Volume 34, Issue 6

The enumeration of reticulocytes is at present a standard test in medical laboratories and is derived from haematology analysers which operate under the principle of flow cytometry. The number of requests to differentiate among different types of anaemias is unsatisfactory, but blood doping with erythropoietin increased interest in this laboratory method. The results of external quality assessment schemes and a multicentric study for reference values showed systematic differences between certain types of instruments and the need for adjusted reference ranges. The material for internal QC differs in relevant aspects from the patient sample and what is important to understand for proper QC. In the same way, it is essential to identify interfering factors that are mostly irrelevant to healthy donors whose blood was used to develop the prototype of the analyser. It is difficult to interpret the results of reticulocyte measurement in the differential diagnosis of anaemia because reference values exist for healthy adults only. The reticulocyte production index has been introduced for this purpose; however, little literature exists for this parameter. A new development is the analysis of so-called reticulated platelets by existing technology; however, the staining behaviour differs. In the present paper, we review the principles of measuring reticulocytes. The interpretation of the results of reticulocyte analysis is given in a different paper.

The introduction of next-generation sequencing technologies in human genetic diagnostics is a challenge to many of its aspects. It is mainly positive, even revolutionary, which will be discussed as well as its critical aspects. It used to take several months or years to complete genetic testing. This already belongs to the past. As sequencing technologies are progressing at an extremely high speed, Germany recently introduced the new Gen Diagnostics Law in February 2010. Four common diseases with genetic contribution (dementia, Parkinson syndrome, epilepsy, and hereditary eye diseases) will be used to exemplify the latest development of human genetic testing.

In the past 5 years, next-generation sequencing (NGS) has been established as a valuable tool for several research applications. Commercially available platforms from Helicos, Illumina, Life Technologies, Pacific Biosciencies, and Roche allow for massively parallel sequencing and analysis in the fields of genomics, transcriptomics, and epigenomics. As in most projects, data throughput of the sequencers is not the limiting factor; genomic DNA samples are directly prepared for sequencing without prior conditioning. However, there are some applications such as targeted resequencing that do not require sequencing of whole genomes. Therefore, a technology called target enrichment was established more than 2 years ago. Different PCR- or hybridization-based approaches were further commercially developed and refined. The combination of this method with NGS can improve analysis of disease-related gene sets in molecular diagnostics by reducing time and costs. By taking advantage of the enormous data output, several genes and patients can be analyzed in parallel in one single instrument run. Zusammenfassung In den letzten 5 Jahren hat sich Next Generation Sequencing zu einer wertvollen Methode für verschiedene Forschungs-anwendungen entwickelt. Die kommerziell von Helicos, Illumina, Life Technologies, Pacific Biosciencies und Roche erhältlichen Plattformen ermöglichen eine massive parallele Sequenzierung und Analysen in den Bereichen Genomik, Transkriptomik und Epigenomik. Da in den meisten Projekten der von den Sequenziergeräten generierte Datendurchsatz nicht limitierend ist, werden genomische DNA Proben ohne weitere Vorbehandlung direkt für die Sequenzierung vorbereitet. In Anwendungen wie der gerichteten Resequenzierung wird jedoch keine Sequenzierung von Gesamtgenomen benötigt. Deshalb wurde vor über 2 Jahren eine Technologie namens Zielregion-Anreicherung (Target Enrichment) entwickelt. Verschiedene PCR- oder Hybridisierungs-basierte Ansätze wurden danach erarbeitet und weiterentwickelt. Die Kombination dieser Methode mit Next Generation Sequen-cing kann die Analyse von krankheitsrelevanten Gensets in der Molekulardiagnostik durch Reduktion von Zeit und Kosten verbessern. Unter Ausnutzung der enormen Daten-ausgabe können mehrere Gene und Patienten gemeinsam in einem Gerätelauf analysiert werden.

Prostate cancer is the most common newly diagnosed malignancy in men and approximately 12,000 men die of prostate cancer per year in Germany. The incidence is increasing and this is related to longer life expectancy and improved diagnostic methods. The meaningful use of prostate specific antigen (PSA)-based diagnosis is very important for the early detection of localized stages which can be treated curatively. However, in elderly men with a limited life expectancy and/or comorbidity, curative treatment will often not be possible and therefore PSA testing will not always be indicated. The problems of the estimation of individual life expectancy and of PSA measurements for the diagnosis and treatment monitoring are discussed along with the consequences for the public healthcare system. New strategies such as active surveillance and watchful waiting are also discussed. Periodical PSA measurements are of importance in patients with prostate cancer without active therapy under surveillance, for the monitoring after curative treatment and for the diagnosis of disease recurrence and/or progression, as well as in patients with metastatic disease under androgen ablation and/or chemotherapy. Special consideration is given to the new joint S3 guideline of the Deutsche Gesellschaft für Urologie and the Deutsche Krebsgesellschaft, which was released in 2009.

