Automated prioritised 3D dose-based MLC segment generation for step-and-shoot IMRT.

Segmentation can degrade a high-quality dose distribution obtained by fluence map optimisation (FMO). A novel algorithm is proposed for generation of MLC segments to deliver an FMO plan with step-and-shoot IMRT while minimising quality loss. All beams are considered simultaneously while generating MLC segments for reproducing the 3D FMO dose distribution. Segment generation is only steered by the 3D FMO dose distribution, i.e. underlying FMO fluence profiles are not considered. The algorithm features prioritised generation of segments, focusing on accurate reproduction of clinical objectives with the highest priorities. The performance of the segmentation algorithm was evaluated for 20 prostate patients, 15 head-and-neck patients, and 12 liver patients. FMO dose distributions were generated by automated multi-criteria treatment planning (Pareto-optimal plans) and subsequently segmented using the proposed method. Various segmentation strategies were investigated regarding prioritisation of objectives and limitation of the number of segments. Segmented plans were dosimetrically similar to FMO plans and for all patients a clinically acceptable segmented plan could be generated. Substantial differences between FMO and segmented fluence profiles were observed. Avoidance of the usual reconstruction of 2D FMO fluence profiles for segment generation, and instead simultaneously generating segments for all beams to directly reproduce the 3D FMO dose distribution is a likely explanation for the obtained results. For the strategies of limiting the number of segments large reductions in number of segments were observed with minimal impact on plan quality.

In vitro detection of cholangiocarcinoma cells using a fluorescent protein-expressing oncolytic herpes virus.

Pathological confirmation is desired prior to high-risk surgery for suspected perihilar cholangiocarcinoma (PHC), but preoperative tissue diagnosis is limited by poor sensitivity of available techniques. This study aimed to validate whether a tumor-specific enhanced green fluorescent protein (eGFP)-expressing oncolytic virus could be used for cholangiocarcinoma (CC) cell detection. Extrahepatic CC cell lines SK-ChA-1, EGI-1, TFK-1 and control cells (primary human liver cells) were exposed to the oncolytic herpes simplex type 1 virus NV1066 for up to 24 h in adherent culture. The technique was validated for cells in suspension and cultured cells that had been exposed to crude patient bile. Optimal incubation time of the CC cells with NV1066 at a multiplicity of infection of 0.1 was determined at 6-8 h, yielding 15% eGFP-expressing cells, as measured by flow cytometry. Cells were able to survive 2-h crude bile exposure and remained capable of producing eGFP following NV1066 infection. Detection of malignant cells was possible at the highest dilution tested (10 CC cells among 2 × 105 control cells), though hampered by non-target cell autofluorescence. The technique was not applicable to cells in suspension due to insufficient eGFP production. Accordingly, as yet the technique is not suitable for standardized clinical diagnostics in PHC.

Computation of mean and variance of the radiotherapy dose for PCA-modeled random shape and position variations of the target.

Radiotherapy dose delivery in the tumor and surrounding healthy tissues is affected by movements and deformations of the corresponding organs between fractions. The random variations may be characterized by non-rigid, anisotropic principal component analysis (PCA) modes. In this article new dynamic dose deposition matrices, based on established PCA modes, are introduced as a tool to evaluate the mean and the variance of the dose at each target point resulting from any given set of fluence profiles. The method is tested for a simple cubic geometry and for a prostate case. The movements spread out the distributions of the mean dose and cause the variance of the dose to be highest near the edges of the beams. The non-rigidity and anisotropy of the movements are reflected in both quantities. The dynamic dose deposition matrices facilitate the inclusion of the mean and the variance of the dose in the existing fluence-profile optimizer for radiotherapy planning, to ensure robust plans with respect to the movements.

Self-regulation in tip-growth: the role of cell wall ageing.

We develop a model to describe the effect of cell wall ageing on the local expansion rate of tip-growing cells. Starting from an exact equation for the stationary age-distribution of the wall material, we propose a generic measure for the local expansion propensity of the wall if the ageing process is described by a constant rate Poissonian decay process. This ageing process may be either interpreted as biochemical in nature describing the finite lifetime of regulatory proteins, or as mechanical in nature describing the gradual "hardening" of the wall through cross-linking or gelation of the wall polymers. In this way we can construct models for tip-growth in which material deposition, evolving wall properties and surface expansion are self-consistently intertwined. As a proof of principle, we implement our ageing approach in two different idealised models of tip-growth, obtaining the stationary tip shapes as a function of the ageing parameter. In the first, the spatial distribution of delivery of growth material is determined by the local curvature of the cell and the growth mode is orthogonal. In the second, the growth material originates from a Vesicle Supply Center, a point-like representation of the Spitzenkörper as found in fungal hyphae, and the growth mode is isometric.

