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PubMed1979

Sickle cell anemia as a syndrome: a review of diagnostic features.

Sickle cell (SS) disease is a complex of various genetic conditions. In some, homozygosity for the beta S gene may be present alone or in combination with the heterozygous or homozygous alpha-thalassemia-2 condition. Such combinations might ameliorate the clinical and hematological condition of the patient. The same may be true for the high levels of Hb F and F-cells observed in many Hb S homozygotes. Howeever, the chemical heterogeneity of Hb F appears not to be related to the clinical status of the Hb S homozygote. Combinations of a Hb S heterozygosity with a heterozygosity for a Hb D-type of variant, for either one of two types of beta-thalassemia, two types of alpha beta- thalassemia, and five types of HPFH are discussed, and data are compared with those obtained for Hb S homozygotes. The use of advanced laboratory procedures and family studies is often necessary for an accurate diagnosis.

Anemia, Sickle Cell↗
PubMed1980

Genetic associates/counselors in genetics services.

A survey of US directors of genetic services showed that about 38% of the non-physician professionals providing such are genetic associates/counselors (GAs). Over 90% of GAs appear to be involved in direct human services, rather than in the performance of laboratory procedures or research. Although they wanted GAs to be competent in communication and inter-personal skills, counseling, and community education, employers and potential employers tended to rank a background preparation in and knowledge of psychosocial principles relatively less important than a knowledge of biological principles. The possible consequences of these attitudes are discussed in light of the fact that GAs represent a new profession which is still evolving and struggling to define its professional role in genetic services. Despite some role ambiguities, employers familiar with their work expressed a strong vote of confidence in these professionals.

Evaluation Studies as Topic↗
PubMedDate not supplied

Factors which contribute to cytogenetic frequency of expression in families of fragile X females.

Frequency of expression in mentally retarded (MR) fragile X (fra[X]) males has been shown to be affected by genetic factors and laboratory procedures. Among MR females, factors that contribute to cytogenetic expression have been more problematic in part due to lower frequency of expression and X-inactivation. We examined fra(X) sisters to determine whether genetic factors affecting frequency of expression in sisters were comparable to those in brothers. Evaluations obtained from 2 data sets for a total of 66 families in which at least 2 affected brothers and/or 2 affected sisters were found cytogenetically positive were selected. Of 166 subjects chosen, 118 were male and 48 were female. Sibs were evaluated using an analysis of covariance (ANCOVA) with 2 factors (family, sex) adjusted for the covariate, age. Results indicated that interactive effects (family-by-sex) and main effects (family, sex, age) were significant but the interactive effect of age-by-sex was not. Consequently, one-way analyses of variance (ANOVA) of the effect of family were calculated separately for brothers and sisters. Correlation coefficients between family and cytogenetic frequency was r = .84 for brothers and r = .79 for sisters. Analysis of these coefficients indicated that they were not significantly different from one another (Z = 0.85, p greater than .40). While other factors may affect cytogenetic expression, whatever familial factors control frequency of expression among brothers appear to affect frequency of expression among sisters as well.

Aging↗
PubMedDate not supplied

Influence of the storage time and the method of stimulation on whole blood chemiluminescence.

The ultra-weak light, chemiluminescence (CL), of stimulated leukocytes is a well-known phenomenon. Parameters of this CL are modified by many factors including laboratory procedures. The order of stimulation and enhancement (two possibilities) and two concentrations of luminol create four types of procedure, which were accomplished in five sample storage 'time points'. We received the strongest signals of CL using higher concentrations of luminol (and DMSO), but only when stimulation (FMLP) was used before enhancement (luminol); luminol used before FMLP strongly inhibited CL. For lower luminol concentration (and DMSO), the order of stimulation and enhancement was of no importance. There were comparable but weaker signals of CL in this case. We received stronger signals with storage time for all procedures. It may be dependent on the priming of phagocytes by releasing cell factors. Stimulation (FMLP) before enhancement (luminol) eliminates the inhibitory effect of DMSO on CL.

Adult↗
PubMed2004

Cardiac vagal regulation across the preschool period: stability, continuity, and implications for childhood adjustment.

