Band 38, Heft s1 - Translations

With increasing life expectancy, neurodegenerative diseases such as Alzheimer’s disease (AD) and Parkinson’s disease (PD), as well as neurovascular diseases such as cerebral ischemia, are becoming a rapidly growing worldwide problem causing an increased burden on society. The development of biomarkers is aimed to enable the identification of at-risk individuals, enable early diagnosis, and reflect the progression of disease. Future challenges refer to the need for standardization of known biomarkers on the one hand and the identification of new biomarkers on the other hand. The former is impressively exemplified by a recent study addressing the impact of various types of cerebrospinal fluid collection tubes for detection of total tau, phosphorylated tau, and β-amyloid 1–42 on the diagnosis of AD. This issue was identified as a key issue by the Alzheimer’s Biomarkers Standardization Initiative (ABSI) and recommendations were listed which should be used to enable better standardization between laboratories and enable results for the AD biomarkers to be comparable between different sites. The latter challenge in particular concerns PD and is addressed by the Parkinson Progression Marker Initiative (PPMI), a comprehensive observational, international, multi-center study designed to identify PD progression biomarkers both to improve understanding of disease etiology and course and to provide crucial tools to enhance the likelihood of success of PD modifying therapeutic trials. With regard to cerebral ischemia, at present there are no clinically validated biomarkers. Nevertheless, the development of a clinically validated biomarker of acute cerebral ischemia would have the potential to facilitate the use of time-sensitive reperfusion strategies, allow for individualization of patient care by predicting relative risk of hemorrhage and volume of penumbral tissue, and add valuable prognostic information for patients presenting with acute stroke. Thereby, it is expected that a panel of biomarkers rather than one single marker will meet these criteria in the future. Unlike cerebral ischemia, cerebral hemorrhage biomarkers will probably be limited to the assessment of prognosis in the course of the disease.

Background: Natalizumab-neutralizing antibodies (NABs) occur in 9% of natalizumab-treated multiple sclerosis (MS) patients. Loss of clinical and biological efficacy in patients with persisting NABs requires termination of natalizumab treatment. Because high-titer NABs are strongly associated with persistence of NABs, we investigated if determination of natalizumab saturation levels of α-4 integrins by flow cytometry has the potential to identify patients early with NABs. Methods: Cell-bound natalizumab and natalizumab saturation of α-4 integrins on T cells were detected by flow cytometry using a monoclonal anti-human IgG4 antibody. Peripheral blood mononuclear cells were enriched from venous blood collected at the start (baseline) and every 4 weeks immediately before subsequent infusions until up to 9 months from natalizumab-treated patients with NABs (n=4) and at the start and after 1 (n=15), 2 (n=14), 3 (n=9), 6 (n=7), and 9 months (n=3) from natalizumab-treated patients without NABs. Natalizumab saturation levels (in %) of T cells were determined by relating median fluorescence intensities (MFIs) of in vivo bound natalizumab to MFIs after in vitro incubation with saturating amounts of natalizumab. Determination of serum NABs was performed by ELISA. Results: In patients without NABs, the median natalizumab saturation level of T cells over 9 months was 75% (confidence interval of 95%: 72–78%). In two of the four patients with NABs, the natalizumab saturation level of T cells only reached approximately 50% after the first infusion and further declined to baseline levels with the second infusion. Low-titer NABs were measured after the first infusion and development of persistent high-titer NABs led to termination of natalizumab treatment after 6 months. In another two patients, the natalizumab saturation level of T cells was 74% and 68% after the first infusion, temporarily decreased to approximately baseline levels and re-increased after approximately 6 months. Transient NABs were detected after 2 and 3 months, which resolved after 5 and 6 months. Conclusions: Monitoring the natalizumab saturation level on T cells is a fast and reliable method to identify patients with a reduced treatment effect due to NABs. Both high- and low-titer NABs were equally effective in reducing cellular natalizumab saturation levels. We were able to show that monitoring the natalizumab saturation levels by flow cytometry, is a sensitive method for detecting a prolonged NAB-mediated reduced treatment effect because NABs are apparently effective longer than suggested by the detection limit of ELISA.

Teardrop erythrocytes (syn. dakryocytes) play a key role in the evaluation of peripheral blood smears in patients with anemia, especially as part of the “leukoerythroblastic picture”. Teardrop-shaped red blood cells can be seen in a wide range of diseases that lead to bone marrow fibrosis, which is often accompanied by extramedullary hematopoesis. The differential diagnoses encompass primary myelofibrosis (PMF), myelodysplastisc syndromes during the late course of the disease, rare forms of acute leucemias and myelophtisis caused by metastatic carcinoma. Rarely, MF associated with post-irradiation, toxins, autoimmune diseases, metabolic conditions, inborn hemolytic anemias, iron-deficient anemia or β-thalassaemia can lead to the formation of teardrops as visualized on peripheral blood smears.

