Using risk calculation to implement an extended relative pair analysis

A new nonparametric method of linkage analysis is described based on identity by descent relationships between all pairs of affected relatives within a pedigree. This approach is an extension of ESPA, which only uses information from pairs of affected siblings. The new method, called ERPA, uses the risk calculation facilities of the LINKAGE programs to obtain the necessary information in a fashion which is simple to implement and which automatically generalizes to allow for marker loci which may be multiple, non-codominant and sex-linked. We have investigated the relative performance of ERPA, ESPA and the lod score method on simulated data. ERPA appears to be more sensitive than ESPA for detecting linkage in pedigrees with small sibships, though both nonparametric methods are inferior to the lod score method when the true mode of transmission can be specified.     

Online measurement of ocular accommodation using a low-cost microcomputer system

The paper describes a low-cost system for the measurement of ocular accommodation. Digitisation of the low-frequency signals obtained from the measurement is straightforward and high-speed methods are not necessary. The methods described are, therefore, of interest to workers involved in the study of low-frequency signals, which are common in biological systems.     

Determination of urea, allantoin and lysine pyroglutamate in cosmetic samples by hydrophilic interaction chromatography

A new HPLC method using a Polyhydroxyethyl A column involving hydrophilic interaction chromatography (HILIC) is described for the simultaneous determination of urea, allantoin and lysine pyroglutamate in a cosmetic cream. Validation of the method was accomplished with respect to linearity, repeatability and limits of detection/quantification. Compound recoveries approach 100% with acceptable RSD values. The method is very simple since no derivatisation is necessary. Furthermore, it allows the rapid and direct chromatographic analysis of urea and hence could provide an alternative to other methods used to determine this compound in biological or cosmetic samples.     

Farmer's lung: a comparison of simple diagnostic techniques and antigen preparation in human and bovine disease

The detection of precipitins to Micropolyspora faeni antigens by simple serological techniques was studied with human and bovine sera. We present data on the relative sensitivities of two gel-diffusion tests, one large-scale, the other using semi-micro methods to conserve antigen in the diagnosis of human and bovine disease. Eight antigenic preparations of M. faeni are described, together with the detailed serology of eight human cases of farmer's lung. The results indicate that the use of more than one antigenic preparation is necessary in any screening programme for the detection of precipitins to M. faeni in either the human or bovine disease.     

Immunoassays and nucleic acid detection with a biosensor based on surface plasmon resonance

A technique based on surface plasmon resonance is described which can be used to detect changes of refractive index that occur when one partner of a molecular binding pair diffuses from solution to bind the other partner which is immobilised on a silver surface. Results for the molecular binding pairs; protein-antibody, hapten-antibody and DNA-DNA are described. Instrumentation necessary for implementation of the technique is detailed. Immunoassay of proteins and haptens is possible in less than one minute with a sensitivity of 10(-9) mol/l. Hybridisation of 10 fmoles of a 97 base target sequence on the 1 mm2 area of detection to an immobilised oligonucleotide probe can be detected in less than five minutes. Advantages of the technique include the ability to record the kinetics of binding reactions in "real time" and the lack of labels in this simple assay format. Methods of improving the sensitivity are discussed.     

Measurement of antibody to influenza virus neuraminidase by single radial hemolysis in agarose gels.

A simple method of assaying anti-influenza neuraminidase antibodies in human sera was described. Suitable antigenic hybrid viruses were adsorbed to sheep erythrocytes, which were then incorporated into agarose gels. When sera were introduced into wells cut in the gels, zones of hemolysis were observed in the neighborhood of those containing neuraminidase antibodies. There was a direct relationship between zone size and antibody titer. No purification of adsorbed viruses was necessary. The test was rapid, required very simple reagents, gave results that agreed well with those given by conventional techniques, and appeared to be the most sensitive of four methods evaluated. Studies of cross-reactions by hyperimmune sera against homologous and heterologous neuraminidases and of absorption of neuraminidase antibodies from human sera indicated a high degree of specificity. The technique seems to be suitable for large-scale epidemiological investigations.     

Correlation of apparent molecular weight and antigenicity of viral proteins: an SDS-page separation followed by acrylamide-agarose electrophoresis and immunoprecipitation.

A simple method is described which combines a sodium dodecyl sulfate polyacrylamide gel electrophoresis (SOS-PAGE) in the first demension with a second electrophoresis, at right angles to the first, into an agarose matrix. The proteins, separated by SDS-PAGE, are exposed to appropriate antisera after the second stage electrophoresis and immunoprecipitates form in the agarose corresponding to the relative electrophoretic mobilities of proteins in the first stage SDS-PAGE separation. The method thus provides a simple, reproducible means for correlating antigenicity with apparent molecular weight of proteins. The technique is qualtitative, but requires smaller quantities of antisera than more conventional immunoelectrophoretic methods such as rocket electrophoresis.     