Stability and continuity of vagal regulation of the heart, operationalized as suppression of respiratory sinus arrhythmia (RSA) during challenge, was examined in a longitudinal study of preschoolers. A sample of 154 two-year-old children was recruited for participation in a study of the effects of emotional and behavioral challenge on cardiac activity and behavioral indices of adjustment and self-regulation. A total of 122 of these children were assessed again at age 4.5 years. At both ages, the children were assessed in a series of laboratory procedures that were intended to be emotionally and behaviorally challenging, during which time heart rate was recorded. To assess vagal regulation, resting measures of RSA and RSA suppression to the challenge task were derived from these procedures. To assess childhood adjustment and self-regulation, a number of parent-report measures were administered when the children were 4.5 years of age. Results indicated that there was high stability in RSA suppression across the challenge tasks within both ages, modest cross-age stability in RSA suppression, and a significant decrease in the magnitude of RSA suppression across age. Second, children who displayed a pattern of stable and high suppression across the preschool period were less emotionally negative, and had fewer behavior problems and better social skills than other children.

Adaptation, Psychological↗
PubMedDate not supplied

Temperature-gradient gel electrophoresis of nucleic acids: analysis of conformational transitions, sequence variations, and protein-nucleic acid interactions.

Temperature-gradient gel electrophoresis (TGGE) is applied to analyze conformational transitions and sequence variations of nucleic acids and protein-nucleic acid interactions. A linear and highly reproducible temperature-gradient is established perpendicular or parallel to the direction of the electrophoresis. The instrument consists of an electrically insulated metal plate, which is heated at one edge and cooled at the other edge by two thermostating baths and is used as an ancillary device for commercial horizontal gel electrophoresis instruments. Biopolymers are separated in TGGE according to size, shape and thermal stability of their conformational transitions. If the temperature-gradient is established perpendicular to the electrophoresis, monomolecular conformational transitions of nucleic acids show up as continuous transition curves; strand-separation leads to discontinuous transitions. In the studies on viroid RNA it was shown that natural circular viroid RNA undergoes one highly cooperative transition detected by TGGE as a drastic retardation in mobility. Oligomeric replication intermediates of viroids exhibit coexisting structures which could not be detected by any other technique. Double-stranded satellite RNA from cucumber mosaic virus is a mixture of sequence variants, all of which have the identical length of 335 nucleotides. In TGGE six different strains were resolved. Sequence variants of viroids were analyzed by hybridizing viroid RNA to (-)strand viroid RNA transcripts from viroid cDNA clones. Sequence variations lead to mismatches in the double strands and thereby to a shift of the transition curve to lower temperature. Mutations in plasmids, particularly in cloned inserts, were detected by mixing plasmids of two different clones, linearizing, denaturing, renaturing, and searching for shifts in the transition curves, which are generated by mismatch-formation during the renaturation of (+)- and (-)strands from different clones. Examples are given for different viroid clones and HIV-clones from one and the same patient. In another example, clones with point mutations from site-directed mutagenesis are analyzed and selected by TGGE. TGGE is also applied to study the effect of amino acid exchanges in the Tet repressor from E. coli on the thermal stability of the repressor and on the mode of binding of the repressor to the operator DNA. The results are discussed under the aspect that TGGE may be applied as routine analytical laboratory procedure.

Base Sequence↗
PubMedDate not supplied

Costing of services of Vietnamese hospitals: identifying costs in one central, two provincial and two district hospitals using a standard methodology.

BACKGROUND: The People's Republic of Vietnam is currently in a period of transition from a purely socialistic country towards a so-called socialist market economy. Since the introduction of the Doi Moi policy in 1987, health care services have been liberalized, medical practitioners have received the right to open private hospitals and private pharmacies are booming. However, the majority of inhabitants strongly depends on governmental hospital services. These services are currently going through a financial crisis. This demands the quest for efficiency and the wise allocation of public funds. RESEARCH QUESTION: The efficient allocation of funds depends on the quotient between costs and health outcomes, but the costs of health services in Vietnamese hospitals are unknown. Therefore the study analysed five hospitals in Vietnam and determined the average costs as well as the main cost drivers. METHODOLOGY: The full costs of five hospitals were analysed, including depreciation and the value of donations. The costs were allocated to cost centres and cost units by a stepping-stone method. MAJOR FINDINGS: As expected, the average costs per inpatient day at a central hospital are about 300% of the costs of provincial hospitals and about 600% of the costs of district hospitals. However, the costs of some laboratory procedures and operations done at district hospitals are higher than those of provincial or even central hospitals. The main reason for the high costs of some procedures at district hospitals was the low quantity of these procedures at that level. This is a strong indicator that some procedures, in particular major operations, should not be performed in Vietnamese district hospitals.

