Volume 36, Issue 5

Cell free fetal DNA (cff-DNA) in maternal blood has given rise to the possibility of new non-invasive approaches in prenatal genetic diagnoses. In contrast to the established invasive techniques chorionic villi sampling and amniocentesis, both associated with specific risk (0.5–1%) for procedure-related abortions, cff-DNA is simply gained by maternal venous blood sampling, without any risk for the embryo or fetus. Therefore, cff-DNA offers the possibility for riskless genetic diagnoses of ongoing pregnancies. Molecular genetic techniques are already used for the qualitative detection of specific fetal sequences, such as paternal inherited or spontaneous originated (de novo) mutations. Until recently, the introduction of digital PCR and next generation sequencing technologies has shown that a reliable quantitative detection of mutant alleles as well as of clinical relevant aneuploidies (Trisomy 13, 18 and 21) from fetal DNA in maternal blood is also possible

Cell free fetal DNA (cff-DNA) in maternal blood has given rise to the possibility of new non-invasive approaches in prenatal genetic diagnoses. In contrast to the established invasive techniques chorionic villi sampling and amniocentesis, both associated with specific risk (0.5%–1%) for procedure-related abortions, cff-DNA is simply gained by maternal venous blood sampling, without any risk for the embryo or fetus. Therefore, cff-DNA offers the possibility for riskless genetic diagnoses of ongoing pregnancies. Molecular genetic techniques are already used for the qualitative detection of specific fetal sequences, such as paternal inherited or spontaneous originated (de novo) mutations. Until recently, the introduction of digital PCR and next generation sequencing technologies has shown that a reliable quantitative detection of mutant alleles as well as of clinical relevant aneuploidies (Trisomy 13, 18 and 21) from fetal DNA in maternal blood is also possible.

After decades of anticipation, non-invasive prenatal testing for aneuploidy is now a reality. Three published methods, described herein, are able to test for certain whole-chromosome abnormalities in the fetus by sequencing and analyzing cell-free DNA in maternal plasma, a fraction of which is fetal. Zusammenfassung Die seit Jahrzehnten erwartete nicht invasive Detektion von fetaler Aneuploidie ist endlich Realität geworden. Ein geringer Anteil der DNA im Plasma der Schwangeren ist fetalen Ursprung. In diesem Artikel werden drei Methoden beschrieben die durch die Analyse mittels Sequenzieren in der Lage sind, nicht-invasiv auf gewisse Chromosomenstörungen im Fetus zu testen.

After decades of research, non-invasive prenatal testing (NIPT) using maternal blood to determine fetal chromosome status has found its way from the research laboratory into clinical practice, triggering a long-awaited paradigm shift in prenatal care. A variety of methods using sequencing of maternal cell-free DNA (cfDNA) have now been studied, primarily demonstrating their ability to detect the most common fetal aneuploidy, trisomy 21 (T21). The focus of this article is on massively parallel sequencing (MPS) with optimized sequence tag mapping and chromosome quantification, which accurately detects T21 as well as multiple other aneuploidies across the genome. The power of this technique resides in its high precision and reduction of variation within and between sequencing runs. Using MPS, classification of aneuploidy status for a given sample can be reliably assigned from the genetic information alone without the need to factor in other maternal pre-test risk or other clinical variables. Performance of this method has been prospectively demonstrated in a rigorous, blinded, multi-center study in the United States. The findings suggest that MPS can be incorporated into existing prenatal screening algorithms to reduce unnecessary invasive procedures. This technology and key considerations for clinical implementation are discussed. Zusammenfassung Nach jahrzehntelanger Forschung hat der Nicht-invasive Pränatal-Test (NIPT) des fetalen Chromosomenstatus im mütterlichen Plasma seinen Weg aus den Forschungslaboren in die klinische Praxis gefunden und löst einen lange herbeigesehnten Paradigmenwechsel in der pränatalen Diagnostik aus. Es wurde bereits eine Vielzahl von Methoden, welche die Sequenzanalyse von mütterlicher zell-freier DNA (cfDNA) verwenden, untersucht. Dabei zeigte sich, dass dieser Ansatz für den Nachweis der häufigsten fetalen Aneuploidie, der Trisomie 21 (T21) genutzt werden kann. Der Fokus dieses Artikels liegt auf der massiv-parallelen Sequenzanalyse (MPS) mit optimiertem „Sequence Tag Mapping“ und Chromosomenquantifizierung, wodurch T21 und zahlreiche andere Aneuploidie im Genom exakt nachgewiesen werden können. Der Vorteil dieser Methode liegt in ihrer hohen Präzision und der Reduzierung der Variation innerhalb eines sowie zwischen mehreren Sequenzierungsläufen. Durch die Verwendung von MPS kann der Aneuploidie-Status einer Probe zuverlässig allein aus der genetischen Information ohne die Fakturierung in anderen mütterlichen Prätest-Risiko oder anderen klinischen Variablen ermittelt werden. Die Eignung dieser Methode konnte prospektiv in einer streng verblindeten, multizentrischen Studie in den USA demonstriert werden. Die Ergebnisse deuten darauf hin, dass MPS in existierende pränatale Screening-Algorithmen integriert werden kann und somit unnötige invasive Eingriffe reduziert werden können. Die Technologie und ihre klinische Implementierung werden diskutiert.

