Spinal anaesthesia--early Australian experience.

Australia in 1902 was a fledgling colony in its second year of Federation with a population of around 3.7 million. European settlement had been largely confined to the coastal margins of this enormous land mass, although some bold adventurers in search of gold and farmland had struggled their way into the interior Horsham, situated 300 km northwest of Melbourne in the state of Victoria, was founded in June 1849. By 1902 the town, with a population of around 2500, had grown to boast a hospital, two doctors, a pharmacist and a dentist. It was at the Horsham Hospital on January 7, 1902 that Dr Robert Ritchie performed Australia's first recorded spinal anaesthetic. Ritchie performed a lumbar puncture at the L3-4 level, injected 2 ml of 2% cocaine solution and waited for a total of 20 minutes before realising that the sensation the patient was feeling when he pinched him was pressure, not pain. The 78-year-old man with a gangrenous right leg, prostatic obstruction and congestive cardiac failure was laid supine, and had his right leg amputated through the thigh while being administered brandy and water Strychnine injections were administered four hourly postoperatively. The adoption of the technique of spinal anaesthesia spread quickly in Australia despite communication difficulties at that time.

Onset and offset pharmacodynamics of propofol.

Propofol whole blood and plasma concentrations at offset of hypnosis in eighteen patients were inversely related to patient age and body fat. The relationship between propofol concentrations and body fat is derived from the relationship between age and body fat and age was the single independent predictor of concentrations at offset of propofol hypnosis.

Detection of HIV-1 DNA and messenger RNA in individual cells by PCR-driven in situ hybridization and flow cytometry.

Human immunodeficiency virus type-1 (HIV-1) DNA and messenger RNA sequences in both cell lines and blood obtained directly from HIV-1-infected patients were amplified by polymerase chain reaction and hybridized to fluorescein-labeled probes in situ, and the individually labeled cells were analyzed by flow cytometry. After flow cytometric analysis, heterogeneous cell populations were reproducibly resolved into HIV-1-positive and -negative distributions. Fluorescence microscopy showed that the cellular morphology was preserved and intracellular localization of amplified product DNA was maintained. Retention of nonspecific probe was not observed. Analysis of proviral DNA and viral messenger RNA in cells in the blood of HIV-1-infected patients showed that the HIV-1 genome persists in a large reservoir of latently infected cells. With the use of this technique it is now possible to detect single-copy DNA or low-abundance messenger RNA rapidly and reproducibly in a minor subpopulation of cells in suspension at single-cell resolution and to sort those cells for further characterization.

Application of automated DNA sizing technology for genotyping microsatellite loci.

Highly polymorphic microsatellite loci offer great promise for gene mapping studies, but fulfillment of this potential will require substantial improvements in methods for accurate and efficient genotyping. Here, we report a genotyping method based on fluorescently labeled PCR primers and size characterization of PCR products using an automated DNA fragment analyzer. We capitalize on the availability of three distinct fluorescent dyes to label uniquely loci that overlap in size, and this innovation increases by threefold the number of loci that can be analyzed simultaneously. We label size standards with a fourth dye and combine these with the microsatellite PCR products in each gel lane. Computer programs provide very rapid and accurate sizing of microsatellite alleles and efficient data management. In addition, fluorescence signals are linear over a much greater range of intensity than conventional autoradiography. This facilitates multiplexing of loci (since signal intensities often vary greatly) and helps distinguish major peaks from artifacts, thereby improving genotyping accuracy.

Ketamine, midazolam and vecuronium infusion. Anaesthesia for Down's syndrome and congenital heart disease.

A ketamine, midazolam and vecuronium infusion was used for total intravenous anesthesia in a patient with Down's syndrome, a ventricular septal defect and pulmonary hypertension. A mixture of ketamine 200 mg, midazolam 5 mg and vecuronium 12 mg in 50 ml of normal saline was infused at 0.5 ml/kg/hour. This simple technique and ventilation with 100% oxygen maintained tissue oxygenation and cardiovascular stability.

Automated genetic analysis.

Automation of several new, non-traditional techniques for genetic analysis has now become possible. A new system is described that performs gel electrophoretic analysis of DNA including VNTRs, gene segments, and restriction enzyme digests. The instrument detects emitted fluorescence from labeled DNA segments in real-time as they electrophore through a gel matrix past a scanning laser beam. Molecular length determination and band quantification is accomplished by comparison to an in-lane standard. Since DNA segments can be labeled and detected with any of four different dyes, the simultaneous analysis of similar length segments from different reactions within a single lane is possible. PCR products are analyzed for research in the areas of human identification and genetic disease. These examples illustrate how automation will play key role in this new era of genetic analysis.

Automation of specific human gene detection.

