physiologic milieu, 3D cultures were established in both the presence and absence of calvariae-derived osteoblasts (OB). Collagen oligomers were polymerized at varying concentration to give rise to matrices of different fibril densities and therefore matrix stiffness (shear storage modulus, 50–800 Pa). Decreased proliferation and increased clonogenicity of LSK cells was associated with increase of matrix stiffness regardless of whether OB were present or absent from the 3D culture system. Also, regardless of whether OB were or were not added to the 3D co-culture system, LSK within 800 Pa collagen oligomer matrices maintained the highest percentage of Lin-Sca1+ cells as well as higher percentage of cells in quiescent state (G0/G1) compared to 50 Pa or 200 Pa matrices. Collectively, these data illustrate that biophysical features of collagen oligomer matrices, specifically fibril density-induced modulation of matrix stiffness, provide important guidance cues in terms of LSK expansion and differentiation and therefore maintenance of progenitor cell function.

Background: Copeptin acts as surrogate marker under stress stimuli, as well as an outcome predictor based on serum or plasma concentration in patients suffering intracranial hemorrhage, aneurysmal subarachnoid hemorrhage (aSAH), and stroke. The aim of this study was to establish a method for quantification of copeptin levels in cerebrospinal fluid (CSF) and to demonstrate its clinical applicability in patients following aSAH. Methods: This assay was validated for CSF samples using a commercial immunoluminometric assay (IMLA). For the control group (10 patients), CSF copeptin levels were determined in patients without signs of acute neurological diseases and who underwent a diagnostic lumbar puncture. The pilot cohort included calculation of copeptin levels in CSF and in serum of patients following aSAH. Results: The control group had CSF copeptin levels lower than 0.78 pmol/L-1. Among patients with aSAH, CSF copeptin values had a mean of 20.1 pmol/L-1 and serum copeptin concentrations had a mean of 61.39 pmol/L-1. Conclusions: This assay provides to best of our knowledge for the first time initial ranges values of CSF copeptin for patients without acute neurological disease and in patients with aSAH. Thus, it opens new doors to develop further calculations and relationships between diseases biomarker and outcome prediction.

Zusammenfassung: Zirkulierende zell-freie Nukleinsäuren (cfNA, meist als cfDNA bezeichnet) werden zunehmend als eine neue Klasse von diagnostischen Markern wahrgenommen. DNA, mRNA und miRNA zirkulieren weniger in „nackter Form“, sondern sind verpackt und entgehen so einem schnellen Abbau im peripheren Blut. Zusammen mit der Tatsache, dass cfNA in verschiedenen wichtigen Erkrankungen sowohl qualitativ als auch quantitativ verändert sind, schafft dies ein völlig neues Universum für die labormedizinische Diagnostik. Erste Anwendungen wie z.B. die sensitive und spezifische Detektion von tumor-spezifischen Mutationen im Plasma erobern die Arena der labordiagnostischen Krankenversorgung und erlauben den Nachweis therapeutisch relevanter molekulargenetischer Tumorprofile aus dem Blut für die frühe Erkennung von Rezidiv oder Therapieversagen. Es bleiben viele Fragen zu klären, darunter die Kreuzvalidierung mit etablierten und wichtigen Parametern der Labormedizin. Auch die Einordnung präanalytischer Aspekte, die metrologische Fragen von Richtigkeit und Präzision, etc. sind für die Einordnung von analytischer und medizinischer Qualität dringend erforderlich. Nicht zuletzt eröffnen die frei zirkulierenden Nukleinsäuren eine völlig neue Biologie von Signalen, die in Gesundheit und Krankheit zwischen Zellen und Organen durch unseren Körper reisen. Es wird eine große wissenschaftliche Herausforderung sein, die biochemischen und pathobiochemischen Implikationen zu verstehen. Für Entwicklung und Implementation dieses neuen diagnostischen Felds ist signifikant, dass die Klinische Chemie die erforderlichen Expertisen sowie das komplementierende Spektrum etablierter Biomarker bereithält, um eine ordentliche Transition des Einsatzes zirkulierender Nukleinsäuren in die Diagnostik zu gewährleisten. Dies wird vorhersehbar das Spektrum der Labormedizin komplementieren, um die therapeutische Medizin bei ihren Entscheidungen in der Patientenbehandlung zu unterstützen.