Since its introduction in the routine diagnostics of patients with mental retardation/developmental delay, array-comparative genomic hybridization (aCGH) has become an indispensable tool for the detection of clinically relevant copy number variants (CNVs). Despite the current tendency for higher resolution arrays, the growing number of public internet databases as well as better calling algorithms allow save reporting and a better classification of CNVs. The application of combined aCGH plus single nucleotide polymorphism (SNP) arrays will increase detection rates by revealing copy number neutral changes, such as uniparental disomy. In the future, next generation sequencing techniques will lead to a further increase in resolution with the simultaneous detection of unbalanced and even balanced chromosomal aberrations.

Since its introduction in the routine diagnostics of patients with mental retardation/developmental delay, array-comparative genomic hybridization (aCGH) has become an indispensable tool for the detection of clinically relevant copy number variants (CNVs). Despite the current tendency for higher resolution arrays, the growing number of public internet databases as well as better calling algorithms allow save reporting and a better classification of CNVs. The application of combined aCGH plus single nucleotide polymorphism (SNP) arrays will increase detection rates by revealing copy number neutral changes, such as uniparental disomy. In the future, next generation sequencing techniques will lead to a further increase in resolution with the simultaneous detection of unbalanced and even balanced chromosomal aberrations.

Evidence is accumulating that vitamin D may play a role in the pathogenesis of cardiovascular diseases. Vitamin D receptors (VDR) and enzymes of vitamin D metabolism are expressed in the vascular system and in the heart. VDR knockout mice suffer from cardiovascular disease (CVD) and selective inactivation of the VDR causes myocardial hypertrophy in cardiac myocytes. Many, but not all, observational studies have shown that vitamin D deficiency is associated with CVD and its risk factors. A low concentration of 25-hydroxyvitamin D (25 [OH] D) is an independent risk factor for CVD, stroke and sudden cardiac death. There are very few randomized controlled trials (RCTs) of the effects of vitamin D supplementation on cardiovascular endpoints, and conclusions can hardly be drawn as in some trials calcium has also been supplemented, which may increase the risk for CVD. Studies in which only vitamin D was given have indicated positive effects on cardiovascular risk factors, such as arterial hypertension. A number of larger studies of the effects of vitamin D supplementation on cardiovascular events and mortality have been undertaken. The design of these studies, however, may be problematic because, for example, individuals with high 25 (OH) D concentration also qualify for inclusion. The current body of evidence is not sufficient to recommend vitamin D supplementation for the prevention and treatment of CVD in general. Prevention and treatment of vitamin D deficiency, however, are both justified by the effects of vitamin D on the musculoskeletal system.

Exactly 100 years ago, Artur Pappenheim published the final version of his staining procedure for blood smears (“panoptische Universalfärbung für Bluttrockenpräparate”), in which he combined the Jenner stain and the May-Grünwald stain with the Giemsa stain. Since then, this stain has been referred to as Pappenheim stain or May-Grünwald-Giemsa stain. It provides a strong and reliable staining of the details of nuclei and cytoplasms, with prominent depiction of their granules. Pappenheim’s stain is used worldwide with various modifications, which lead to inconsistencies in staining results that are not compatible with modern quality standards. There are no reports on quality assurance of hematological stains in the literature. Therefore, the task force Laboratorium of the German Society of Hematology and Oncology publishes this “Standard Procedure for the Pappenheim -Stain”. This standard stain reliably produces inter-laboratory identical results in all cytological specimens of body fluids as well as tissue. The staining results parallel those published by Pappenheim in words and images. This standard procedure describes pre-analytical aspects, the staining in jars, and has associated quality assurance issues. Using a similar protocol, automated smear preparation and staining machines produce an identical staining result. We did not investigate staining on racks. We report on preliminary colorimetric data of correct and incorrect Pappenheim stains and describe correct staining using visual and colorimetric terms.

Currently, automated microscopes are increasingly entering clinical and private labs, making necessary a critical review of their performance. Within two trials, the two most advanced systems were compared with a hematology analyzer, a manual whole blood differential and an immunophenotypic approach. The diverse handling of nuclear shadows leads to the largest differences, followed by different classification schemes for lymphocytes and judgement of red cells and platelets. The implementation of these instruments into hematological step-by-step diagnostic schemes will be discussed. Because of poor low count statistics, an automated hematology analyzer differential and an immunological differential were included for comparison.

Currently, automated microscopes are increasingly entering clinical and private labs, making necessary a critical review of their performance. Within two trials, the two most advanced systems were compared with a hematology analyzer, a manual whole blood differential and an immunophenotypic approach. The diverse handling of nuclear shadows leads to the largest differences, followed by different classification schemes for lymphocytes and judgement of red cells and platelets. The implementation of these instruments into hematological step by step diagnostic schemes will be discussed. Because of poor low count statistics, an automated hematology analyzer differential and an immunological differential were included for comparison.