A description of the coagulopathy characteristics in amniotic fluid embolism: a case report.

Amniotic fluid embolism is frequently associated with coagulopathy. However, the exact nature and evolution of the bleeding disorder is incompletely understood. We report a case of clinically diagnosed amniotic fluid embolism associated with major haemorrhage and coagulopathy. We measured sequential levels of all individual clotting factors, thrombin generation, fibrinogen, and D-dimer levels over the course of the event, beginning shortly after the patient's initial collapse and during the subsequent resuscitation, to identify the specific abnormalities of coagulation from stored blood samples. A better understanding of amniotic fluid embolism and the associated coagulopathy is an important area of research to inform targeted treatment of the coagulopathy and improve outcomes for patients.

[Discharge medication - what do patients know about their medication on discharge?]. / Entlassungsmedikation - Was weiß der Patient bei Entlassung über seine Arzneimittel?

BACKGROUND: Patients' lack of knowledge about their discharge medication can endanger patients' safety after their hospital stay. This is especially the case with regard to medications that were newly prescribed during the hospital stay and are intended to be used after discharge or medications with an increased risk for adverse drug reactions (high-risk drugs). The aim of this study was to analyse the patients' level of knowledge about their discharge medication and to identify influence factors. METHODS: In a bicentric survey patients were interviewed prior to their discharge from an acute and a geriatric rehabilitation hospital. They were asked about their discharge medication in a structured interview. Influence factors were statistically analysed by Tobit regression. RESULTS: In total, 179 patients were interviewed. On average, patients named 48% of their discharge medication correctly (95% CI: 46-50%). Influence factors for knowledge deficits were the lack of a medication plan, an older age, the hospitalization in a rehabilitation hospital and a long hospitalization. 81% of the patients had at least one drug in their discharge medication, which was newly prescribed during the hospital stay. 11% of those drugs were named correctly, the potency was named correctly in 6%, the indication in 8%. For almost two-thirds of the patients at least one high-risk drug was recommended in the discharge letter, among them most frequently oral anticoagulants and opioid analgesics. 38% of these high-risk drugs were named correctly. CONCLUSION: Our results demonstrate an urgent need to train patients about their discharge medication, especially if medications are included that were newly prescribed during the hospital stay and recommended for further use after discharge or medications with an increased risk of adverse drug reactions. Particularly older patients and patients of a rehabilitation hospital after long hospitalization should be intensively counselled and obtain a medication plan upon discharge.

[General hospital patients with alcohol problems welcome counselling]. / Krankenhauspatienten mit riskantem Alkoholkonsum sind offen für Beratung.

AIMS: The aim of this study was to determine predictors for openness to alcohol-related counselling among general hospital patients with alcohol problems. We wanted to test whether those with less severe alcohol problems and those with a lack of motivation for behaviour charge or with a lack of motivation to seek help would agree to participate in alcohol-related counselling. METHOD: A total of 1150 hospital patients with alcohol dependence, alcohol abuse, at-risk drinking or excessive drinking were interviewed. They were asked about their attitude towards alcohol-related counselling, about their motivation to change their drinking and about their motivation to seek professional help. A multi-variable logistic regression analysis was calculated to determine predictors for agreement to counselling. RESULTS: A total of 66 % of all participants agreed to receive information on professional help and on how they could help themselves. Among these were 77 % of the alcohol dependent participants and 56 % of the non-dependent participants. Motivation to change and motivation to seek help were identified as the most significant predictors for agreement to counselling. However, 63 % of the participants open for counselling were not yet ready to change their habits and 62 % were not yet ready to seek profession help. CONCLUSION: The majority of hospital patients with less severe alcohol problems as well as the majority of hospital patients not ready to seek more intensive professional help were open for alcohol-related counselling. Given a systematic screening, this opens up the opportunity for addiction counsellors, hospital physicians or nurses to actively offer counselling.

FTIR spectroscopy demonstrates biochemical differences in mammalian cell cultures at different growth stages.