Diagnostic assays for detection and monitoring of virus infections have become more and more important and are widely used in routine diagnostics. In particular, the detection of newly emerging infectious diseases is challenging. In addition, travel-associated diseases caused by dengue viruses, chikungunya viruses, influenza viruses, or other viruses draw the attention of physicians. Therapeutic treatment regimens and prevention strategies can also influence the need for diagnostic assays, such as, in the case of the human immunodeficiency virus (HIV) and human papillomavirus (HPV). Moreover, well-known virus infections, such as hepatitis E virus infections, can gain new clinical relevance.

This article discusses relevant and up-to-date issues for the clinical laboratory concerning arthritis activity and monitoring of rheumatoid arthritis, details for the new disease entity of IgG4-related diseases and Sjogren’s syndrome. The article puts the focus on the data of 2013.

Owing to the high prevalence and associated complications of liver fibrosis, of any etiology, and nonalcoholic fatty liver disease (NAFLD), both have become important public health issues. Liver biopsy is considered the gold standard for diagnosis and staging of liver fibrosis, as well as NAFLD. Recent studies have discovered and validated several non-invasive biochemical biomarkers and imaging procedures for the diagnostics of liver fibrosis and NAFLD. In comparison to patented tests (FibroTest ® , Fibrometer ® , and Hepascore ® ), non-patented tests (APRI, ELFG, FIB-4, Forns Index, and MP3) tend to have a lower diagnostic performance, especially for the diagnosis of significant fibrosis (METAVIR stage F2). The difference in performance is less pronounced for the diagnosis of cirrhosis (METAVIR stage F4). Elastography is superior to biomarkers in the diagnosis of cirrhosis (F4) but not fibrosis (F2). However, in 20% of patients elastography cannot be performed or evaluated due to anatomical reasons. Cytokeratin 18 (CK-18) is the most promising single biomarker for the diagnosis of non-alcoholic steatohepatitis (NASH). Scores and algorithms have been less extensively validated for their diagnostic performance in diagnosing and staging of NAFLD and NASH as compared with fibrosis in chronic hepatitis. Data are promising. Patented scores as well as CK-18 appear slightly superior to freely available scores including the NAFLD fibrosis score, which is recommended by American guidelines. In conclusion, non-invasive biomarkers and elastography appear promising as prescreening tools to limit the number of liver biopsies.

Background: General screening for gestational diabetes mellitus (GDM) is recommended in Austria since 2010. As a result of the guidelines, pregnant women are tested between 24 and 28 weeks of gestation with the 75 g/2 h-oral glucose tolerance test (75 g/2 h-OGTT). The aim of this study was to evaluate the prevalence of GDM in our laboratory retrospectively. Furthermore, we wanted to study the pattern of abnormal 1 h- and 2 h-glucose values from 75 g/2 h-OGTTs compared with fasting plasma glucose values. Further testing of GDM patients after delivery is recommended. As a result of this issue we analyzed all follow-up screening. Methods: Standardized 75 g/2 h-OGTTs were assessed in 3963 pregnant women. The cut-off value for fasting plasma glucose (FPG) is ε 5.1 mmol/L, for 1 h value ε 10.0 mmol/L, and for 2 h value ε 8.5 mmol/L. One or more abnormal values were considered as GDM, respectively. Results: GDM was detected in 8.5% (n=335) of the tested pregnant women. Elevated FPG values were measured in 5.1% (n=201). These are 60% of all GDM patients. After delivery we analyzed 14 out of 335 GDM patients (4.2%) to reevaluate postpartum glucose tolerance with the standard OGTT (World Health Organization criteria). Conclusions: GDM is a common disease, and in our study 8.5% of pregnancies were affected. When and how to screen is still a matter of discussion. One strategy to become more cost-effective is to use a two-step screening algorithm including FPG measurement and a risk estimation model. In postpartum follow-up, there is still considerable potential to reduce diabetes-associated illness and costs.

Immunological diagnostics is a rapidly developing area in laboratory medicine. Most recently, major developments in the area of immunodeficiency and the monitoring of chronic inflammatory diseases have been observed. Regarding immuno-monitoring, recently a consensus panel for basic flow cytometry has been published together with age-related reference values. In the USA, the search for severe inherited immunodeficiency diseases (such as severe combined immunodeficiency disease, SCID) is part of neonatal screening procedures. Recently, several US states published first results which are based on the measurement of T-cell receptor excision circles (TRECs). Furthermore, age-dependent reference values for the measurement of IgG subclasses and subclass-specific vaccination antibodies have been published. Monitoring of chronic inflammatory disease focuses on asthma. A novel classification based on the cellular distribution of neutrophils versus eosinophils in induced sputum has been developed. Furthermore, novel biomarkers, such as periostin, are currently under evaluation. Such novel approaches of phenotyping are now the basis of individualized therapeutic approaches in patients with (severe) asthma, who respond to certain biologicals.