Despite recent advances in management of patients in intensive care units, sepsis and septic shock are the major causes of morbidity and mortality. Prompt and adequate antibiotic therapy accompanied by surgical removal of the infectious material are the first-line therapy of choice. In addition, various immunomodulatory treatments have been investigated during the past decades. However, despite promising results in studies with animal models, studies in humans with antibodies against lipopolysaccharide (LPS), tumor necrosis factor and interleukin-1 have not been successful. In addition, high doses of steroids, immunoglobulins or antibodies against LPS and cytokines did not reduce mortality, probably owing to timing and dosage of these drugs. Prophylactic administration of immunomodulatory drugs cannot be recommended due to severe adverse effects. However, owing to pleiotropic effects of statins this class of cholesterol lowering drugs has been suggested to be beneficial as adjuvant therapy for sepsis. The present review summarizes the pathophysi-ology of sepsis as well as experimental and clinical evidence for the use of statins in sepsis.

Neuromyelitis optica (NMO), an inflammatory demyelinating disease, is characterized by the occurrence of optic neuritis and longitudinally extensive transverse myelitis and is easily misdiagnosed as a severe variant of multiple sclerosis (MS). The discovery of AQP4-antibodies in serum of NMO and high-risk (HR) patients enables early diagnosis and specific treatment. In this review, we will give an overview of AQP4-antibodies in serum and cerebrospinal fluid of NMO and HR patients. In addition, the diagnostic and therapeutic relevance of these AQP4-antibodies with regard to the disease course will be discussed.

Subarachnoid haemorrhage (SAH) is a neurological emergency. One of the major complications is the development of delayed cerebral ischaemia due to cerebral vasospasm (CV) that significantly affects clinical outcome and accounts for up to 23% of disability and mortality related to SAH. Here, we review the literature on endothelin and nitric oxide related components as potential cerebrospinal fluid (CSF) biomarkers and discuss their clinical relevance to act as surrogate for CV detection and for prediction of clinical outcome. We classified recommendations for these markers as levels A to C according to the scheme approved for the European Federation of Neurological Societies guidelines. We identified significantly elevated CSF endothelin-1 concentrations in patients with SAH, especially in those who developed CV, as well as the potential role of nitrite/nitrate for clinical outcome prediction. Owing to mainly methodological shortcomings, none of the above biomarkers reached a level of recommendation that would justify implementation in clinical routine. Further prospective studies under standardised conditions are required. Zusammenfassung Die Subarachnoidalblutung (SAB) ist ein neurologischer Notfall. Eine der wichtigen Komplikationen ist die Entwicklung cerebraler Ischämien aufgrund eines cerebralen Vasospasmus (CV), der das klinische Outcome signifikant beeinflusst und für bis zu 23% von Behinderung und Mortalität verantwortlich ist. In diesem Artikel geben wir einen Überblick über die Evidenz von Endothelin und Stickstoffmonoxid assoziierten Proteinen als Liquorbiomarker im Hinblick auf die Detektion von CV und Prädiktion von klinischem Outcome. Wir klassifizierten die Marker als Level A bis C gemäß dem Schema der European Federation of Neurological Societies. Es zeigte sich sowohl eine signifikant erhöhte Konzentrationen von Endothelin-1 im Liquor cerebrospinalis bei Patienten mit SAB, insbesondere wenn diese CV entwickelten, als auch eine potentielle Rolle von Nitrit/Nitrat zur Prädiktion von klinischem Outcome. Aufgrund vorwiegend methodologischer Probleme kann für keinen der beschriebenen Marker eine Empfehlung für die klinische Routine abgegeben werden. Prospektive Studien zur Bestätigung und genaueren Evalueriung unter standardisierten Rahmenbedingungen wären notwendig.

The progress that has been made in the field of high-throughput sequencing is such that even experts in the field are impressed. At the time of writing, it is possible to sequence an entire genome within a few days for <€50,000. Thus, we are rapidly entering the era of the “thousand-Euro genome” or even the “hundred-Euro genome”, a price at which whole-genome sequencing can be considered for inclusion in routine diagnostic workups. The current uses of high-throughput sequencing include research in epigenomics, population and medical genetics, tumor genetics, and in the analysis of gene expression. The emphasis of our conference in 2009 was on the possible uses of “next-generation” sequencing in medical diagnostics in the future. Other topics include the bioinformatic analysis of very large datasets and techniques in gene expression analysis. Other high-throughput methods of gene analysis, such as mass spectrometry, are also discussed.