Evaluation of the simultaneous estimation of anti-dsDNA and anti-ssDNA antibodies for clinical purposes.

The double-antibody solid-phase assay for DNA antibodies permits simultaneous and quantitative determination of antibodies to dsDNA and ssDNA. Using this method, 170 sera, mainly ANA-positive, were examined for the presence of anti-dsDNA and anti-ssDNA to assess the role of these antibodies in the ANA reaction. It was found that in the SLE group of patients, their ability to respond to dsDNA was correlated with the multiorgan symptomatology of disease. Anti-ssDNA titres are also highest in this group. However, anti-ssDNA titres predominate over anti-dsDNA in other collagen diseases. This predominance increases as we progress from the SLE group to undefined mild collagenosis, because the response to dsDNA decreases more than the response to ssDNA. This observation suggests that the clinical manifestation of the collagen diseases and multiorgan manifestation of SLE is linked with the pattern of response to DNA in the majority of cases. In conclusion, it appears that the determination of both ssDNA and dsDNA antibodies can be of value for the prognosis and management of patients with connective tissue disease.     

Equipment for the quantification of motor performance for clinical purposes

An apparatus is described for the quantitative assessment of important parameters that characterise motor performance in normal subjects and in patients with different types of motor disorders. The apparatus has a handle that can be moved along a straight horizontal track either by the subject (to study voluntary movements) or by a torque motor (to study reflex activity). During voluntary movements the mass and friction of the mechanical part of the equipment are eliminated by feedback of the force exerted at the handle by the subject. The computer program that controls the apparatus gives a choice of four different tests that characterise different aspects of the motor system: the reflex organisation, the regulation of viscoelastic properties mediated in part by reflex activity, the control of fast goal-directed movements, and the performance in a tracking task. The results of a pilot study to the tracking behaviour of clumsy children show that the group of clumsy children differs from a group of normal children in the latency of the tracking response, in the ability to track high-frequency components and in the fact that clumsy children introduce relatively more frequency components in the response that are not present in the tracking signal.     

A method for paraplegic upper-body posture estimation during standing: a pilot study for rehabilitation purposes

This paper presents a contribution for restoring standing in paraplegia while using functional electrical stimulation (FES). Movement generation induced by FES remains mostly open looped and stimulus intensities are tuned empirically. To design an efficient closed-loop control, a preliminary study has been carried out to investigate the relationship between body posture and voluntary upper body movements. A methodology is proposed to estimate body posture in the sagittal plane using force measurements exerted on supporting handles during standing. This is done by setting up constraints related to the geometric equations of a two-dimensional closed chain model and the hand–handle interactions. All measured quantities are subject to an uncertainty assumed unknown but bounded. The set membership estimation problem is solved via interval analysis. Guaranteed uncertainty bounds are computed for the estimated postures. In order to test the feasibility of our methodology, experiments were carried out with complete spinal cord injured patients.

The resistance of glyoxylic acid induced catecholamine fluorescence to sodium borohydride reduction

The borohydride reduction of glyoxylic acid induced fluorescence in noradrenergic and DOPA-minergic nervous structures and in amines in model experiments was studied. Both DOPAmine and noradrenaline fluorescences were resistant to borohydride reduction differing thus from the formaldehyde-induced fluorescence. Thus when the specificity of glyoxylic acid induced fluorescence is in doubt, other tests than borohydride reduction of the fluorescence must be employed.     

Selected technologies to control genes and their products for experimental and clinical purposes

"On-demand" regulation of gene expression is a powerful tool to elucidate the functions of proteins and biologically-active RNAs. We describe here three different approaches to the regulation of expression or activity of genes or proteins. Promoter-based regulation of gene expression was among the most rapidly developing techniques in the 1980s and 1990 s. Here we provide basic information and also some characteristics of the metallothionein-promoter-based system, the tet-off system, Muristerone-A-regulated expression through the ecdysone response element, RheoSwitch, coumermycin/novobiocin-regulated gene expression, chemical dimerizer-based promoter activation systems, the "Dual Drug Control" system, "constitutive androstane receptor"-based regulation of gene expression, and RU486/mifepristone-driven regulation of promoter activity. A large part of the review concentrates on the principles and usage of various RNA interference techniques (RNAi: siRNA, shRNA, and miRNA-based methods). Finally, the last part of the review deals with historically the oldest, but still widely used, methods of temperature-dependent regulation of enzymatic activity or protein stability (temperature-sensitive mutants). Due to space limitations we do not describe in detail but just mention the tet-regulated systems and also fusion-protein-based regulation of protein activity, such as estrogen-receptor fusion proteins. The information provided below is aimed to assist researchers in choosing the most appropriate method for the planned development of experimental systems with regulated expression or activity of studied proteins.     