Costs and Cost Analysis↗
PubMed2003

Detection of trisomy 21 by quantitative fluorescent-polymerase chain reaction in uncultured amniocytes.

Prenatal diagnosis of fetal trisomy 21 is usually performed by cytogenetic analysis. This requires lengthy laboratory procedures, high costs and is unsuitable for large-scale screening of pregnant women. Today, trisomy 21 can be rapidly diagnosed within 24 h by molecular analysis of uncultured fetal cells using the semi-quantification of fluorescent PCR products from short tandem repeat (STR) polymorphic markers. The aim of our study was to test a chromosome quantification method on the basis of the analysis of fluorescent PCR products derived from non-polymorphic target genes. Co-amplification of a portion of DSCR1 (Down syndrome Critical Region 1) and the reference gene, CFTR (cystic fibrosis transmembrane regulator) enabled molecular detection of trisomy 21. Our method was successfully tested on a total of 154 amniotic fluids in a blind prospective study. Calculation of the DSCR1/CFTR ratio allowed us to distinguish between 152 normal amniotic fluids (mean ratio 0.99) and 2 amniotic fluids presenting a trisomy 21 status (DSCR1/CFTR ratio of 1.53 and 1.61, respectively). The results obtained by conventional cytogenetic analysis and our quantitative PCR method were concordant in every case. Our gene-based fluorescent PCR approach represents an alternative molecular method for rapid and reliable detection of trisomy 21, which can be helpful in the prenatal diagnosis of women at high risk of fetal trisomy 21.

Adult↗
PubMedDate not supplied

Spatial distribution of neurons in tissue culture wells: implications for sampling methods to estimate population size.

Many laboratory procedures require the counting of cells in culture. While many cultured cells may be counted by automated methods, neuronal cultures often require manual cell counting methods that are prohibitively time-consuming. This paper examines methods of sampling from tissue culture wells for estimating total cell counts. Performance of sampling and estimation schemes will depend in part on how the cells distribute themselves within a well. Spatial statistical analysis techniques are applied to the known total number and distribution of neurons in two wells counted in a grid scheme to demonstrate some important features of the neuron distributional patterns. Based on these two wells and simulated realizations from other point processes, a new sampling and estimation technique using open wedge-shaped sampling regions radiating from the centre of the well is proposed. This method is shown to result in more accurate estimates of the total number of neurons in the well than standard methods.

Animals↗
PubMed1997

A microtiter-based assay for hyaluronidase activity not requiring specialized reagents.

A sensitive, rapid microtiter-based assay for hyaluronidase activity is described that does not require highly specialized biological reagents, as required heretofore. The free carboxyl groups of hyaluronan are biotinylated in a one-step reaction using biotin-hydrazide. This substrate is then covalently coupled to a 96-well microtiter plate. At the completion of the enzyme reaction, residual substrate is detected with an avidin-peroxidase reaction that can be read in a standard ELISA plate reader. Because the substrate is covalently bound to the microtiter plate, artifacts such as pH-dependent displacement of the biotinylated substrate do not occur. The sensitivity permits rapid measurement of hyaluronidase activity from cultured cells and biological samples with an interassay variation of less than 5%. Using this new assay, we measured the distribution profile of plasma hyaluronidase levels in normal human sera. A 1-microl sample of plasma was sufficient for assays in triplicate. Hyaluronidase activity in human foreskin primary keratinocyte cultures was also quantitated. A 25-fold increase in hyaluronidase activity was observed in keratinocyte cultures induced to differentiate in high calcium (1.5 mM), compared to levels in low calcium (0.05 mM) media. The microtiter-based assay may be used as a routine clinical laboratory procedure.

Biotin↗
PubMed1999

Optimized multiplex PCR: efficiently closing a whole-genome shotgun sequencing project.

A new method has been developed for rapidly closing a large number of gaps in a whole-genome shotgun sequencing project. The method employs multiplex PCR and a novel pooling strategy to minimize the number of laboratory procedures required to sequence the unknown DNA that falls in between contiguous sequences. Multiplex sequencing, a novel procedure in which multiple PCR primers are used in a single sequencing reaction, is used to interpret the multiplex PCR results. Two protocols are presented, one that minimizes pipetting and another that minimizes the number of reactions. The pipette optimized multiplex PCR method has been employed in the final phases of closing the Streptococcus pneumoniae genome sequence, with excellent results.

Algorithms↗
PubMed2004

Recurrent aphthous ulcers today: a review of the growing knowledge.