A population-based model to describe geometrical uncertainties in radiotherapy: applied to prostate cases.

Local motions and deformations of organs between treatment fractions introduce geometrical uncertainties into radiotherapy. These uncertainties are generally taken into account in the treatment planning by enlarging the radiation target by a margin around the clinical target volume. However, a practical method to fully include these uncertainties is still lacking. This paper proposes a model based on the principal component analysis to describe the patient-specific local probability distributions of voxel motions so that the average values and variances of the dose distribution can be calculated and fully used later in inverse treatment planning. As usually only a very limited number of data for new patients is available; in this paper the analysis is extended to use population data. A basic assumption (which is justified retrospectively in this paper) is that general movements and deformations of a specific organ are similar despite variations in the shapes of the organ over the population. A proof of principle of the method for deformations of the prostate and the seminal vesicles is presented.

Multi-site, multi-country evaluation of analytical and operational performance of a low-mid volume chemiluminescent immunoassay analyzer.

BACKGROUND: A new automated immunoassay low-mid volume (< or = 250 immunoassays/day) chemiluminescent analyzer, Abbott Architect i1000sR, was evaluated by seven laboratories around the world (4 in Europe, one each in Canada, Japan, and the U.S.A.) to demonstrate equivalent performance for key operating characteristics (e.g., precision, turn around time, limit of detection, functional sensitivity, and linearity). METHODS: The laboratories followed standard protocols to assess precision, limit of detection (LoD), functional sensitivity, assay linearity, method comparison, and sample carryover. Turn around time for three stat assays (beta-hCG, BNP, and CK-MB) and the time required to complete workloads of 50 and 100 tests with a mixture of 75% routine tests and 25% stat tests was also evaluated. RESULTS: Total precision was typically < 5% CV for nine immunoassays. Analytical performance met design goals and demonstrated equivalency to package insert data for assays on market and in use for an existing high volume immunoassay system. Stat turn around times were consistent with the fixed analytical time of 15.6 minutes and met the expectations of the laboratories. Measured test throughput ranged from 47 - 54 tests per hour and demonstrated that the analyzer was fit for the intended purpose of supporting a laboratory that performs < or = 250 immunoassays per day. CONCLUSIONS: A multisite, international analyzer familiarization study is a practical means of confirming that a new instrument meets both a manufacturer's design specifications and users' real world expectations and provides a pragmatic test for the system. The experience of investigators at seven sites around the world indicates that a new fully automated chemiluminescent system is suitable for use.

Structural heterogeneity in DNA: temperature dependence of 2-aminopurine fluorescence in dinucleotides.

The fluorescent base analogue 2-aminopurine is a sensitive probe for local dynamics of DNA. Its fluorescence is quenched by interaction with the neighboring bases, but the underlying mechanisms are still under investigation. We studied 2-aminopurine fluorescence in dinucleotides with each of the natural bases. Consistently, two of the four fluorescence-decay components depend strongly on temperature. Our results indicate that these components are due to the excited-state dynamics of a single conformational state. We propose a variation of the gating model in which transient unstacking occurs in the excited state.

Effects of high-fat diets with different carbohydrate-to-protein ratios on energy homeostasis in rats with impaired brain melanocortin receptor activity.