A comprehensive system for monitoring the foetal heartrate and uterine contractions

Intensive labour-room management has created a need for instrumental monitoring of foetal heartrate and uterine contraction. To fulfil this need, a versatile instrument has been designed and built, which provides transvaginal and external abdominal methods of monitoring both variables. For the foetal heartrate, these are, respectively, foetal-scalp electrocardiography (a simple scalp electrode which is atraumatic and efficient is described) and Doppler sonar at 2 MHz. For uterine contraction, the methods are direct pressure measurement via intrauterine catheter and indirect measurement by guard-ring dynamometer. A new combined transducer is described that incorporates the ultrasonic Doppler Sonicaid D205 foetal heart-beat detector in a development of Smyth's guard-ring principle of measuring uterine contraction (UK Patent Application 40642/71). The clinical application and results are briefly reviewed.     

Reliable and Precise Identification of R-Waves in the EKG with a Simple Peak Detector

A simple instrument is described which reliably and accurately identifies R-waves in the EKG signal. These R-waves are obtained by “Modified Lead 2′’and other similar methods. By using a peak detector instead of a Schmitt trigger, three important advantages are obtained. If the instrument is connected to a time interval counter, reliable and accurate acquisition of heart beat interval and heart rate data may be achieved with a relatively simple operation.     

Microbiology--laboratory examinations for bacterias

As it has been required to identify pathogenic microbes in shorter times, simple and rapid methods have been developed and used. Here, we summarized the present situation of rapid diagnostic testing in clinical microbiology in Japan, and also presented our results on PBP2' detection. The rapid test kits available in Japan for E. coli, Helicobacter pylori, Salmonella, Streptococcus and Staphylococcus aureus were described. Rapid examination methods are based mainly on immunologic reactions, which included slide agglutination using latex particle, immunochromatography and ELISA. Times required for the identification are 10 to 15 minutes. Moreover, rapid test kits employing PCR are also marketed. Further, we evaluated MRSA-LA "Seiken" which is a rapid detection kit for PBP2' produced by MRSA. The test was shown to be highly sensitive and specific. For the rapid identification of pathogenic microbes, simple and rapid test kits described here will be used more in clinical diagnosis.     

Oligonucleotide microarray design for detection and serotyping of human respiratory adenoviruses by using a virtual amplicon retrieval software

The rapid development of new molecular biology methods has improved infectious disease diagnosis, which is increasingly important to clinical management and public health. A wide variety of new methods which are more specific, sensitive and robust, such as combination of PCR and microarray technology, has gradually replaced the conventional assays usually used in routine diagnosis. Both methods have the advantage of speed and sensitivity but tend to be expensive and technically demanding. Therefore, it is necessary to develop more simple assays that could be available for all diagnostic laboratories. To this aim, a simple microarray assay for detection and typing of adenoviruses causing acute respiratory disease in humans was developed. The absence of effective therapeutic or alternative prophylactic treatment for this infection makes essential its rapid diagnosis to implement fast control measures. Moreover, the family Adenoviridae includes numerous serotype groups and constitutes an ideal model system to develop diagnostic methods for other human pathogens. For their precise identification, an amplicon retrieval software that simplifies and accelerates the processing of all sequences necessary to perform this type of experiments has also been developed. This new technology was applied for the simultaneous detection and serotyping of acute respiratory disease-causing adenoviruses in laboratory and clinical samples with efficiency and accuracy.     

Supplementary procedures in embedding tissues in paraffin

Recommendations for paraffin homogenization which should preferentially be performed in a thermostat at 85-90 degrees C, and the methods of preparations of a protein-glycerol mixture are given. A simple set for a regulated heating of water used for paraffin section reexpansion is described.     

Automated quality control for the haematology laboratory

Two simple methods of using sophisticated statistical techniques to analyse intralaboratory quality control data are described. One method uses a plastic grid to assess the cumulative sum (cusum) graph. An equivalent procedure, which may be carried out by a laboratory computer, gives automated on-line quality control of the Coulter S blood counter. The possible development of simple apparatus to carry out the same function is discussed.     

A simple routine method for mono and diamine oxidase estimation in human serum

The advantages and disadvantages of previous methods for the routine estimations of serum mono and diamine oxidase are reviewed. 4-hydroxy-3-methoxy phenyl acetic acid reacts wtih hydrogen peroxide and peroxidase to form an intensely fluorescent substance, and a simple method using this reaction as a basis for the estimation is described. The results are tabulated and discussed.     