Recurrent aphthous ulcers represent a very common but poorly understood mucosal disorder. They occur in men and women of all ages, races and geographic regions. It is estimated that at least 1 in 5 individuals has at least once been afflicted with aphthous ulcers. The condition is classified as minor, major, and herpetiform on the basis of ulcer size and number. Attacks may be precipitated by local trauma, stress, food intake, drugs, hormonal changes and vitamin and trace element deficiencies. Local and systemic conditions, and genetic, immunological and microbial factors all may play a role in the pathogenesis of recurrent aphthous ulceration (RAU). However, to date, no principal cause has been discovered. Since the aetiology is unknown, diagnosis is entirely based on history and clinical criteria and no laboratory procedures exist to confirm the diagnosis. Although RAU may be a marker of an underlying systemic illness such as coeliac disease, or may present as one of the features of Behcet's disease, in most cases no additional body systems are affected, and patients remain otherwise fit and well. Different aetiologies and mechanisms might be operative in the aetiopathogenesis of aphthous ulceration, but pain, recurrence, self-limitation of the condition, and destruction of the epithelium seem to be the ultimate outcomes. There is no curative therapy to prevent the recurrence of ulcers, and all available treatment modalities can only reduce the frequency or severity of the lesions.

Adolescent↗
PubMed1999

Reducing the number of microlocations in oligonucleotide microchip matrices by the application of degenerate oligonucleotides.

The application of degenerate oligonucleotides to DNA Sequencing by Hybridisation with Oligonucleotide Matrix (SHOM) is proposed. The use of degenerate oligonucleotides is regarded as an example of pooling methods that are suitable for various laboratory procedures requiring numerous samples to be assayed. As each DNA sequence coded by four letters (A, G, C, T) may be defined by two sequences: a sequence coded by W and S (W-weak-A or T, S-strong-G or C) and a sequence coded by R and Y (R-purine-A or G, Y-pirymidine-T or C), n4n -nucleotide sequences may be defined with the help of 2xn2sequences. In the place of the originally described microchip matrix composed of all possible unambiguous octanucleotides (4(8)=65 536) attached to the equal number of 65 536 microlocations a matrix composed of 512 microlocations containing 256 2(8)-degenerate octanucleotides is proposed. The matrix contains all 256 possible octanucleotides coded by W and S variations and all 256 possible octanucleotides coded by R and Y variations. The 512 256-degenerate octanucleotides allows to retrieve the same information as 65 536 unambiguous octanucleotides. A variant of the DNA sequence reconstruction method applicable to this system is presented. The use of degenerate oligonucleotides also gives the possibility to apply matrices composed of longer oligonucleotides without increasing the number of microlocations in matrices, which would enable increasing the length of unambiguously reconstructed sequence, e.g. a matrix comprising 131 072 16-mer oligonucleotides i.e. 65 536 65 536-fold degenerate oligonucleotide coded by W and S variations and 65 536 65 536-fold degenerate oligonucleotide coded by R and Y variations could replace one matrix comprising all possible unambiguous 16-mer oligonucleotides (ca. 4.3x10(9)).

Animals↗
PubMed1988

Incidence and clinical presentation of chlamydial keratoconjunctivitis: a preliminary study.

Using a direct immunofluorescent technique and monoclonal antibody, chlamydia trachomatis was detected in the conjunctival smears of 13 out of 47 patients presenting with conjunctivitis or keratoconjunctivitis (an incidence of 28%). Two patients presented with acute symptoms of few days duration, whereas the condition was chronic and of long duration in 11 patients. Conjunctival changes noted were upper and lower palpebral conjunctival follicles and papillae (11 patients), chemosis (5 patients), upper tarsal scar (2 patients) and pseudomembrane (one patient). Corneal involvement was detected in 9 patients and was manifested as micropannus (6 patients), multiple small epithelial punctate stains (3 patients), extensive pannus affecting the upper third of the cornea (2 patients), subepithelial punctate infiltrates similar to that of adenovirus infection (2 patients) and upper limbal follicles (one patient). Identification of chlamydia trachomatis in conjunctival smears by use of the monoclonal antibody is a simple, rapid and reliable laboratory procedure.

Acute Disease↗
PubMed1976

The essentials of data sources in eye epidemiology.