We have observed differences in the infrared spectra of viable fibroblast cells depending on whether the cells were in the exponential (proliferating) or plateau (nonproliferating) phase of growth. The spectral changes were observed even after correcting for cell number and volume, ruling out these trivial explanations. Several of the changes occurred for both transformed and normal cell lines and were greater for the normal cell line. The biochemical basis of the spectral changes was estimated by fitting the cell spectra to a linear superposition of spectra for the major biochemical components of mammalian cells (DNA, RNA, protein, lipids, and glycogen). The ratios of RNA/lipid and protein/lipid increased when the cells were in the exponential phase compared to the plateau phase of growth. The fits of cell spectra to individual biochemical components also demonstrated that DNA is a relatively minor spectroscopic component as would be expected biochemically. Contrary to other reports in the literature, our data demonstrate that determining DNA content or structure using Fourier transform infrared spectroscopy data is difficult due to the relatively small amount of DNA and the overlap of DNA bands with the absorption bands of other biochemical components.

Polarized angular dependent spectroscopy of epithelial cells and epithelial cell nuclei to determine the size scale of scattering structures.

An understanding of the relationship between tissue structures and light scattering from tissue will help facilitate the development and acceptance of noninvasive optical diagnostics including elastic scattering spectroscopy, diffuse reflectance, and optical coherence tomography. For example, a quantitative model of the structures that scatter light in epithelial cells would allow determination of what structures control the characteristics of in vivo light transport measurements and subsequently could provide a detailed relationship between cellular structures and optical measurements. We have determined the size distribution of refractive index structure variations in epithelial cells as well as in nuclei isolated from epithelial cells from measurements of the angular dependence of polarized light scattering. The quantitative size distributions we obtained for both whole cells and isolated nuclei include particles with effective radii of 2 microm to 10 nm or less and contain orders of magnitude more small particles than large particles. These results demonstrate that not only are biological cells very heterogeneous, but so are the nuclei within them. Light scattering is likely sensitive to structures smaller than those commonly investigated by standard pathology methods.

Angular dependent light scattering from multicellular spheroids.

We demonstrate that the effects of cell-cell contact and of changes in cell shape have only a minor effect on the angular distribution of light scattering from mammalian fibroblast cells. This result is important for the development of light scattering as a noninvasive tool for tissue diagnostics such as cancer detection. Changes in cell organization that are not accompanied by changes in internal cellular structure may not be measurable. On the other hand, changes in internal cellular structure should be measurable without interference from changes in overall cellular organization. The second major result of this work is that there are small but significant differences between light scattering of tumorigenic and nontumorigenic cells grown in a three-dimensional culture system. The cause of the differences in light scattering are likely due to the nontumorigenic cells arresting in the G1 phase of the cell cycle, while the tumorigenic cells continue to proliferate.

Impact of proliferative activity and tumorigenic conversion on mitochondrial function of fibroblasts in 2D and 3D culture.

The purpose of the present study was to examine mitochondrial function in differently transformed cells relative to their tumorigenic state and proliferative activity in vitro. An established two-step carcinogenesis model consisting of immortal and tumorigenic rat embryo fibroblasts that can be cultured as monolayers and multicellular spheroids was investigated. Flow cytometric measurements were carried out using the two mitochondrial-specific fluorochromes rhodamine 123 (Rh123) and 10-N-nonyl acridine orange (NAO), in combination with the DNA dye Hoechst 33342 for simultaneous cell cycle analysis. Since the accumulation of Rh123 depends on mitochondrial membrane potential, Rh123 fluorescence intensity gives an estimate of mitochondrial activity per cell, as determined by both overall mitochondrial function and mass. In contrast, NAO uptake reflects mitochondrial mass only, as it binds to cardiolipin in the inner mitochondrial membrane independently of membrane potential. Aliquots of cell suspensions derived from exponential monolayer, confluent monolayer, and a range of sizes of multicellular spheroids were stained with either Rh123 or NAO and Hoechst 33342, then mitochondrial mass and activity per unit cell volume and cellular DNA content were measured by flow cytometry. Differences in the average mitochondrial activity per cell in different cell lines and culture conditions were primarily due to alterations in cell volume. Importantly, tumorigenic conversion by ras-transfection did not consistently change mitochondrial activity per unit cell volume. The mitochondrial mass per unit cell volume increased for all cells when cellular quiescence was induced, either in monolayers or spheroids. However, mitochondrial function (activity/mass) decreased when cells became quiescent, resulting in a positive correlation between mitochondrial function and S-phase fraction, independent of transformation status or culture condition. We conclude that mitochondrial function reflects proliferative activity rather than tumorigenic conversion.