Diagnostic lab results are the basis of all rational medical actions. Within diagnostic medicine, there has been a long-standing trend in clinical chemistry/laboratory medicine that it employs the far greatest number of different technologies, analytical laboratory methods and parameters in preparing medical in vitro lab results. It is foreseeable that the sustained rapid technological developments over the past few years, as well as the concept of their application on a more personalized form of medicine, will further intensify the significance of laboratory findings. Already in the second half of the 20th century, the profession recognized, based on its involvement with analytical techniques, that a key prerequisite for properly performing a clinical-chemical analysis and subsequent assessment of its results is first based on the definition of technical measuring equipment terms. We owe today’s cornerstones of quality assurance to the desire for comparability of results through standardization of analytic methods and systematic development of their regularities. As a scientific professional association, today’s Deutsche Vereinte Gesellschaft fuer Klinische Chemie und Laboratoriumsmedizin (DGKL) plays a central role here in the establishment and expansion of a reference method concept for clinical-chemical benchmarks as well as in the establishment and dissemination of so-called “Standardized Methods of the Deutsche Gesellschaft fuer Klinische Chemie” (DGKC, one of the two precursor organizations). A logical outgrowth of the external quality assurance tests performed since the 1960s for standardization of laboratory tests was the first Directive of the German Medical Association (Rili-BAEK) in 1971. Amendments to the directive, the promulgation of EU directives on medical devices (IVDD) and national regulations in the medical products act (MPG) and the Medical Products Operator Ordinance (MP-BetreibV) have further solidified the obligation towards quality assurance for quantitative medical laboratory tests in the Rili-BAEK since 2002. Thoughts on the formulation of an analogous Rili-BAEK “for the performance of qualitative medical laboratory tests” derived their direct impetus from daily experience in the increasing presence of molecular diagnostics: While legally binding rules for the quantitative tests applied in medical laboratories according to the Rili-BAEK, quality assurance of nominal or ordinal findings, as is typical for research findings from the field of molecular and cytogenetics, only exists on a voluntary basis. In particular, against the backdrop of the permanence of genetic findings, an increasing number of laboratories felt the need for a set of rules analogous to the existing Rili-BAEK. This was also impressively manifest in the double-digit percentage growth rates of participation in molecular external quality assurance tests from 1998 by the former DGKC. After acceptance of the advisory board’s concept for a comprehensive revision of the Rili-BAEK by the management board of the German Medical Association (BAEK) in 2007, the BAEK advisory board, under the leadership of its Chairman Wolfgang Vogt and BAEK consultant Manfred Brüggemann, established interdisciplinary working groups to formulate special components of qualitative laboratory tests. Upon the recommendation of the German Medical Association (BAEK), and based on the method-oriented classification system of the Rili-BAEK, in 2007 a working group of the Deutsche Vereinte Gesellschaft fuer Klinische Chemie und Laboratoriumsmedizin (DGKL) prepared an initial recommendation of a directive for “Quality Assurance of Qualitative Medical Laboratory Tests”. Due to the Genetic Diagnostics Act that came into effect in the year 2010 and its regulations concerning genetic analytics, it had become necessary, however, to regulate the moleculargenetic tests contained in B2 in a separate Section B5. As a result of the interdisciplinary effort, the new Rili-BAEK is complete and encompasses all the working areas of laboratory medicine. In addition to a section on “Qualitative Medical Laboratory Tests” (B2, published in 2011) with the additional special directive sections “Ejaculate Tests” (B4, published in 2011), it also includes “Molecular and Cytogenetic Tests” (B5, published in 2011) and “Medical Laboratory Tests for the Direct Detection and Characterization of Infectious Agents” (B3, published in 2013). Now that an initial English translation is available, it is our hope that this set of rules will also be perceived as helpful and inspiring in other countries and will thus facilitate the strengthening of the high quality of laboratory diagnostics in diagnostic medicine.

