Volume 38, Issue 1

Within a few years, 25-hydroxyvitamin D (25-OHD) 1 has emerged as a high volume test in many regions. Several analytical issues have been recognized regarding this analyte, in particular variable co-detection of metabolites (25-hydroxyvitamin D 2 ; 3-epi-25-hydroxyvitamin D; 24,25-dihydroxyvitamin D), variable release of the analyte from its protein bonds and matrix effects in automated ligand binding tests, contributing to an often unsatisfactory correlation of high-throughput assays with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Reference methods based on LC-MS/MS as well as reference materials have been introduced only very recently, achieving a truly significant improvement in the standardization of serum 25-OHD measurement. However, beyond these analytical issues in relation to 25-OHD, it should be scrutinized how biologically appropriate this inactive intermediate metabolite can actually describe vitamin D status as a surrogate marker – individually and at a population level. There is, for example, at present little knowledge regarding individual vitamin D requirement in relation to dietary calcium supply; the impact of genetic variation of vitamin D binding protein on serum concentrations; the impact of genetic variation in downstream metabolism and signaling of vitamin D on individual vitamin demand. Furthermore, there is no accepted approach to assess functional whole year vitamin D status, addressing the fundamental seasonal variation of endogenous generation of vitamin D in many regions. Consequently, additional functional markers should be considered when describing vitamin D status (parathyroid hormone, corrected serum calcium and phosphate, urinary calcium, well selected bone markers, etc.), with season-adapted sampling strategies. In conclusion, it should be recognized that there is substantial uncertainty in the currently used approach to characterize vitamin D status by singular measurement of 25-OHD using mainstream assays. It seems questionable to focus the worldwide debate on a widespread vitamin supplementation merely on cut-off results of this marker. Zusammenfassung Innerhalb weniger Jahre hat sich 25-Hydroxy-Vitamin D (25-OHD) in vielen Regionen zu einer massenhaft angeforderten Messgröße entwickelt. Eine Reihe von analytischen Problemen wurden bezüglich dieses Analyten erkannt, insbesondere eine variable Miterfassung von Metaboliten (25-Hydoxyvitamin D 2 , 3-epi-25-Hydroxyvitamin D 3 , 24,25-Dihydroxy-Vitamin D), die variable Ablösung des Analyten aus seiner Proteinbindung und Matrixeffekte bei Ligandenbindungs-Tests. All dies trägt zu einer oft unbefriedigenden Korrelation von Hochdurchsatz-Methoden mit der LC-MS/MS bei. Referenzmethoden auf Basis der LC-MS/MS sowie Referenzmaterialien wurden erst vor relativ kurzer Zeit eingeführt; in der Tat hat dies zu einer wesentlichen Verbesserung der Standardisierung der 25-OHD-Messung beigetragen. Über diese analytischen Probleme bezüglich 25-OHD hinaus sollte jedoch eingehend hinterfragt werden, wie biologisch valide dieser inaktive Stoffwechselmetabolit als Surrogat-Marker tatsächlich den Vitamin D-Status zu beschreiben vermag, sowohl individuell als auch auf Populationsebene. So ist zum Beispiel wenig über den Zusammenhang zwischen individuellem Vitamin D-Bedarf und diätetischer Kalzium-Aufnahme bekannt, sowie über den Einfluss genetischer Variablen des Vitamin D-Bindeproteins auf die Serum-Konzentrationen, beziehungsweise über den Einfluss von genetischer Polymorphismen nachgeschalteter Verstoffwechslungs- und Signalwege auf den individuellen Vitamin D-Bedarf. Des Weiteren existiert kein allgemein anerkannter Ansatz zur Erfassung des Ganzjahres-Vitamin D-Status, der die erhebliche jahreszeitliche Variation der endogenen Vitamin D-Bildung – wie in vielen Regionen gegeben – berücksichtigen würde. Entsprechend sollten zusätzliche, funktionelle Marker bezüglich der Beschreibung des Vitamin D-Status geprüft werden (v.a., Parathormon, korrigiertes Serum-Kalzium, Serum-Phosphat, Urin-Kalzium sowie ausgewählte Knochenmarker), jeweils mit einer Jahreszeit-angepassten Strategie der Probengewinnung. Insgesamt sollte berücksichtigt werden, dass eine erhebliche Unschärfe im gegenwärtig überwiegend gewählten Ansatz der Beschreibung des Vitamin D-Status anhand der einmaligen Messung von 25-OHD mittels Standardmethoden besteht. Es erscheint fragwürdig, die weltweite Diskussion über eine flächendeckende Vitamin D-Supplementation ausschließlich auf Entscheidungsgrenzen dieses Markers zu basieren.

Analyses of cerebrospinal fluid (CSF) samples place high demands on the executing laboratory. Apart from the directive of the Federal German Medical Association of Medical Doctors for the quality assurance of medical laboratory investigations (RiLiBÄK), the guidelines and method catalogue of the German Society for Liquor Diagnostics and Clinical Neurochemistry (DGLN) need to be used. Historically, CSF diagnostics are carried out in laboratories tightly connected to neurological departments. In general, CSF diagnostics are offered by central laboratories, partly for the reason that independent neurological laboratories do not exist and partly for the need to supply diagnostics at night-time and at weekends in acute patient care. This may result in a double structure with differences in methods used, evaluation, or reporting. The execution of CSF diagnostics in an interdisciplinary CSF laboratory merges the expertise of both medical disciplines, which results in increased quality of patient care and better economic conditions simultaneously.

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 (in %) of T cells was 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 of T cells over 9 months was 75% (confidence interval of 95%: 72–78%). In two of the four patients with NABs, natalizumab saturation 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, natalizumab saturation 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 natalizumab saturation 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. We were able to show that natalizumab saturation, as detected 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.

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.

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.

New trends in diagnostic approaches in hematology are impressive. Information is fostered in many aspects due to techniques such as next generation sequencing. This can lead to the discovery of molecular markers in benign and malignant diseases. Furthermore, this not only unravels the molecular backbone in acute leukemia but also in myelodysplastic syndromes. In addition, the characterization and classification of lymphomas is changing. Moreover, approaches to the common problem of anemia have also improved.