Volume 33, Issue 5

The German Reference Laboratory for meningococci is responsible for laboratory surveillance of meningococcal disease in Germany and collaborates closely with the Robert Koch Institute. Bacterial typing beyond serogrouping is conducted exclusively using DNA sequence-based techniques, in consensus with the European Monitoring Group on Meningococci. Typing data are used in computer-assisted detection of spatio-temporal clusters of disease. They are visualized using the EpiScanGIS geographic information system. Data from the reference laboratory are matched to national notification data at the Robert Koch Institute to provide a comprehensive data set of high quality for submission to the TESSy database of the European Centre for Disease Control and Prevention. The estimated coverage of notified cases is satisfactory because of the efforts of laboratories that submit data. In the present review, diagnosis of Neisseria meningitidis , antimicrobial resistance testing, and fine typing are discussed from the viewpoint of the reference laboratory, including issues related to laboratory safety management.

The German Reference Laboratory for meningococci is responsible for laboratory surveillance of meningococcal disease in Germany and collaborates closely with the Robert Koch Institute. Bacterial typing beyond serogrouping is conducted exclusively using DNA sequence-based techniques, in consensus with the European Monitoring Group on Meningococci (EMGM). Typing data are used in computer-assisted detection of spatio-temporal clusters of disease. They are visualized using the EpiScanGIS geographic information system. Data from the reference laboratory are matched to national notification data at the Robert Koch Institute to provide a comprehensive data set of high quality for submission to the TESSy database of the European Centre for Disease Control and Prevention (ECDC). The estimated coverage of notified cases is satisfactory because of the efforts of laboratories that submit data. In the present review, diagnosis of Neisseria meningitidis , antimicrobial resistance testing, and fine typing are discussed from the viewpoint of the reference laboratory, including issues related to laboratory safety management.

In the last few years, array comparative genomic hybridisation (aCGH) has become a valuable genome-wide screening tool for the detection of chromosomal aberrations in the form of copy number variants (CNVs). Commercially available platforms cover the subtelomeric regions and all known microdeletion/microduplication syndrome regions, as well as the rest of the genome, with resolution ranging from 8 kb to 1 Mb. Besides detecting clearly pathogenic or benign CNVs, aCGH can uncover CNVs with unknown clinical significance, thus complicating the interpretation of aCGH results. The main indications include children with mental retardation, developmental delay, congenital anomalies and neuropsychiatric disorders such as autism. In this patient group, aCGH will gradually replace conventional chromosome analysis as the frontline test, and its implementation in prenatal diagnostics is in discussion. Another promising field is cancer diagnosis, for which aCGH will enable clinicians to classify and prognose different tumours more accurately in the near future. Zusammenfassung In den letzten Jahren hat sich die Array-basierte genomische komparative Hybridisierung (aCGH) zu einer wertvollen, genom-weiten Screeningmethode zur Aufdeckung chromosomaler Veränderungen in Form von Kopienzahlvarianten (CNV) entwickelt. Die kommerziell erhältlichen Plattformen beinhalten die Subtelomerregionen sowie die bekannten Mikrodeletions- und Mikroduplikationssyndromregionen, das restliche Genom wird mit unterschiedlichen Auflösungen von 8 kb bis 1 Mb abgedeckt. Neben der Aufdeckung eindeutig pathogener oder harmloser CNVs kann die aCGH auch CNVs mit unklarer klinischer Signifikanz aufdecken, welche die Interpretation einer aCGH-Analyse erschweren. Ihre Hauptindikationsstellungen umfassen Kinder mit mentaler Retardierung, Entwicklungsverzögerung, angeborenen Fehlbildungen und neuropsychiatrischen Erkrankungen wie Autismus. In dieser Patientengruppe wird die aCGH zunehmend die klassische Chromosomenanalyse als Ersttest ersetzen und ihr Einsatz in der Pränataldiagnostik steht derzeit in der Diskussion. Ein weiteres viel versprechendes Einsatzgebiet ist die Tumordiagnostik, wo die aCGH dem Behandler die Klassifizierung und Prognosestellung verschiedener Tumorentitäten erleichtern wird.

During the last 3 years the sequencing market has completely changed. New technologies in the so-called next-generation sequencers can produce gigabases of sequence raw data in only a few days. In the past, data production represented the greatest cost factor in a sequencing project. At present, bioinformatic analysis represents a challenge. The main difference between technologies in influencing bioinformatic analysis is the read length. The classical Sanger system produces a read length of 1100 bases on average, whereas that of next-generation instruments is 250–400 bases (Roche/454 GS FLX sequencer) or 36–100 bases (Illumina/Solexa genome analyzer; Applied Biosystems SOLiD). To ensure the success of a sequencing project and to maximize the information obtained, it is necessary to choose optimal next-generation technology or a combination of technologies followed by bioinformatic analysis in a sequential approach using state-of-the-art analysis tools.