In 2011, the German Diabetes Association (DDG) and the German Society of Obstetrics and Gynecology (DGGG) published a new, inter-disciplinary, evidence-based S3-guideline (AWMF guideline 057/008) for gestational diabetes mellitus (GDM, ICD-10: 024.4G) [1]. It contains recommendations on the diagnostic procedure, treatment and follow-up care of GDM. This guideline also makes it possible for the first time for the attending physician to use POCT glucose measurement systems with unit-use reagents in the initial diagnostic procedure of GDM. At the same time, a corresponding manufacturer's recommendation is a requirement for this use [1] [2]: "For the initial diagnostic procedure of diabetes manifested in pregnancy or gestational diabetes when using unit-use reagents and related measurement systems, these systems are explicitly intended for physician use in the diagnostic procedure according to the manufacturer's recommendations (instructions for use)."

Zusammenfassung Die DDG und die Deutsche Gesellschaft für Gynäkologie und Geburtshilfe haben im Jahr 2011 für den Gestationsdiabetes mellitus eine neue S3-Leitlinie veröffentlicht. In dieser Leitlinie wird erstmals auch der Einsatz von POCT-Glukose-Messsystemen mit Unit-use-Reagenzien bei der Erstdiagnostik des GDM durch den betreuenden Arzt ermöglicht. Da diese Leitlinie keine detaillierten Vorgaben für die geforderten Herstellerempfehlungen enthielt, wurden im Jahr 2012 die Anforderungen an die Messqualität und die Qualitätssicherung von POCT-Glukose-Messsystemen mit Unit-use Reagenzien für die primäre GDM-Diagnostik durch eine gemeinsame Stellungnahme der Deutschen Vereinten Gesellschaft für Klinische Chemie und Laboratoriumsmedizin (DGKL) und der DDG unter Berücksichtigung der zu diesem Zeitpunkt verfügbaren technischen Möglichkeiten konkretisiert und im Jahr 2015 wegen der zwischenzeitlichen technologischen Fortschritte dieser POCT-Systeme aktualisiert. Damit soll den Herstellern, den Zulassungs- und Regulierungsbehörden, den Nutzern bei klinischen Studien und im Praxisalltag, sowie den Kostenträgern eine evidenzbasierte Orientierung gegeben werden.

Antibiotic resistance is an unsolved healthcare problem with increasing impact on patient management in the last years. In particular, multidrug resistance among Gram-negative bacterial strains has become the most pressing challenge. In order to deliver the most efficacious antimicrobial therapy with minimum delay, rapid diagnostic tests are required in order to detect multidrug resistant pathogens early during infection. In line with these efforts, we have developed a mass spectrometry-based assay for the rapid determination of ampicillin and cefotaxime resistance. The assay quantifies beta-lactamase activities towards ampicillin and cefotaxime within a turnaround time of 150 min, which is substantially faster than classical susceptibility testing.