Automated standardized pupillometry with optical method for purposes of clinical practice and research.

The aim of the current study was the introduction and standardization of two experimental conditions for dynamic pupillometry. Pupillometry is a method that can provide valuable data concerning the functioning of the autonomous nervous system. The system for recording the pupil reaction was developed in the Laboratory of Clinical Neurophysiology of the 1st Department of Neurology of Aristotle University of Thessaloniki, in co-operation with the Laboratory of Fluid Mechanics of the Aristotle University of Thessaloniki. This system is fully automated. It includes an infra-red video camera, which has the capacity to record in complete darkness, and an SLE (clinical photic stimulator) lamp. A software application automatically performed all the procedures. During the first experiment, one flash was administered. During the second experiment, a series of 25 flashes (1 Hz frequency) was administered. Fifty physically and mentally healthy subjects aged 23-48 years took part in the study. Means, standard deviations and ranges for all variables characterizing normal subjects during both experimental conditions are reported. Test/re-test results and comparisons of the two eyes are also reported. The combined use of these two experimental conditions in dynamic pupillometry may be a very useful tool in medical research. There are already reports on the usefulness of pupillometry in the research of various diseases, including depression and Alzheimer's disease. It is expected that it will also be a valuable research tool in the study of diabetes, alcoholism, myasthenia gravis, cancer, multiple sclerosis, etc.     

Evaluation of flow cytometry and fluorescence microscopy for the estimation of bovine mononuclear phagocytes

Bovine blood mononuclear cells were isolated by density gradient centrifugation on Ficoll-Paque. Phagocytic mononuclear cells were characterized functionally by ingestion of fluorescent latex beads. After incubation with beads the cells were treated with Triton X-100 and propidium iodide (PI) to stain DNA. Cells were analyzed with a FACS-III instrument connected to a Nuclear Data-6660 multiparameter computer system. The computer was used to evaluate the 2 parameter histograms in order to enumerate the percentage of cells with different numbers of associated beads. With this system we also obtained information about cell concentration and number of beads per cell. Results from flow cytometry and manual counting by fluorescence microscopy were compared and good correlation (r = 0.91) was obtained. During the first hours of incubation latex beads adhered to cell surfaces as demonstrated by FCM histograms and fluorescence microscopy. Blood mononuclear phagocytes have to be incubated for several hours before significant phagocytotic activity can be detected.     

Bone marrow aspiration for diagnostic purposes

Examination of bone marrow aspiration is an important tool in the diagnosis of haematological diseases. First attempts of bone marrow sampling took place at the beginning of the twentieth century. Thereafter, numerous methods were proposed and different materials were described. The commonly accepted sites for sampling are sternum and the iliac crest. We describe here a sampling procedure for each site. Bone marrow aspiration is a safely investigation, but not recommended for patients with impaired haemostasis. The physician must be aware of its side effects and complications which could occur. The consequence of the complications varies according to the type of iatrogenic injury. Prevention and rapid diagnosis are a crucial point in the management of bone marrow aspiration accidents. To avoid malpractice, the procedure should be taught by senior physicians including theoretical as well as practical learning. The purpose of the learning is a high quality of care to ensure patients the best comfort in subsequent bone marrow examinations, this point being particularly important in paediatrics.     

Identifying psychiatric suicides for research purposes

Unusual accidental deaths recorded for former public psychiatric patients during a 3-year period in Missouri were compared with data previously published on suicide and undetermined deaths. Demographic and diagnostic data suggested that undetermined deaths resemble suicides, while the remaining unusual accidents do not. For purposes of estimating incidence or prevalence, it is recommended that "undetermined deaths" might well be combined with suicides, while the remaining accidents should not.     