Changes in dietary macronutrient composition and/or central nervous system neuronal activity can underlie obesity and disturbed fuel homeostasis. We examined whether switching rats from a diet with high carbohydrate content (HC; i.e., regular chow) to diets with either high fat (HF) or high fat/high protein content at the expense of carbohydrates (LC-HF-HP) causes differential effects on body weight and glucose homeostasis that depend on the integrity of brain melanocortin (MC) signaling. In vehicle-treated rats, switching from HC to either HF or LC-HF-HP feeding caused similar reductions in food intake without alterations in body weight. A reduced caloric intake (-16% in HF and LC-HF-HP groups) required to maintain or increase body weight underlay these effects. Chronic third cerebroventricular infusion of the MC receptor antagonist SHU9119 (0.5 nmol/day) produced obesity and hyperphagia with an increased food efficiency again observed during HF (+19%) and LC-HF-HP (+33%) feeding. In this case, however, HF feeding exaggerated SHU9119-induced hyperphagia and weight gain relative to HC and LC-HF-HP feeding. Relative to vehicle-treated controls, SHU9119 treatment increased plasma insulin (2.8-4 fold), leptin (7.7-15 fold), and adiponectin levels (2.4-3.7 fold), but diet effects were only observed on plasma adiponectin (HC and LC-HF-HP

Pseudoplatelets: a retrospective study of their incidence and interference with platelet counting.

AIMS: Spurious platelet counts can be found in acute leukaemias, as a result of the fragmentation of blood cells. Microscopic examination of a blood smear should be performed to detect the presence of these so called pseudoplatelets. When present, the platelet count should be corrected because of the important clinical consequences that a lower platelet count may have in these patients. METHODS: K(3)EDTA anticoagulated blood was measured on an automated blood cell counter, and a blood smear was made and stained according the May Grünwald-Giemsa method for microscopic observation. A 500 cell/particle differentiation was performed and the automated platelet count was corrected. RESULTS: The incidence of pseudoplatelets in 169 patients with acute leukaemia was studied. Pseudoplatelets were detected in 43 patients (25.4%), and seven patients (4.1%) were re-classified as having a major bleeding risk (platelet count, < 15 x 10(9)/litre). CONCLUSIONS: Platelets should be determined morphologically in patients with acute leukaemia and a routine screening method for the detection of pseudoplatelets should be developed.

Capillary blood gas analysis in complex regional pain syndrome: a pilot study.

BACKGROUND: The pathophysiology of complex regional pain syndrome type 1 (CRPS 1) is still a matter of debate. An inflammatory reaction may cause the syndrome. Increasing evidence points to a role for impairment of oxygen metabolism in the affected limb. METHODS: In this pilot study (16 patients) we performed capillary blood gas analysis in extremities with acute CRPS 1, in order to assess oxygen saturation and lactate concentrations. Comparison was made with the unaffected limb for capillary blood pH, pO(2), SaO(2), and lactate and glucose concentrations. RESULTS: No statistically significant differences could be found. CONCLUSIONS: Capillary blood gas analysis is not useful to detect changes in oxygen saturation and lactate concentrations in CRPS 1.

Pseudothrombocytopenia: a report of a new method to count platelets in a patient with EDTA- and temperature-independent antibodies of the IgM type.

Pseudothrombocytopenia is usually associated with blood specimens anticoagulated with ethylenediamine tetraacetic acid (EDTA) or other anticoagulants. It may be caused by temperature-independent, EDTA-dependent antibodies of the immunoglobulin-M (IgM) type. Here a patient with EDTA-independent and temperature-independent pseudothrombocytopenia mediated by IgM or IgM-containing immune complex is reported, and a reliable method is described for a proper counting of platelets in such cases.

Automated counting of nucleated red blood cells in blood samples of newborns.

Nucleated red blood cells (NRBC) in blood samples interfere with the white blood cell (WBC) count on many types of automated haematology analysers. This makes it necessary to correct the WBC count by counting NRBC microscopically. This report describes the evaluation of two analysers, the Cell-Dyn 4000 and the Sysmex XE-2100, which use new techniques to recognize and enumerate NRBC. We conclude that both the Cell-Dyn 4000 and the Sysmex XE-2100 give an accurate WBC count in the presence of NRBC. Furthermore, they can enumerate NRBC correctly when compared with microscopic observation.

Bacteria in blood smears: overwhelming sepsis or trivial contamination.

It is unusual to find microorganisms in peripheral blood smears, and their presence is frequently associated with overwhelming sepsis and consequently a poor prognosis. In this report, we demonstrate 4 cases with bacteria in blood smears. Two of them had a fatal outcome, but the other 2 were caused by a contamination either via the central venous catheter or in vitro, both without dramatic outcome. The finding of bacteria in blood smears has to be interpreted carefully, and thorough examination of peripheral blood smears may be of great importance in the early diagnosis of bacteremia; however, in vitro contamination must be excluded.

