Volume 30, Issue 6

Zusammenfassung Die Durchführung von Immunfluoreszenz-Untersuchungen zum Nachweis von antinukleären Antikörpern (ANA) auf HEp-2-Zellen stellt nach wie vor die Basis des immunologischen Autoantikörper- Screenings dar. Mittlerweile sind über 30 verschiedene Muster, zytoplasmatische und nukleäre, beschrieben. Diese Muster repräsentieren den Nachweis von über 100 verschiedenen Autoantikörpern. Der Vorteil der Verwendung eines solchen „unspezifischen” Substrates, der HEp-2-Zelle, liegt in der hohen Sensitivität bei der Identifizierung von ANAs. Auch nach jetzt fast 50 Jahren der ANA-Diagnostik hat diese, sicher etwas altertümliche Technik, nichts von ihrer Wichtigkeit und Faszination in der Labordiagnostik verloren.

During the past two decades, flow-cytometric immunophenotyping of lymphocytes evolved from a research technique into routine laboratory diagnostics. Extensive studies in healthy individuals resulted in detailed age-related reference values for different lymphocyte subpopulations in peripheral blood. This is an important tool for the diagnosis of hematological and immunological disorders. Similar albeit less detailed information is now available for other lymphoid organs, e.g., normal bone marrow, lymph nodes, tonsils, thymus and spleen. Flow-cytometric immunophenotyping forms the basis of modern classification of acute and chronic leukemias and is increasingly applied for initial diagnostic work-up of non-Hodgkin's lymphomas. Finally, with multiparameter flow cytometry it is now possible to identify routinely and reliably low numbers of leukemia and lymphoma cells (minimal residual disease).

Since bacterial infection of the recipient has become the most frequent infection risk in transfusion medicine, suitable methods for bacteria detection in blood components are of great interest. Platelet concentrates are currently in the focus of attention as they are stored under temperature conditions, which enable the multiplication of most bacteria species contaminating blood donations. Rapid methods for bacteria detection allow testing immediately before transfusion in a bedside-like manner. This approach would overcome the sampling error observed in early sampling combined with culturing of bacteria and would at least prevent the transfusion of highly contaminated blood components leading to acute septic shock or even death of the patient. Flow cytometry has been demonstrated to be a rapid and feasible approach for the detection of bacteria in platelet concentrates. The general aim of the current study is to develop protocols for the application of this technique under routine conditions. The effect of improved test reagents on practicability and sensitivity of the method is evaluated. Furthermore, the implementation of fluorescent absolute count beads as an internal standard is demonstrated. A simplified pre-incubation procedure has been worked out in order to diminish the detection limit in a pragmatic manner. Additionally, the application of bacteria detection by flow cytometry (BDFC) as a culture method is shown, i.e., the transfer of samples from PCs into a satellite bag, incubation of the latter at 37°C, and measuring the contaminating bacteria in a flow cytometer.

Although blood component transfusion is currently regarded to be safe, adverse events may occur in recipients of these products. Among those, blood borne viral, bacterial and parasitic infections are best known. For detection of bacterial contamination in platelet concentrates various methods are available or under investigation. One of these methods, real-time polymerase chain reaction (PCR) with particular focus on real-time 16S rDNA detection, will be discussed in this review.

Bacterial contamination of blood components, especially of platelet concentrates (PCs), represents the highest infectious risk in blood transfusion. Although the incidence of platelet bacterial contamination is approximately one per 2000 units, the urgent need for a method for the routine screening of PCs to improve safety for patients had not been considered for a long time. Besides the culturing systems, which will remain the gold standard, rapid methods for sterility screening will play a more important role in transfusion medicine in the future. In particular, the nucleic acid amplification techniques (NAT) are powerful tools in bacterial screening assays. The combination of excellent sensitivity and specificity, low contamination risk, ease of performance and speed, has made real-time PCR technology an appealing alternative to conventional culture-based testing methods. When using real-time PCR for the detection of bacterial contamination, several points have to be considered. The main focus refers to the choice of the target gene, the assay format, the nucleic acid extraction method, depending on the sample type, and the evaluation of an ideal sampling strategy. However, several factors like the bacterial-derived nucleic acid contamination of amplification reagents or laboratory materials, the impracticability and the costs have limited the use of NAT until now. Attempts to reduce the amount of contaminating nucleic-acids from reagents in real-time PCR have been described, but none of these methods have proved to be very effective or may lower the sensitivity of the assay. Recently, a number of broad-range NAT assays targeting the 16S rDNA or 23S rRNA for detection of bacteria based on real-time technology have been reported. This review will give a short survey of current approaches to and limitations of the application of real-time PCR for bacterial detection in blood components, with emphasis on PCs.