During the last 3 years the sequencing market has completely changed. New technologies in the so-called next-generation sequencers can produce gigabases of sequence raw data in only a few days. In the past, data production represented the greatest cost factor in a sequencing project. At present, bioinformatic analysis represents a challenge. The main difference between technologies in influencing bioinformatic analysis is the read length. The classical Sanger system produces a read length of up to 1100 bases, whereas that of next-generation instruments is 250–400 bases (Roche/454 GS FLX sequencer) or 50–100 bases (Illumina/Solexa Genome Analyzer; Applied Biosystems SOLiD). To ensure the success of a sequencing project and to maximize the information obtained, it is necessary to choose optimal next-generation technology or a combination of technologies followed by bioinformatic analysis in a sequential approach using state-of-the-art analysis tools.

With the introduction of molecular cytogenetic and genetic techniques, the laboratory diagnosis of hematological neoplasias has become more complex and meaningful over the past 10 years. Depending on the clinical phenotype, application of the entire spectrum of laboratory methods is required in many cases. However, to ensure a cost-effective, targeted and individualized approach to a given patient, application of techniques such as blood count, morphology, cytochemistry, histology, immunophenotyping, cytogenetics [karyotyping, fluorescence in situ hybridization (FISH), array comparative genomic hybridization (CGH)], and molecular genetics should be carefully examined. Array CGH is a comparatively new technique that provides additional information on novel deletions and amplifications. It is particularly useful if conventional chromosome anlysis cannot be interpreted or if a conventional FISH panel fails to detect abnormalities. Like any other technique, array CGH has diagnostic shortcomings, in particular for the detection of balanced translocations. Optimal diagnostic safety can therefore only be achieved by combined application of the currently available techniques.

Deep vein thrombosis is a common medical condition and investigation of clotting status or thrombophilia is a common task in the medical laboratory. Differential diagnosis is based on the clinical features and a combination of clotting function, biochemical, and genetic tests. Therefore, investigation of thrombophilia represents a good example of the role of molecular genetic testing in laboratory medicine. The aim of the present review is to outline the current state of the art in investigating thrombophilia with particular reference to molecular genetic testing. One cause of thrombophilia that is often overlooked is the presence of antiphospholipid antibodies, so this topic also receives particular attention.

Deep vein thrombosis is not a rare medical condition, and the investigation of a clotting disposition or thrombophilia is one of the most common tasks required of the medical laboratory. In this complex approach, the diagnosis is achieved on the basis of the clinical features backed up by the combination of investigations of clotting function, biochemical analyses, and genetic tests. Therefore, the investigation of thrombophilia represents as a good example of the role of molecular genetic testing in laboratory medicine. The aim of the present review is to outline the current state of the art in investigating thrombophilia with particular reference to molecular genetic testing. One cause of thrombophilia that is often overlooked is the presence of antiphospholipid antibodies. Accordingly, they also receive detailed attention.

The selective estrogen receptor modulator tamoxifen is approved for treatment of hormone receptor-positive breast cancer in pre- and postmenopausal patients. The main active metabolite of tamoxifen is endoxifen, which has high affinity towards the receptor and reaches high plasma concentrations. Endoxifen results from cytochrome P450 enzyme CYP2D6 action. CYP2D6 is subject to genetic polymorphism, with a prevalence of enzyme deficiency of approximately 7% in most European populations. Enzyme deficiency is reliably predicted by genotyping of known CYP2D6 deficiency alleles. Poor metabolizers (homozygote carriers of deficiency alleles) exhibit lower endoxifen plasma concentrations. Retrospective analyses of tamoxifen study data according to CYP2D6 genotypes reveal a poorer oncological outcome for subjects with deficiency alleles in most studies (level 3 evidence). Data from randomized controlled studies (level 1 evidence) on the use of CYP2D6 typing for tamoxifen therapy are lacking. However, CYP2D6 genotyping before initiating tamoxifen therapy and avoidance of tamoxifen in postmenopausal women with a CYP2D6 enzyme deficiency seem warranted. Prescribing information does not list CYP2D6 status as a contraindication for tamoxifen. Pharmacological supression of hot flashes by comedication with CYP2D6 inhibitors (e.g., fluoxetine, paroxetine) must be avoided because such patients will become functional Poor metabolizers with lower endoxifen levels.