Characterizing Mammalian cells and cell phantoms by polarized backscattering fiber-optic measurements.

Fiber-optic, polarized elastic-scattering spectroscopy techniques are implemented and demonstrated as a method for determining both scatterer size and concentration in highly scattering media. Measurements of polystyrene spheres are presented to validate the technique. Measurements of biological cells provide an estimate of the average effective scatterer radius of 0.5-1.0 mum. This average effective scatterer size is significantly smaller than the nucleus. In addition, to facilitate use of polarization techniques on biological cells, polarized angular dependent scattering from cell suspensions was measured. The light scattering from cells has properties similar to those of small spheres.

Light scattering from cells: the contribution of the nucleus and the effects of proliferative status.

As part of our ongoing efforts to understand the fundamental nature of light scattering from cells and tissues, we present data on elastic light scattering from isolated mammalian tumor cells and nuclei. The contribution of scattering from internal structures and in particular from the nuclei was compared to scattering from whole cells. Roughly 55% of the elastic light scattering at high-angles (> 40 degrees) comes from intracellular structures. An upper limit of 40% on the fractional contribution of nuclei to scattering from cells in tissue was determined. Using cell suspensions isolated from monolayer cultures at different stages of growth, we have also found that scattering at angles greater than about 110 degrees was correlated with the DNA content of the cells. Based on model calculations and the relative size difference of nuclei from cells in different stages of growth, we argue that this difference in scattering results from changes in the internal structures of the nucleus. This interpretation is consistent with our estimate of 0.2 micron as the mean size of the scattering centers in cells. Additionally, we find that while scattering from the nucleus accounts for a majority of internal scattering, a significant portion must result from scattering off of cytoplasmic structures such as mitochondria.

Decreased mitochondrial function in quiescent cells isolated from multicellular tumor spheroids.

Cells in the inner region of multicellular spheroids markedly reduce their oxygen consumption rate, presumably in response to their stressful microenvironment. To determine the mechanism behind this metabolic adaptation, we have investigated relative mitochondrial mass and mitochondrial function in cells isolated from different regions of tumor spheroids by using a combination of mitochondrial-specific fluorescent stains and flow cytometric analysis. Uptake of rhodamine 123 (R123) is driven by the mitochondrial membrane potential and thus reflects mitochondrial activity. Uptake of 10-nonyl-acridine orange (NAO) reflects total mitochondrial mass independently of activity because this compound binds to cardiolipin in the inner mitochondrial membrane. NAO fluorescence per unit cell volume only decreased 10-20% for cells from the inner spheroid region compared with those near the surface. There was greater than a twofold reduction in R123 fluorescence in the inner region cells, however. Thus, tumor cells in spheroids alter their rate of respiration predominately by downregulating mitochondrial function as opposed to degradation of mitochondria. There was a correlation between R123 staining per unit cell volume and the growth fraction of the cells from spheroids, but not for monolayer cultures. We also show a linear correlation between R123 staining and the rate of oxygen consumption for both monolayer- and spheroid-derived cells. After separating the inner region cells from the spheroid and replating them in monolayer culture, the R123 uptake recovered to normal levels prior to entry of the cells into S-phase. This reduction in mitochondrial function in quiescent cells from spheroids can explain the long period required for these cells to re-enter the cell cycle and may have important implications for the regulation of tumor cell oxygenation in vivo.

Differential regulation of cyclin-dependent kinase inhibitors in monolayer and spheroid cultures of tumorigenic and nontumorigenic fibroblasts.