Laboratory testing is crucial for the successful medical treatment of many patients. Laboratory tests should neither be ordered too infrequently nor too frequently (in the form of repeat testing). These recommendations summarize the intervals for repeat testing based on studies, pathophysiology and consensus, in regard to both the time intervals between two tests and the additional criteria for the repeat testing. These recommendations are complemented by general principles for the indication and testing frequency of laboratory testing.

Abstract: The use of whole genome sequencing in translational research not only holds promise for finding new targeted therapies but also raises several ethical and legal questions. The four main ethical and legal challenges are as follows: (1) the handling of additional or incidental findings stemming from whole genome sequencing in research contexts; (2) the compatibility and balancing of data protection and research that is based on broad data sharing; (3) the responsibility of researchers, particularly of non-physician researchers, working in the field of genome sequencing; and (4) the process of informing and asking patients or research subjects for informed consent to the sequencing of their genome. In this paper, first, these four challenges are illustrated and, second, concrete solutions are proposed, as elaborated by the interdisciplinary Heidelberg EURAT project group, as guidelines for the use of genome sequencing in translation research and therapy in Heidelberg.

NMR spectroscopy is a modern analytical method which is extremely suitable for the analysis of various body fluids. In addition to small metabolite quantification, the method is capable of differentiated lipoprotein subfraction measurements. The technique can be well standardized and allows extensive automation and good sample throughput compared with lipoprotein fractionation via ultracentrifugation. Because all evaluated parameters are determined simultaneously in one measurement, this method is especially suitable for metabolomics approaches in which even subtle changes in metabolite ratios may be relevant. This review gives an overview of the current methods of NMR spectroscopy, analysis strategies, and practical applications in various studies concerning lipid analysis as well as metabolomics. Over the course of the past years, NMR spectroscopy has heavily evolved from a mere research method into a tool that can be expected to play an important role in routine diagnostic testing in the future.

A number of legal issues arise with regard to genome analyses. In the context of medical treatment, some of these issues are being dealt with by the German Gendiagnostikgesetz. In the context of science, the right to self-determination over personal data should be observed. A legislative act would be necessary to create a reliable basis for scientists, doctors, and patients.

The success of transplantation medicine is closely associated with the introduction of potent immunosuppressive drugs. Therapy guidance of the calcineurin inhibitors cyclosporine and tacrolimus as well as the mammalian target of rapamycin (mTOR) inhibitors sirolimus and everolimus is achieved by therapeutic drug monitoring (TDM). For other immunosuppressive drugs such as mycophenolic acid, TDM is not established. It has been suggested that a better individualization of pharmacotherapy could be helpful in avoiding over- and under-immunosuppression, which have been associated with poor long-term outcome of grafts and patients. Therefore, a search for biomarkers that could complement TDM, thereby allowing a better individualization of therapy, is ongoing worldwide. Pharmacodynamic biomarkers in transplantation medicine can be either non-drug-specific, aiming at assessing the global effect on the immune system, or drug-specific, aiming at recording the pharmacologic effect on a drug target. Non-specific pharmacodynamic biomarkers can reflect the degree of immune activation by measuring T- or B-cell activation, the development of operational tolerance by monitoring, e.g., regulatory T-cells, or the graft damage by measuring cell-free DNA released by the graft in response to injury. Drug-specific pharmacodynamic biomarkers have been published for mycophenolic acid, calcineurin, and mTOR inhibitors. The field of biomarker research to complement TDM for a better individualization of immunosuppression is still in its infancy, and controlled prospective clinical trials are needed to unravel or dismiss the potential of particular biomarkers or biomarker combinations in various patient cohorts with different grafts.

If there is a suspicion of a systemic autoimmune disease, a two-step assessment of autoantibodies (AAb) is recommended for the serological diagnosis thereof. First, AAb will be determined using sensitive, cell-based indirect immunofluorescence. Then, a positive result must be confirmed with a more specific test due to the possibility of false-positive results. This gradual approach is necessary because there is currently no assay technique that fulfills the requirements for a one-stage procedure for sensitivity and specificity. For effective AAb analysis, simultaneous determination of several AAb with multiparametric confirmatory assays significantly shortens serological diagnosis, compared with conventional monoparametric testing. Yet, currently available multiparametric AAb detection techniques do not offer the combination of screening and confirmatory testing. Thus, a new approach based on digital fluorescence was developed by applying a novel CytoBead technology that is presented here. The aim was to combine the recommended stepwise approach consisting of sensitive screening and confirmation of specific diagnosis in a reaction environment and thereafter the possibility of adaptation to the serological diagnosis of several autoimmune diseases. Using standard microscopic glass slides and the combination of native cellular or tissue substrates with autoantigen-loaded fluorescent microparticles (beads) in a reaction environment, along with the possibility of manual and automatic evaluation by IIF and the quantitative measurement of fluorescent signals, the disadvantages of currently existing test systems could be overcome. This novel concept is applicable for the determination of various multiparametric AAb, e.g., the determination of antinuclear antibodies and the corresponding AAb in molecular cytoplasmic and nuclear autoantigenic structures. Further, this becomes the basis for the simultaneous multiparametric AAb determination for the serology of celiac disease or ANCA-associated vasculitides.