The selective estrogen receptor modulator tamoxifen is approved for treatment of estrogen receptor-positive breast cancer in pre- and postmenopausal patients. The main active metabolite of tamoxifen is endoxifen, which has high affinity towards the receptor and reaches high plasma concentrations. Endoxifen results from cytochrome P450 enzyme CYP2D6 action. CYP2D6 is subject to genetic polymorphism, with a prevalence of enzyme deficiency of approximately 7% in most European populations. Enzyme deficiency is reliably predicted by genotyping of known CYP2D6 deficiency alleles. Poor metabolizers (homozygote carriers of deficiency alleles) exhibit lower endoxifen plasma concentrations. Retrospective analyses of tamoxifen study data according to CYP2D6 genotypes reveal a poorer oncological outcome for subjects with deficiency alleles in most studies (level 3 evidence). Data from randomized controlled studies (level 1 evidence) on the use of CYP2D6 typing for tamoxifen therapy are lacking. However, CYP2D6 genotyping before initiating tamoxifen therapy and avoidance of tamoxifen in postmenopausal women with a CYP2D6 enzyme deficiency seem warranted. Prescribing information does not list CYP2D6 status as a contraindication for tamoxifen. Pharmacological supression of hot flashes by comedication with CYP2D6 inhibitors (e.g., fluoxetine, paroxetine) must be avoided because such patients will become functional Poor metabolizers with lower endoxifen levels.

In haematology, blood count analyses are often performed on samples obtained by capillary puncture. However, only venous values are well accepted. This paper reviews the data available and highlights this topic from the viewpoint of laboratory medicine and clinical practice. We also present our own data. Mean capillary-venous differences were +0.3 g/dL or +0.2 mmol/L (+2.1%) for haemoglobin, +1.5% or +0.02 L/L (+3.6%) for haematocrit, +0.2×10 3 /μL (+2.7%) for white blood cells, +0.22×10 3 /μL (+4.7%) for absolute neutrophil counts, –19×10 3 /μL (–8.3%) for platelets and +0.1×10 6 /μL (+1.8%) for red blood cells. Differences were greater in children than in adults: +1.1 g/dL or +0.7 mmol/L (+6.3%) for haemoglobin, +3.1% or +0.03 L/L (+6.0%) for haematocrit, +2.0×10 3 /μL (+14.6%) for white blood cells, +0.91×10 3 /μL (+11.0%) for absolute neutrophil counts, –33×10 3 /μL (–14.1%) for platelets and +0.3×10 6 /μL (+6.0%) for red blood cells. The capillary values correlated very well with the venous values. Capillary blood count analysis is safe, reliable and advantageous compared to venous analysis and has high probability for true -positive and true -negative values for patients with anaemia, polyglobulia, thrombocytopenia and neutrocytopenia in comparison to venous blood count analysis. Therefore, at least in adults, capillary blood count analysis can be used as a substitute for venous blood count analysis in clinical practice.

In haematology, blood count analyses are often performed on samples obtained by capillary puncture. However, only venous values are well accepted. This paper reviews the data available and highlights this topic from the viewpoint of laboratory medicine and clinical practice. We also present our own data. Mean capillary-venous differences were +0.3 g/dL or +0.2 mmol/L (+2.1%) for haemoglobin, +1.5% or +0.02 L/L (+3.6%) for haematocrit, +0.2×10 3 /μL (+2.7%) for white blood cells, +0.22×10 3 /μL (+4.7%) for absolute neutrophil counts, –19×10 3 /μL (–8.3%) for platelets and +0.1×10 6 /μL (+1.8%) for red blood cells. Differences were greater in children than in adults: +1.1 g/dL or +0.7 mmol/L (+6.3%) for haemoglobin, +3.1% or +0.03 L/L (+6.0%) for haematocrit, +2.0×10 3 /μL (+14.6%) for white blood cells, +0.91×10 3 /μL (+11.0%) for absolute neutrophil counts, –33×10 3 /μL (–14.1%) for platelets and +0.3× 10 6 /μL (+6.0%) for red blood cells. The capillary values correlated very well with the venous values. Capillary blood count analysis is safe, reliable and advantageous compared to venous analysis and has high probability for true -positive and true -negative values for patients with anaemia, polyglobulia, thrombocytopenia and neutrocytopenia in comparison to venous blood count analysis. Therefore, at least in adults, capillary blood count analysis can be used as a substitute for venous blood count analysis in clinical practice.