The Ras proto-oncogene has been implicated in the in vivo development of tumors and in the in vitro transformation of cultured cell lines. In both of these conditions, Ras-mediated disruption of cell cycle-regulatory mechanisms leads to unregulated cellular proliferation, although the exact mechanisms by which Ras accomplishes this are not clear. Using as a model the M1 and MR1 rat fibroblast cell lines, which differ in the expression of a regulated Ras (M1 cells) versus a constitutively active Ras (MR1 cells), we examined the role of Ras in the control of cellular proliferation in two-dimensional (monolayer) and three-dimensional (spheroid) cell cultures. These cell lines are very similar in their monolayer growth characteristics, but M1 cells will arrest their cell cycle progression in aggregate culture, whereas MR1 cells proliferate normally as small spheroids. We report here that G1-phase arrest in plateau-phase monolayer cultures of both M1 and MR1 cells correlates with up-regulated expression of the cyclin-dependent kinase (CDK) inhibitor p18INK4c. Enhanced p18INK4c expression was also observed in G1-arrested M1 cells cultured as multicellular spheroids but was not induced in small proliferating MR1 multicellular spheroids. The kinetics of G1 arrest in M1 cells after inoculation into aggregate culture correlated well with the induction of p18INK4c expression. Conversely, resumption of proliferation in monolayer culture of arrested M1 cells isolated from spheroids coincided with the loss of expression of p18INK4c. After extended culture, cells in the inner region of MR1 spheroids arrested in the G1 phase without any up-regulation of p18INK4c expression. In this case, the CDK inhibitor p21(Cip1/Waf1) was selectively induced in the inner regions of large MR1 spheroids, concomitant with a decrease in cyclin and CDK expression. Thus, Ras-dependent regulation of p18INK4c expression seems to control the ability of rat embryo fibroblasts to proliferate as small multicellular aggregates, whereas p21(Cip1/Waf1) expression seems to regulate the G1-phase arrest induced by the stressful microenvironment found within the inner region of large spheroids.

Evidence of intrinsic differences in the light scattering properties of tumorigenic and nontumorigenic cells.

BACKGROUND: The objective of this study was to determine whether there are intrinsic differences in the light scattering properties of tumorigenic and nontumorigenic cells from a multistep carcinogenesis model. METHODS: Wavelength-dependent and polarization-dependent light scattering properties of cell suspensions were measured. RESULTS: Statistically significant differences were found between the tumorigenic and nontumorigenic cells. CONCLUSIONS. Differences in the light scattering properties of tumorigenic and nontumorigenic cells are attributed to a change in the average size of the scattering centers on the order of a few ten of nanometers. This work is relevant to the development of noninvasive optical methods for cancer diagnosis.

Mechanisms of light scattering from biological cells relevant to noninvasive optical-tissue diagnostics.

We have studied the optical properties of mammalian cell suspensions to provide a mechanistic basis for interpreting the optical properties of tissues in vivo. Measurements of the wavelength dependence of the reduced scattering coefficient and measurements of the phase function demonstrated that there is a distribution of scatterer sizes. The volumes of the scatterers are equivalent to those of spheres with diameters in the range between ~0.4 and 2.0 mum. Measurements of isolated organelles indicate that mitochondria and other similarly sized organelles are responsible for scattering at large angles, whereas nuclei are responsible for small-angle scattering. Therefore optical diagnostics are expected to be sensitive to organelle morphology but not directly to the size and shape of the cells.

Mitochondrial function in oncogene-transfected rat fibroblasts isolated from multicellular spheroids.

Two mitochondrion-specific fluorochromes, 10-N-nonyl acridine orange (NAO) and rhodamine 123 (Rh123), were used to determine the mechanism responsible for alterations in energy metabolism of transformed rat embryo fibroblast cells isolated from different locations within multicellular spheroids. Accumulation of Rh123 depends on intact mitochondrial membrane potential, whereas NAO is taken up by mitochondria independently of their function and thus represents mitochondrial distribution only. A reproducible selective dissociation procedure was used to isolate cells from different locations within the spheroids. After isolation, cells were simultaneously stained with one mitochondrial stain and the DNA dye Hoechst 33342, and several parameters, including cell volume, were monitored via multilaser-multiparameter flow cytometry. Our data clearly show a decrease in the uptake of Rh123 in cells from the periphery to the inner regions of the tumor spheroids, reflecting a persistent alteration in mitochondrial function. However, NAO staining experiments showed no reduction in the total mitochondrial mass per unit cell volume. Because cells were exposed to stain under uniform conditions after isolation from the spheroid, these data indicate the downregulation of mitochondrial function is associated with cell quiescence rather than a transient effect of reduced nutrient availability. This result, which is in accordance with data from two other cell lines (EMT6 and 9L), might reflect a general phenomenon in multicellular spheroids, supporting the hypothesis that quiescent cells in the innermost viable spheroid layer stably reduce their mitochondrial function, presumably to compensate for lower nutrient supply and/or decreased energy demand.