Hyperhomocysteinemia as a risk factor for venous thrombosis.

Classical homocystinuria is associated with arterial vascular diseases and venous thrombosis. In the last decade, several studies have been published indicating that even mild hyperhomocysteinemia is a risk factor for venous thrombosis. The 677C-->T mutation in the methylenetetrahydrofolate reductase (MTHFR) gene is an important cause of mild hyperhomocysteinemia, but this polymorphism does not seem to be a risk factor for venous thrombosis. Studies on the interaction between hyperhomocysteinemia and other thrombotic risk factors are conflicting. Little is known about the pathophysiology of venous thrombosis in hyperhomocysteinemia. Several mechanisms proposed for vascular disease may be applied to venous thrombosis as well. However, up to now there is no satisfying model which might explain a thrombophilic state at plasma homocysteine concentrations in the range of mild hyperhomocysteinemia. The results of a first clinical intervention study are expected in 2002. As the results are pending, clinicians could perform homocysteine measurements in patients with venous thrombosis if screening for thrombophilia is indicated. Vitamin supplementation could be prescribed if homocysteine levels are elevated. However, the patient should be informed about the uncertainty of the benefits of vitamin supplementation.

Peripheral blood lymphocyte appearance in a case of I cell disease.

In general, peripheral blood smears are performed to obtain information with regard to various morphological features as an aid in the diagnosis of infection or malignancy. This report presents a patient with I cell disease (inclusion cell disease), a fatal lysosomal storage disorder caused by a defect in an enzyme responsible for the transfer of mannose-6-phosphate ligands to precursor lysosomal enzymes. As a consequence, most lysosomal enzymes are transported outside the cell instead of being correctly targeted into the lysosomes, resulting in the storage of macromolecules in lysosomes. I cell disease, with its heterogeneous clinical presentation, can be diagnosed by the presence of intracellular vacuole-like inclusions in lymphocytes and fibroblasts, high serum lysosomal enzyme activities, and a defect of N-acetylglucosamine-1-phosphotransferase. This report describes the morphological aspects of peripheral lymphocytes in a blood smear of a patient, the first clue to the final diagnosis of I cell disease. The observed vacuole-like inclusions in lymphocytes of this patient were negative for periodic acid Schiff (PAS) and Sudan black B staining, in contrast to earlier reports.

Point mutation in the stalk of angiotensin-converting enzyme causes a dramatic increase in serum angiotensin-converting enzyme but no cardiovascular disease.

BACKGROUND: Angiotensin-converting enzyme (ACE) metabolizes many small peptides and plays a key role in blood pressure regulation. Elevated serum ACE is claimed to be associated with an increased risk for cardiovascular disease. Previously, two families with dramatically increased serum ACE were described, but no systematic survey of affected individuals was performed, and the molecular background of this trait is unknown. METHODS AND RESULTS: Eight families were identified with autosomal dominant inheritance of a dramatic (5-fold) increase of serum ACE activity. Strikingly, no clinical abnormalities were apparent in the affected subjects. Isolated blood cells were used for genetic and biochemical analysis. The level of ACE expression on the blood leukocytes and dendritic cells and total cell-associated ACE of the affected individuals was similar to that in nonaffected relatives; however membrane-bound mutant ACE was much more efficiently clipped from the cell surface compared with its wild-type counterpart. A point mutation causing Pro1199Leu in the stalk region of the ACE molecule cosegregates with the increase in serum ACE (LOD score, 6.63). CONCLUSIONS: A point mutation in the stalk region of the ACE protein causes increased shedding, leading to increased serum ACE, whereas cell-bound ACE is unaltered, and affected individuals exhibit no clinical abnormalities. These findings qualify the importance of serum ACE and establish a new determinant of ACE solubilization.

Optimization of multileaf collimator settings for radiotherapy treatment planning.

Multileaf collimators have become available in many radiotherapy treatment centres. The cross section of a beam can be shaped to a projection of the target area by moving the leaves of the multileaf collimator into the beam. In this paper, a method is described to optimize the positions of the individual leaves automatically, once the beam directions and weights have been chosen. The individual positions of the leaves are optimized using the variable metric method. Changes in dose resulting from small leaf movements are computed efficiently using a special method. The optimization method was tested on a treatment plan for a phantom patient. It was found that the unnecessary edges of the beams were trimmed efficiently.