The extensive literature on eye epidemiology was selectively reviewed to provide a concise and comprehensive summary on the current status of available data sources. The topics being covered were epidemiologic data evaluation, data handling, card registration, computer analysis, geographic factors, socio-economic conditions, personal attributes, genetic backgrounds, twin analysis and laboratory procedures for host defense factors including humoral and cellular immunity. Also, the scope of this article encompassed such major practical issues as glaucoma, cataract and other blinding diseases as well as traffic and occupational hazards. Epidemiologic information thus obtainable through data searching may prove tobe instrumental in the identification of any potential preventive measures in ophthalmology and public health.

Data Display↗
PubMed1978

Genetic exchange in Bacillus subtilis in soil.

Genetically labelled strains of Bacillus subtilis have been shown to exchange blocks of linked genes while growing together in soil. After eight days of incubation, 79% of unselected colony-forming units exhibited a phenotype containing markers from both parents; the parental strains were not detected after the first day of incubation. High frequencies of transformation were also obtained by adding genetically labelled deoxyribonucleic acid to single-strain soil cultures. Observed linkage of genetic markers was greater in soil transformation than in standard laboratory procedures. The results indicate that transformation may play an important role in the adaptation of the Bacilli to their natural habitat.

Bacillus subtilis↗
PubMed1989

New in vivo model to assess venous endothelial cell functions. Effect of defibrotide.

In the past few years there has been increasing interest in the role of the vascular endothelium as an active modulator of biological responses. Endothelial cells exert antithrombotic activity by the release of prostacyclin [23] and adenine nucleotides [16], the availability on the cell surface of heparin-like substances [3], and thrombomodulin-mediated activation of protein C [8]. In addition, endothelium is involved in the regulation of fibrinolysis by releasing soluble factors, such as tissue plasminogen activator (tPA; [10]) and plasminogen activator inhibitor (PAI; [22, 11]), as well as in the control of vascular responsiveness by the production of smooth muscle relaxing and contracting factors. Endothelial cells have also been shown to synthesize and to express procoagulant activities [18]. Many data on endothelial cell functions has been obtained from two experimental models, namely endothelial cell cultures and perfused segments of animal and human vessels. Both are subject to methodological criticism since they only represent in part in vivo conditions, and the necessary experimental manipulations and laboratory procedures greatly modify the naturally occurring cellular functions. In order to overcome such difficulties as far as possible, a new in vivo model has been employed to provide easily assessable and reliable data on the properties of endothelial cells in man. A venous segment was isolated functionally by cannulating a dorsal vein in the hand and a cubital vein in the same arm. Changes observed ex vivo in blood from the cubital vein following infusion into the hand vein of an active drug, can mainly be attributed to its local effect on the venous wall. At the same time, a cubital vein in the other arm was cannulated in order to provide information to distinguish systemic from regional effects.

6-Ketoprostaglandin F1 alpha↗
PubMed2000

Clinical Experience with the NIR Stent: Retrospective Observation on Acute Results and Follow-up in 709 Patients.

The NIR stent is an expandable cellular coronary prosthesis with high flexibility and excellent trackability. The very low crossing profile, associated to the high flexibility, makes this stent suitable for complex and distal lesions. The aim of this study was to evaluate the feasibility, safety, and long-term (clinical follow-up) efficacy of elective and/or urgent deployment of the NIR stent in a broad patient group with coronary artery disease. Between June 1996 and May 1997, 986 NIR stents were implanted in 781 vessels (de novo or restenotic lesions in native vessels, saphenous vein grafts and internal mammary conduits) of 709 consecutive patients (498 men, 211 women) undergoing coronary angioplasty in two Villa Maria Group Catheterization Laboratories. Procedural angiographic success was achieved in 98.4% of all the lesions treated, ranging from 100% success rate in type A lesion to 97.6% in type C lesions (overall procedural success, 98.4%). Major adverse cardiac events (MACE) were considered from PTCA/stenting time to the patient discharge. The in-hospital MACE were limited and occurred in 15 patients: urgent surgical revascularization 0.8%, death 0.7%, sub-acute stent thrombosis 0.5% (overall in-hospital MACE rate 2.1%). Clinical follow-up data were obtained at 8.6 +/- 2.8 months following the stenting procedure. The event-free survival rate was 87.9%. The late MACE rate evaluation showed death, target lesion revascularization 9.9% (over-all late MACE rate 12.1%). The NIR stent performances in this broad patient population were excellent, showing very high procedural success rate both in normal and complex coronary anatomy. On the basis of the previous data, we can define NIR stent a safe multifunctional device suitable both for easy and complex situations. The clinical late results are very promising in term of event-free survivals and late MACE.

Journal Article↗