Review of pathology data for regulatory purposes

This paper describes the review process for pathology data submitted to the Division of Pathology, Center for Food Safety and Applied Nutrition of the Food and Drug Administration. The Division of Pathology independently evaluates the pathology data submitted in support of the safety of a given compound. The submissions are examined for agreement between summarized information and data from individual animals, appropriateness of terminology applied to lesions, and adequacy of information (distribution and severity of observed lesions). Problems and concerns encountered during review sometimes require examination of microscopic slides. The slide review provides an independent characterization of the lesions and a verification of their incidence. We present some problems commonly encountered in our review of the pathology data and describe some recent pathology evaluations. Finally, we suggest some considerations for reporting pathology data that may facilitate regulatory review.     

Calibration standards for multicenter clinical trials of fluorescence spectroscopy for in vivo diagnosis

In the context of clinical trials, calibration protocols for optical instruments that ensure measurement accuracy and the ability to carry out meaningful comparisons of data acquired from multiple instruments are required. A series of calibration standards and procedures are presented to assess technical feasibility of optical devices for cervical precancer detection. Measurements of positive and negative standards, and tissue are made with two generations of research grade spectrometers. Calibration accuracy, ability of standards to correct and account for changes in experimental conditions, and device components are analyzed. The relative frequency of measured calibration standards is investigated retrospectively using statistical analysis of trends in instrument performance. Fluorescence measurements of standards and tissue made with completely different spectrometers show good agreement in intensity and lineshape. Frequency of wavelength calibration standards is increased to every 2 h to compensate for thermal drifts in grating mount. Variations in illumination energy detected between standards and patient measurements require probe redesign to allow for simultaneous acquisition of illumination power with every patient measurement. The use of frequent and well-characterized standards enables meaningful comparison of data from multiple devices and unambiguous interpretation of experiments among the biomedical optics community.     

Twin zygosity testing for medical purposes

After being poisoned by eating the mushroom species Cortinarius speciosissimus, a twin developed interstitial nephritis with acute renal failure. He received a renal transplant from his living twin brother, who was presumed dizygotic on phenotypic grounds. Fifteen years later, the twins were zygosity tested by DNA “fingerprint analysis” and found to be monozygotic, despite important phenotypic discordances. The recipient has discontinued immunosuppression therapy and remains well after 9 months. We suggest that, for medical and other reasons, zygosity should be determined at birth on all like-sexed twins. Am. J. Med. Genet. 77:412–414, 1998. © 1998 Wiley-Liss, Inc.     

Preclinical validation of fluorescence in situ hybridization assays for clinical practice.

PURPOSE: Validation of fluorescence in situ hybridization assays is required before using them in clinical practice. Yet, there are few published examples that describe the validation process, leading to inconsistent and sometimes inadequate validation practices. The purpose of this article is to describe a broadly applicable preclinical validation process. METHODS: Validation is performed using four consecutive experiments. The Familiarization experiment tests probe performance on metaphase cells to measure analytic sensitivity and specificity for normal blood specimens. The Pilot Study tests a variety of normal and abnormal specimens, using the intended tissue type, to set a preliminary normal cutoff and establish the analytic sensitivity. The Clinical Evaluation experiment tests these parameters in a series of normal and abnormal specimens to simulate clinical practice, establish the normal cutoff and abnormal reference ranges, and finalize the standard operating procedure. The Precision experiment measures the reproducibility of the new assay over 10 consecutive working days. To illustrate documentation and analysis of data with this process, the results for a new assay to detect fusion of IGH and BCL3 associated with t(14;19)(q32;q13.3) in lymphoproliferative disorders are provided in this report. RESULTS: These four experiments determine the analytic sensitivity and specificity, normal values, precision, and reportable reference ranges for validation of the new test. CONCLUSION: This report describes a method for preclinical validation of fluorescence in situ hybridization studies of metaphase cells and interphase nuclei using commercial or home brew probes.