In the human organism more than 500 proteases have been described so far. Many of them are essential in the regulation of physiological processes, as inflammation, immune response, coagulation or growth. A dysregulation in protease activity corresponds to severe malfunctions and causes numerous pathophysiological diseases, as neurodegenerative disorders, cardiovascular diseases and cancer. When it comes to cancer, proteases play an important role in progression and metastasis. Some are secreted from the tumor and can be found in the extracellular matrix of the tumor microenvironment and also in the bloodstream. Functional protease profiling aims at discovering tumor associated protease activity in clinical specimens (serum, plasma and tissue), which could be used for diagnostic and prognostic purposes. Therefore, it is necessary to find substrates, which are specifically cleaved by cancer-associated proteases. Various approaches using antibody based antigen detection or MS-based techniques, are limited in the number of samples, which can be screened in parallel. To overcome these problems, peptide microarrays were used. Compared to Ronald Frank´s SPOT-synthesis, micro-particle solid phase peptide synthesis (mpSPPS) allows much higher peptide densities with up to 1000 different peptides per cm2, dependent on the layout. This PhD thesis dealt with the development of a high-throughput screening assay platform based on in-situ synthesized peptide microarrays. As first step a model system, using known proteases (trypsin, thrombin, proteinase k etc.), was developed. To check for general applicability of the PEGMA/MMA surface, on which the peptide synthesis takes place, the manufactured peptide microarrays, containing N-terminal antibody recognition sequences (FLAG- & HA-tags), were used without further chemical modification. After proteases incubation, the respective fluorescently labeled anti-FLAG- & anti-HA antibodies will only bind to peptides, bearing the intact tag-sequence, leading to a decrease in fluorescence intensity, where the enzymes were active. After demonstrating on-chip proteolysis, using indirect antibody labeling, the biotin-streptavidin system was introduced to minimize the peptide label to a smaller tag. This allowed greater sequence variability and avoided false positive cleavage, as when using a proteinogenic tag sequence. Together with the PEPperPRINT Company, a biotin toner was developed, to integrate this labeling reagent into the in-situ synthesis process, which turned out to be advantageous compared to in-solution modification of the peptide content. To further overcome limitations on the part of the solid support, the polymer film was optimized, by introducing a new dextran surface. Preliminary experiments in lab-scale showed good proteolytic cleavages with model proteases and spotted peptides. The transfer to production scale however, showed the requirement of optimization, regarding polymer composition and peptide density, which is an ongoing process.

AimsTo evaluate the diagnostic and prognostic value of osteopontin in patients with acute dyspnoea and/or peripheral oedema suspected of having acute congestive heart failure (aCHF).Methods and resultsA total of 401 patients presenting with acute dyspnoea and/or peripheral oedema to the emergency department were prospectively enrolled and followed up for up to 5 years. Blood samples for biomarker measurements were collected on admission to the emergency department. Osteopontin combined with NT‐proBNP vs. NT‐proBNP alone for diagnosis of aCHF was tested. Additionally, osteopontin vs. NT‐proBNP for prognostic outcomes (i.e. all‐cause mortality, aCHF‐related rehospitalization, and both in combination) was tested. The diagnostic and prognostic capacity of osteopontin was tested by C‐statistics, reclassification indices, and multivariable Cox prediction models. Osteopontin plus NT‐proBNP improved the diagnostic capacity for aCHF diagnosis [accuracy 76%, 95% confidence interval (CI) 72–80%; specificity 74%, 95% CI 69–79%, net reclassification improvement (NRI) +0.10] compared with NT‐proBNP alone in the emergency department (P = 0.0001). Osteopontin independently predicted all‐cause mortality and aCHF‐related rehospitalization after 1 and 5 years. Compared with NT‐proBNP, osteopontin was of superior prognostic value, specifically in aCHF patients and for the prognostic outcome of aCHF‐related rehospitalization.ConclusionOsteopontin improves aCHF diagnosis when combined with NT‐proBNP. Osteopontin identifies aCHF patients with high 1‐ and 5‐year mortality and rehospitalization risk, and adds prognostic value to NT‐proBNP.Trial registrationNCT00143793

Early targeted antimicrobial therapy helps decrease costs and prevents the spread of antimicrobial resistance, including in Escherichia coli, the most frequent Gram-negative bacterium that causes sepsis. Therefore, rapid susceptibility testing represents the major prerequisite for knowledge-based successful antimicrobial treatment. To accelerate testing for antibiotic susceptibility, we have developed a new mass spectrometry-based assay for antibiotic susceptibility testing (MAAST). For proof of principle, we present an ampicillin susceptibility test for E. coli with a turnaround time of 90 min upon growth detection.