Skin testing and food challenges in allergy and immunology practice

Summary Skin tests by prick technique offer considerable guidance in the diagnosis of food allergy. Negative prick skin tests are powerful evidence against food allergy. Positive food skin tests are slightly to moderately predictive of reaction to a food on DBPCFC. Oral food challenge is necessary for confirmation of food allergy, except where the history is overwhelmingly convincing. Open, incremental food challenge as described is diagnostic if negative, but only 50% of all positive open challenges are confirmed on blinded challenge. DBPCFC can be designed for any food with simple blinding techniques. The technique of DBPCFC can be modified for investigation of atypical symptoms.     

Pressure-radius relationships for elastic tubes and their application to arteries: Part 1-Theoretical relationships

The displacement relationships describing the deformation of an elastic vessel under excess internal pressure which are derived from different theories of elasticity are compared. The main result of the comparison is that theories which take account of the thickness of the wall of the vessel produce a significantly better representation than those theories which treat the wall as a membrane. The classical and statistical theories of thick-walled tubes result in complicated pressure-radius relationships. It is shown that there is little difference between the results of the more exact theories and those for a thin membrane corrected by means of a simple thickness factor. A review of the different theories is necessary to decide which pressure-displacement relationship to apply as an approximation for the elastic properties of arteries. An indication is given of the manner in which the relationship is used in numerical computations. In Part 2 the experimental determination of the pressure-radius relationship for a rubber tube is described. The results are in agreement with the conclusions of the comparison of theoretical treatments in Part 1.     

Facilitated endogenous repair: making tissue engineering simple, practical, and economical

Facilitated endogenous repair is a novel approach to tissue engineering that avoids the ex vivo culture of autologous cells and the need for manufactured scaffolds, while minimizing the number and invasiveness of associated clinical procedures. The strategy relies on harnessing the intrinsic regenerative potential of endogenous tissues using molecular stimuli, such as gene transfer, to initiate reparative processes in situ. In the simplest example, direct percutaneous injection of an osteogenic vector is used to stimulate bone healing. If necessary, additional progenitor cells and space-filling scaffolds can be provided by autologous bone marrow, muscle, fat, and perhaps other tissues. These can be harvested, processed, and reimplanted by simple, expedited, intraoperative procedures. Examples of repair of experimental osseous and osteochondral lesions in laboratory animals are described. If successful, these strategies will provide methods for tissue regeneration that are not only effective but also inexpensive, safe, and clinically expeditious. Although orthopaedic examples are given here, the technology should be more generally applicable.     

Use of the average antibody-antigen bond concept and probability theory to simplify modeling of linear and circular antibody-antigen complex formation

We illustrate use of a simple approach to describe the equilibrium interactions of mixtures of monoclonal antibodies and antigens. This procedure is based on elementary concepts in probability theory and is readily suited to describing interactions of antibodies and antigens which form circular as well as linear complexes. The method is also suited to describing the inhibitory effects of antibodies which compete for overlapping epitopes and an example is provided to show how the procedure can be used to describe the interactions of antibodies which inhibit circular complex formation. We also outline simple strategies for preparing computer programs to simulate binding of antigens to defined antibody mixtures. The methods described should facilitate design of immunoassay procedures based on the use of defined mixtures of monoclonal antibodies.     

Use of the ELISA to screen for anti-thymocyte and anti-beta 2-microglobulin antibodies in leprosy and SLE.

A report is given of the use of the enzyme-linked immunosorbent assay to measure antibody to preparations of human thymocyte membranes (HTMA) and to beta 2-microglobulin. The assay described is simple and rapid, and requires only small quantities of an easily stored membrane preparation. The advantages of this technique over conventional methods involving cytotoxicity are discussed. Raised levels of IgM antibody to beta 2-microglobulin were detected in sera from SLE patients. Raised levels of IgG and IgM antibody to HTMA were found in sera from most active lepromatous cases. Two of eight sera from SLE patients showed raised IgG anti-HMTA, but not raised IgM. An attempt was made to study the subclass of the IgG antibodies found, but when checked against purified human IgG myeloma proteins, the available anti-subclass sera were found to lack the necessary degree of specificity in this assay.     

Polarimetric assay for the determination of the native collagen content of soluble collagen

Two standardized assays for determining the amount of native collagen in a collagen solution are described which are based on polarimetric measurements. Native collagen content is calculated from the loss of optical rotation during transition from the original to the denatured conformation using constants which were obtained from standard collagen of highest available native collagen content. While Method I gives information on the content of helical collagen, Method II estimates collagen which is resistant to peptic degradation. Both methods are simple to perform and suitable for routine quality control.