Despite the progress in the establishment of specific autoantibody assays, screening for antinuclear antibodies (ANA) by indirect immunofluorescence on HEp-2 cells for quality-oriented laboratory diagnosis of ANA associated rheumatic diseases (AARD) remains indispensable. Research results on the relevance of the dense fine speckled (DFS) pattern and DFS70 antibodies disclosed novel possibilities to optimize the serological stepwise diagnostics of AARD. The DFS pattern on HEp-2 cells is well differentiated from the classic “homogeneous” ANA pattern associated with dsDNA antibodies. In DFS pattern positive sera the most important detectable ANA specificity is the DFS70 antibody (synonym LEDGF antibody). This antibody is also the most frequent ANA specificity in ANA positive healthy persons. The prevalence of DFS70 antibodies in AARD patients is significantly lower compared with the prevalence in ANA-positive healthy individuals. There is a negative association between DFS70 antibodies and AARD, especially if no concomitant AARD-specific autoantibodies are found. Isolated DFS70 antibodies are detectable in <1% of AARD, but are detectable in 5%–11% of healthy individuals. In the presence of an isolated DFS70 antibody, the posttest probability for AARD is reduced significantly. DFS70 antibodies are valuable novel biomarkers for the improved interpretation of positive ANA but without detectable AARD associated autoantibodies and should be integrated in modified test algorithms to avoid unnecessary referrals and examinations of ANA-positive subjects.

Reimbursement of medical laboratory services may initially seem self-explanatory to all involved – doctors, hospitals, and laboratories. However, recurrent questions on how and to whom a particular test has to be invoiced, or whether a particular mode of billing is correct, demonstrate the great complexity in this field. These and other related issues emerge if laboratory orders involve different laboratory sectors, medical tariffs, and invoice recipients. The correct invoice practice – often evident only at second glance – has fundamental legal and financial significance not just for the laboratory service provider but also for the medical client. Influencing nearly all business processes from order to invoice, the correct invoice practice deserves special attention and diligence. In this review, we cover all relevant aspects of billing medical laboratory services in the context of ambulatory care, hospital treatment, and other models on the basis of the German health care law. We focus not only on laboratory medicine and microbiology but also include human genetics and histopathology. In addition, a schematic representation of common rules of invoicing laboratory services is provided with references to the appropriate legal text sources.

Hematological laboratory diagnostics of platelets is faced with technical difficulties and requires preanalytical considerations. The handling of platelet agglutination needs to be addressed in the daily routine. This article summarizes the current state of platelet counting and subsequent differential diagnosis.

Abstract: Cryoproteins still remain largely undiagnosed despite their clinical relevance. Cryoglobulins, cryofibrinogen and cold agglutinins respectively show a significant association with autoimmune diseases and non-Hodgkin’s lymphoma. The analytical part is relatively straightforward; however, the preanalytical phase is a challenge. This article briefly summarizes the relevant aspects to improve this situation.

Therapeutic drug monitoring of psychiatric medication as well as pharmacogenetic testing is performed more and more frequently in numerous laboratories. In this review, a summary of the literature in the years 2011 and 2012 has been completed. The guidelines of the German AGNP (Association for Neuropsychopharmacology and Pharmacopsychiatry) contain all the information needed for the interpretation of drug concentrations. The determination of serotonin in urine could be a marker for the assessment of the response of antidepressants, and correlations between the occupancy of the target receptors in the brain and drug concentration have been established using positron emission tomography. The influence of age on drug concentrations has been controversially described, and additionally females have always showed a slower metabolism and higher serum concentrations. Several liquid chromatography-mass spectrometry (LC-MS)/MS multi-analyte procedures for the quantification of psychiatric medication have been described. All methods showed good validation data, but there have always been some compounds with less good validation results due to the fact that not all compounds of a multi-analyte procedure can be analyzed optimally. Pharmacogenetic testing is not routinely performed prior to the prescription of psychiatric medication. This relies, among other things, on missing large randomized trials and the absence of standardized analytical methods, which allow the identification of the whole genetic variability.