Diffuse backscattering Mueller matricesof highly scattering media.

We report on the development of a method that records spatially dependent intensity patterns of polarized light that is diffusely backscattered from highly scattering media. It is demonstrated that these intensity patterns can be used to differentiate turbid media, such as polystyrene-sphere and biological-cell suspensions. Our technique employs polarized light from a He-Ne laser (l=543nm), which is focused onto the surface of the scattering medium. A surface area of approximately 4x4 cm centered on the light input point is imaged through polarization-analysis optics onto a CCD camera. One can observe a large variety of intensity patterns by varying the polarization state of the incident laser light and changing the analyzer configuration to detect different polarization components of the backscattered light. Introducing the Mueller-matrix concept for diffusely backscattered light, a framework is provided to select a subset of measurements that comprehensively describe the optical properties of backscattering media.

In vivo 31P MRS evaluation of ganciclovir toxicity in C6 gliomas stably expressing the herpes simplex thymidine kinase gene.

Phosphorus MRS was evaluated as a monitor of tumour therapeutic response to the herpes simplex virus thymidine kinase suicide gene therapy paradigm. In vivo 31P spectra were obtained from subcutaneous rat C6 gliomas constitutively expressing the HSVtk gene post treatment with ganciclovir (GCV, 15 mg/kg i.p., twice-daily). Significant regression (p < 0.1) of tumour volume was observed 10 days after beginning GCV administration. However, no changes in tumour pH or energy metabolites from pre-treatment values were observed. High-resolution 31P spectra of tumour extracts revealed a statistically significant reduction in the phosphocholine to phosphoethanolamine ratio six days post-GCV administration. These results indicate that the HSVtk/GCV-induced killing of tumours is not associated with corresponding changes in 31P MRS-observable energy metabolites and pH. The observed reduction in the PE/PC ratio may provide a non-invasive in vivo indicator of therapeutic efficacy.

Tumor growth in vivo and as multicellular spheroids compared by mathematical models.

In vivo volume growth of two murine tumor cell lines was compared by mathematical modeling to their volume growth as multicellular spheroids. Fourteen deterministic mathematical models were studied. For one cell line, spheroid growth could be described by a model simpler than needed for description of growth in vivo. A model that explicitly included the stimulatory role for cell-cell interactions in regulation of growth was always superior to a model that did not include such a role. The von Bertalanffy model and the logistic model could not fit the data; this result contradicted some previous literature and was found to depend on the applied least squares fitting method. By the use of a particularly designed mathematical method, qualitative differences were discriminated from quantitative differences in growth dynamics of the same cells cultivated in two different three-dimensional systems.

Analysis of growth of multicellular tumour spheroids by mathematical models.

We wished to determine the applicability of previously proposed deterministic mathematical models to description of growth of multicellular tumour spheroids. The models were placed into three general classes: empirical, functional and structural. From these classes, 17 models were applied systematically to growth curves of multicellular tumour spheroids used as paradigms of prevascular and microregional tumour growth. The spheroid growth curves were determined with uniquely high density of measurements and high precision. The theoretical growth curves obtained from the models were fitted by the weighted least-squares method to the 15 measured growth curves, each corresponding to a different cell line. The classical growth models such as von Bertalanffy, logistic and Gompertz were considered as nested within more general models. Our results demonstrate that most models fitted the data fairly well and that criteria other than statistical had to be used for final selection. The Gompertz, the autostimulation and the simple spheroid models were the most appropriate for spheroid growth in the empirical, functional and structural classes of models, respectively. We also showed that some models (e.g. logistic, von Bertalanffy) were clearly inadequate. Thus, contrary to the widely held belief, the sigmoid character of a three or more parameter growth function is not sufficient for adequate fits.