Anja Mittag

Aneuploidy and DNA Replication in the Normal Human Brain and Alzheimer's Disease

Reactivation of the cell cycle, including DNA replication, might play a major role in Alzheimers disease (AD). A more than diploid DNA content in differentiated neurons might alternatively result from chromosome mis-segregation during mitosis in neuronal progenitor cells. It was our objective to distinguish between these two mechanisms for aneuploidy and to provide evidence for a functional cell cycle in AD. Using slide-based cytometry, chromogenic in situ hybridization, and PCR amplification of alu-repeats, we quantified the DNA amount of identified cortical neurons in normal human brain and AD and analyzed the link between a tetraploid DNA content and expression of the early mitotic marker cyclin B1. In the normal brain, the number of neurons with a more than diploid content amounts to ∼10%. Less than 1% of neurons contains a tetraploid DNA content. These neurons do not express cyclin B1, most likely representing constitutional tetraploidy. This population of cyclin B1-negative tetraploid neurons, at a reduced number, is also present in AD. In addition, a population of cyclin B1-positive tetraploid neurons of ∼2% of all neurons was observed in AD. Our results indicate that at least two different mechanisms need to be distinguished giving rise to a tetraploid DNA content in the adult brain. Constitutional aneuploidy in differentiated neurons might be more frequent than previously thought. It is, however, not elevated in AD. In addition, in AD some neurons have re-entered the cell cycle and entirely passed through a functional interphase with a complete DNA replication.

Microfluidic impedance‐based flow cytometry

Microfabricated flow cytometers can detect, count, and analyze cells or particles using microfluidics and electronics to give impedance‐based characterization. Such systems are being developed to provide simple, low‐cost, label‐free, and portable solutions for cell analysis. Recent work using microfabricated systems has demonstrated the capability to analyze micro‐organisms, erythrocytes, leukocytes, and animal and human cell lines. Multifrequency impedance measurements can give multiparametric, high‐content data that can be used to distinguish cell types. New combinations of microfluidic sample handling design and microscale flow phenomena have been used to focus and position cells within the channel for improved sensitivity. Robust designs will enable focusing at high flowrates while reducing requirements for control over multiple sample and sheath flows. Although microfluidic impedance‐based flow cytometers have not yet or may never reach the extremely high throughput of conventional flow cytometers, the advantages of portability, simplicity, and ability to analyze single cells in small populations are, nevertheless, where chip‐based cytometry can make a large impact.

Polychromatic (eight-color) slide-based cytometry for the phenotyping of leukocyte, NK, and NKT subsets.

Natural killer (NK) and NK T (NKT) cells are important in innate immune defense. Their unequivocal identification requires at least four antigens. Based on the expression of additional antigens, they can be further divided into functional subsets. For more accurate immunophenotyping and to describe multiple expression patterns of leukocyte subsets, an increased number of measurable colors is necessary. To take advantage of the technologic features offered by slide‐based cytometry, repeated analysis was combined with sequential optical‐filter changing.

Hyperchromatic cytometry principles for cytomics using slide based cytometry

Polychromatic analysis of biological specimens has become increasingly important because of the emerging new fields of high‐content and high‐throughput single cell analysis for systems biology and cytomics. Combining different technologies and staining methods, multicolor analysis can be pushed forward to measure anything stainable in a cell. We term this approach hyperchromatic cytometry and present different components suitable for achieving this task. For cell analysis, slide based cytometry (SBC) technologies are ideal as, unlike flow cytometry, they are non‐consumptive, i.e. the analyzed sample is fixed on the slide and can be reanalyzed following restaining of the object.

Iterative Restaining as a Pivotal Tool for n-Color Immunophenotyping by Slide-Based Cytometry

Slide‐based cytometry (SBC) allows to “ask a cell a second time.” We used this tool for detailed immunophenotyping of peripheral blood leukocytes (PBLs).

Basics of standardization and calibration in cytometry – a review

Standardization, calibration, and controls (negative and positive controls) are essential for quality assurance. Cytometers are capable of reliable and repeatable cellular analyses. However, a prerequisite is instrument calibration and standardized preanalytics. Calibration is often done by beads. Beads are available for different quality control applications, e.g. calibration of size and measuring scale, compensation, absolute cell counting, and laser alignment. Results can be standardized by converting MFI values into MESF or ABC values. Standardized data allow comparison of experiments over a long period of time and between different instruments and laboratories. Alterations in the sensitivity of the cytometer can be detected by routinely performing quality control. The process of quality assurance quantifies and helps manage the variance from the desired value. Results can thus be compared objectively with those of other laboratories. Standardization is the basis of cytometry and a prerequisite for obtaining reliable data.

Changes in neuronal DNA content variation in the human brain during aging

The human brain has been proposed to represent a genetic mosaic, containing a small but constant number of neurons with an amount of DNA exceeding the diploid level that appear to be generated through various chromosome segregation defects initially. While a portion of these cells apparently die during development, neurons with abnormal chromosomal copy number have been identified in the mature brain. This genomic alteration might to lead to chromosomal instability affecting neuronal viability and could thus contribute to age‐related mental disorders. Changes in the frequency of neurons with such structural genomic variation in the adult and aging brain, however, are unknown. Here, we quantified the frequency of neurons with a more than diploid DNA content in the cerebral cortex of normal human brain and analyzed its changes between the fourth and ninth decades of life. We applied a protocol of slide‐based cytometry optimized for DNA quantification of single identified neurons, which allowed to analyze the DNA content of about 500 000 neurons for each brain. On average, 11.5% of cortical neurons showed DNA content above the diploid level. The frequency of neurons with this genomic alteration was highest at younger age and declined with age. Our results indicate that the genomic variation associated with DNA content exceeding the diploid level might compromise viability of these neurons in the aging brain and might thus contribute to susceptibilities for age‐related CNS disorders. Alternatively, a potential selection bias of “healthy aging brains” needs to be considered, assuming that DNA content variation above a certain threshold associates with Alzheimer′s disease.

Laser Scanning Cytometry in Human Brain Slices

The Laser Scanning Cytometry (LSC) offers quantitative fluorescence analysis of cell suspensions and tissue sections.

Sequential Photobleaching of Fluorochromes for Polychromatic Slide-Based Cytometry

Slide‐based cytometry is a key technology for polychromatic cytomic investigations. Here we exploit the relocalization and merge feature of Laser Scanning Cytometry for distinguishing fluorochromes of comparable emission spectra but different photostabilities.

Assay validation of phosphorylated S6 ribosomal protein for a pharmacodynamic monitoring of mTOR-inhibitors in peripheral human blood.

Therapeutic drug monitoring (TDM) of immunosuppressive drugs after organ transplantation is based on measuring blood levels alone, which often results in under‐ or over‐immunosuppression. Previous studies have shown the potential of measuring pharmacodynamic drug effects for TDM, but assessment of biomarkers for individual drugs is still not clinical routine. Therefore, we validated a specific assay to measure the pharmacodynamic effects of mammalian target of rapamycin (mTOR)‐inhibitors on phosphorylated S6 ribosomal protein (p‐S6RP), a downstream target of mTOR.