An instrument/chemistry system is described that automates a new chemical procedure functionally equivalent to Southern blotting. A fluorescence gel scanner that detects migrating DNA fragments in real-time analyzes the samples produced by a prototype liquid-handling instrument that automates a solution-phase hybridization/solid-phase capture chemistry for DNA analysis. The combination of this chemistry, the gel scanner, and robotic automation eliminates the tedium encountered in traditional manual methods for specific gene detection and reduces analysis time from days to hours. Restriction fragment lengths are measured with high precision by comparison with in-lane standards to minimize effects attributable to migration anomalies. The utility of this automated system is demonstrated by executing a clinical research application involving hybridization to a multi-copy repeat sequence on the Y chromosome and its detection.

Automated DNA sequencing methods involving polymerase chain reaction.

Polymerase chain reaction (PCR) as a method for preparing DNA templates has been used for several DNA sequencing applications. An in situ procedure for directly sequencing PCR products by the dideoxy-termination method has been developed by using fluorophore-labeled sequencing primers. Completed sequence reactions were combined and loaded into a single electrophoretic lane of a fluorescence-based DNA sequence analyzer. DNA targets devoid of a universal primer sequence could be sequenced with labeled universal primers by incorporating a universal primer sequence into the PCR product. With this method, the sequence of a 351-bp region in the bacteriophage lambda genome was fully analyzed in a single lane with automatic base identification accuracy of greater than 99%. An unknown sequence, 1.7 kb long, also was sequenced by this procedure, in combination with a "PCR gene walking" strategy. Comparison of the 1110 bases in overlapping sequence data from both strands yielded only two single-base ambiguities. Automated DNA sequence analysis of the highly polymorphic HLA-DQA-1 (alpha) region in the human genome can be performed with this simple methodology. Use of this PCR-sequencing method to analyze DNA extracted from a one-month-old blood sample from an individual who is heterozygous at this locus allowed unambiguous assignment of genotype.

Identification of mutations leading to the Lesch-Nyhan syndrome by automated direct DNA sequencing of in vitro amplified cDNA.

The Lesch-Nyhan (LN) syndrome is a severe X chromosome-linked disease that results from a deficiency of the purine salvage enzyme hypoxanthine phosphoribosyltransferase (HPRT). The mutations leading to the disease are heterogeneous and frequently arise as de novo events. We have identified nucleotide alterations in 15 independently arising HPRT-deficiency cases by direct DNA sequencing of in vitro amplified HPRT cDNA. We also demonstrate that the direct DNA sequence analysis can be automated, further simplifying the detection of new mutations at this locus. The mutations include DNA base substitutions, small DNA deletions, a single DNA base insertion, and errors in RNA splicing. The application of these procedures allows DNA diagnosis and carrier identification by the direct detection of the mutant alleles within individual families affected by LN.

High-performance liquid chromatographic analysis of oligodeoxyribonucleotide base composition.

A significantly improved method for base composition analysis of synthetic oligodeoxyribonucleotides is presented. This highly accurate and sensitive method used enzymatic digestion followed by high-resolution HPLC of the nucleosides to determine the empirical base composition of the parent compound. The enzymatic digestion reaction is quantitative and is not blocked by modified bases, thus allowing the degree of base deprotection and chemical modification to be assessed. Digestion data are presented for oligodeoxyribonucleotides which range from 18 to 150 bases in length with excellent agreement of experimental and theoretical composition. The method is also applicable to high-molecular-weight genomic DNA.

Traction--a surgical/orthodontic procedure.

Maxillary crowding may cause failure of eruption of the canine. The teeth so displaced are directed buccally or palatally. In both cases, surgical intervention is indicated after space has been made in the arch. Palatally displaced canines are treated by surgical excision of palatal mucosa to promote eruption. Wide excision is indicated to prevent the healing process from covering the exposed tooth crown. Because of the anatomy of the buccal mucosa, however, surgical excision of the crowns of buccally displaced canine frequently results in periodontal problems. An alternative technique is therefore presented; this involves the surgical exposure of the crowns of buccally displaced teeth to allow the attachment of a wire traction hook. The crown is then recovered. The traction hook provides a point of attachment, so that orthodontic forces may be applied to the unerupted tooth to guide its eruption. The preservation of the mucosal flap ensures a normal epithelial attachment develops on the buccal surface of the tooth, and the normal gingival anatomy of the buccal mucosa is maintained. The procedure may also be used in cases where upper incisors have been prevented from erupting because of the presence of supernumerary teeth. Even after surgical removal of the supernumerary teeth, the permanent incisors often fail to erupt. In such cases, the placement of traction hooks will enable the orthodontist to bring the unerupted teeth into their correct positions in the arch.