Zusammenfassung Durch ihre zunehmende Anwenderfreundlichkeit und hohe Sensitivität sowie Spezifität findet die Flüssigkeitschromatographie-gekoppelte (Tandem-) Massenspektrometrie (LC/MS/MS) immer weitere Verbreitung in der Klinischen Chemie und der therapeutischen Arzneimittelspiegel-Bestimmung. Allerdings bieten die Gerätehersteller bisher keine Komplettlösungen zur Bestimmung definierter Parameter im Sinne eines automatisierten labordiagnostischen Systems an, sondern die Methode zur Bestimmung eines definierten Parameters mittels Massenspektrometrie ist vom Anwender selbst zu etablieren, zu validieren und zu zertifizieren. In der Validation der Methode für die LC/MS/MS für den Routinebetrieb und der Qualitätssicherung liegen die wesentlichen Herausforderungen für den Anwender. Einige wesentliche Tücken der quantitativen Nutzung von LC/MS/MS-Systemen sind in der Literatur beschrieben. Auf den wenig erfahrenen Anwender kommen jedoch eine Reihe anderer Fehlerquellen zu. Einige wichtige Aspekte und Fallstricke in der Methodenentwicklung und Qualitätssicherung für den Routinebetrieb der LC/MS/MS werden im vorliegenden Artikel zusammen mit in der Literatur beschriebenen Störfaktoren erörtert.

Zusammenfassung Der Dihydropyrimidindehydrogenase (DPD)-Mangel in heterozygoter Ausprägung ist ein pharmakogenetisches Syndrom, welches bei Fluorpyrimidinexposition im Rahmen einer Chemotherapie eine Prädisposition für verstärkte Toxizität darstellen kann. Die molekulargenetische Grundlage der DPD-Defizienz ist heterogen, wobei ein bestimmtes Mangelallel ( DPYD*2A , Exon-14-Skipping-Variante) quantitativ im Vordergrund steht. Die in retrospektiven Untersuchungen gezeigten Zusammenhänge zwischen partieller DPD-Defizienz und verstärkter Toxizität bis zu Todesfällen bei Applikation von 5-Fluorpyrimidin-Antimetaboliten sind als Grundlage für die Empfehlung eines prätherapeutischen Screenings allerdings nicht ausreichend, da eine individuelle Risikoprädiktion mit der isolierten DPYD*2A -Analyse nicht gelingt. Diese sollte folglich derzeit Studien vorbehalten bleiben.

In addition to the monographs which were published last year by the working group “Drug Monitoring” of the Swiss Society of Clinical Chemistry (SSCC) [1], new monographs have been written. The aim of these monographs is to give an overview of the most important information necessary for ordering a drug analysis or interpreting the results. Therefore, the targeted readers comprise laboratory health professionals and all receivers of laboratory reports. There is information provided on the indication for therapeutic drug monitoring, protein binding, metabolic pathways and enzymes involved, elimination half-life and elimination routes, and on therapeutic or toxic concentrations. Preanalytical considerations are of particular importance for therapeutic drug monitoring. Therefore, information is provided regarding a reasonable timing for the determination of drug concentrations as well as steady-state concentrations after changing the dose. Furthermore, the stability of the drug and its metabolite(s) after blood sampling is described. For readers with a specific interest in drug analysis, references to important publications are given. The number of monographs will be continuously enlarged. The updated files are presented on the homepage of the SSCC (www.sscc.ch). We hope that these monographs are helpful and look forward to receiving comments from the audience.

A constant extracellular Ca 2+ concentration is required for numerous physiological functions at tissue and cellular levels. This suggests that minor changes in Ca 2+ will be corrected by appropriate homeostatic systems. The system regulating Ca 2+ homeostasis involves several organs and hormones. The former are mainly the kidneys, skeleton, intestine and the parathyroid glands. The latter comprise, amongst others, the parathyroid hormone, vitamin D and calcitonin. Progress has recently been made in the identification and characterisation of Ca 2+ transport proteins CaT1 and ECaC and this has provided new insights into the molecular mechanisms of Ca 2+ transport in cells. The G-protein coupled calcium-sensing receptor, responsible for the exquisite ability of the parathyroid gland to respond to small changes in serum Ca 2+ concentration was discovered about a decade ago. Research has focussed on the molecular mechanisms determining the serum levels of 1,25(OH)2D3, and on the transcriptional activity of the vitamin D receptor. The aim of recent work has been to elucidate the mechanisms and the intracellular signalling pathways by which parathyroid hormone, vitamin D and calcitonin affect Ca 2+ homeostasis. This article summarises recent advances in the understanding and the molecular basis of physiological Ca 2+ homeostasis.

Background: The mass action law-based calculation methods for free testosterone (FT) and “bioavailable” testosterone (BAT) – routinely used for assessing androgen disorders – rely on the supposition that the sex hormone binding globulin (SHBG) molecule contains one steroid binding domain (SBD). However, recent biochemical investigations revealed that this molecule actually comprises two SBDs. This necessitates new equations (Calc2) for FT and BAT calculation. Methods and results: Calc2 was deduced using the recently published SHBG-testosterone association constant and was compared to other FT and/or BAT determination methods (e.g., a conventional calculation version [CalcV], ammonium sulfate precipitation [for measuring BAT] relying on total testosterone [TT] by mass spectrometry, and a FT radioimmunoassay [RIA] proving satisfactory). In contrast to CalcV BAT, Calc2 BAT fitted ammonium sulfate precipitation BAT. An analogous result was found by means of a comparison of FT ratios determined by RIA, Calc2, and CalcV, respectively. Additionally, Calc2 data mostly appear to meet literature data (measured by methods such as equilibrium dialysis) better than CalcV data do. Conclusions: There is evidence that Calc2 affords reliable FT and BAT derivation from assayed TT, SHBG, and albumin making direct FT and BAT measurements unnecessary in most cases. Finally, Calc2 was formatted as a preliminary nomogram for convenient clinical use.