Harmonisation of Measurement Procedures: how do we get it done?

Clinical laboratory measurement results must be comparable among different measurement procedures, different locations and different times in order to be used appropriately for identifying and managing disease conditions. Harmonisation in the broad sense is the overall process of achieving comparability of results among clinical laboratory measurement procedures that measure the same measurand. The term standardisation is used when comparable results among measurement procedures are based on calibration traceability to SI using a reference measurement procedure of the highest available order. When there is no higher order reference measurement procedure available, and it is unlikely that one can be developed, the term harmonisation refers to any process for achieving comparable results among measurement procedures for an individual measurand.This review explains calibration traceability and focuses on the principles of harmonisation for those measurands for which a reference measurement procedure does not exist. We discuss the value of harmonisation, the importance of commutable reference materials, the barriers to harmonisation that exist today, and conclude with a discussion of a current global effort to improve the state of harmonisation.     

Traceability of measurement results

* Trueness must be independent of analytical platform and measurements comparable regardless of the analytical procedure used. * Traceability requirements for the clinical laboratory are via National Metrology Institutes, Reference (Calibration) laboratories and finally the routine laboratory. * Traceability information required by today's clinical laboratory may be requested from the manufacturer of the analytical kits and the internet. * Traceable laboratory results will greatly enhance the role of the laboratory in patient management.     

Multiple uses of liquepipets in Southern, northern, and western blots

Individually wrapped, sterile disposable transfer pipets can be used in the isolation of ds-DNA and ds-RNA fragments from gels as well as in the screening of multiple samples in Southern, Northern, and Western blots without potential contamination by exogenous nucleases and proteases. The sensitivity and results obtained by this method are comparable to those obtained by conventional methods. All the prehybridization, blocking, hybridization, and detection processes can be performed within the transfer pipet. The isotopically labeled probes used in hybridization can easily be recovered, stored for reuse, or disposed of as waste with no potential contamination of personnel or laboratory equipment. Strip blots are stable in appropriate buffers within the liquepipets which can be shipped easily worldwide for comparative analyses by collaborative investigators. This method is simple, time saving, and inexpensive and is particularly suitable for multiple sample screening. Other potential applications of this procedure are discussed.     

The muskrat in biomedical research

Muskrats are aquatic rodents of moderate size which are plentiful throughout North America, but are not used commonly in the laboratory. Recently, we tested the feasibility of muskrats as experimental models and have found them to be acquired and cared for easily in conventional laboratory animal facilities. Some of their natural characteristics and diseases are described. The husbandry techniques that we used are presented and form a base for the preparation of future guidelines for the maintenance and use of feral animals in research. The results of some initial experiments testing the muskrat's utility for investigations of cardiorespiratory control mechanisms also are presented. Our data show that even anesthetized muskrats possess brisk and dramatic cardiovascular and respiratory reflexes. Our findings that their brains possess the cytoarchitectural and myeloarchitectural features comparable to other mammals, combined with their relative uniformity in size, has allowed us to locate specific neuronal loci stereotaxically. We suggest that the muskrat be considered as an experimental animal model for studies of the neural control of cardiorespiratory systems.     

Development of a shaking bioreactor system for animal cell cultures

The feasibility of using shake flasks to culture animal cells was evaluated using various sizes of cylindrical shaped vessels as bioreactors. It was found that conditions can be optimized so that hybridoma, Chinese Hamster Ovary cells, and insect cells can be efficiently cultured in the shaking reactors to cell densities comparable to that obtained with stirred-jar bioreactors, and the system is scalable to larger volumes for the production of recombinant proteins or cell mass production in the laboratory.     

Assessing accuracy and precision for field and laboratory data: a perspective in ecosystem restoration

Unlike most laboratory studies, rigorous quality assurance/quality control (QA/QC) procedures may be lacking in ecosystem restoration (“ecorestoration”) projects, despite legislative mandates in the United States. This is due, in part, to ecorestoration specialists making the false assumption that some types of data (e.g. discrete variables such as species identification and abundance classes) are not subject to evaluations of data quality. Moreover, emergent behavior manifested by complex, adapting, and nonlinear organizations responsible for monitoring the success of ecorestoration projects tend to unconsciously minimize disorder, QA/QC being an activity perceived as creating disorder. We discuss similarities and differences in assessing precision and accuracy for field and laboratory data. Although the concepts for assessing precision and accuracy of ecorestoration field data are conceptually the same as laboratory data, the manner in which these data quality attributes are assessed is different. From a sample analysis perspective, a field crew is comparable to a laboratory instrument that requires regular “recalibration,” with results obtained by experts at the same plot treated as laboratory calibration standards. Unlike laboratory standards and reference materials, the “true” value for many field variables is commonly unknown. In the laboratory, specific QA/QC samples assess error for each aspect of the measurement process, whereas field revisits assess precision and accuracy of the entire data collection process following initial calibration. Rigorous QA/QC data in an ecorestoration project are essential for evaluating the success of a project, and they provide the only objective “legacy” of the dataset for potential legal challenges and future uses.     

Technical note: compatibility of microtomographic imaging systems for dental measurements

Modern micro-computed tomography techniques allow the accurate visualization of internal dental structures, and are becoming widely used within (paleo-) anthropological dental studies. There exist several types and name brands of microtomographic systems, however, which have been demonstrated to produce images that vary in resolution and signal-to-noise ratio. As a growing body of dental research using disparate microtomographic techniques is likely to continue accumulating, it is imperative that different systems are compared to ensure that results are comparable and not machine-dependent. In the present study, we compare volume, surface area, and linear measurements recorded on a sample of modern and fossil teeth using four microtomographic systems (three laboratory scanners, and the ID19 beamline of the European Synchrotron Radiation Facility). Results indicate that measurements are comparable between systems (within 3%), but that synchrotron radiation is superior to the other systems because its monochromatic X-rays prevent beam hardening and its parallel beam prevents geometric artifacts in the resultant images, making it easier to record measurements and see fine details at the enamel cervix or dentine horn tips. Although the synchrotron produces higher resolution images with less artifacts, results indicate that for gross morphological measurements (e.g., enamel cap volume, intercuspal distances), each of the scanners produces approximately the same measurements. Combining measurements of teeth from multiple microCT systems presupposes that measurements from each system are comparable; the research presented here indicates that this is the case when teeth are not severely diagenetically remineralized.     

From Lab to Field Conditions: A Pilot Study on EEG Methodology in Applied Sports Sciences

Although neurophysiological aspects have become more important in sports and exercise sciences in the last years, it was not possible to measure cortical activity during performance outside a laboratory due to equipment limits or movement artifacts in particular. With this pilot study we want to investigate whether Electroencephalography (EEG) data obtained in a laboratory golf putting performance differ from a suitable putting task under field conditions. Therefore, parameters of the working memory (frontal Theta and parietal Alpha 2 power) were recorded during these two conditions. Statistical calculations demonstrated a significant difference only for Theta power at F4 regarding the two putting conditions “field” and “laboratory”. These findings support the idea that brain activity patterns obtained under laboratory conditions are comparable but not equivalent to those obtained under field conditions. Additionally, we were able to show that the EEG methodology seems to be a reliable tool to observe brain activity under field conditions in a golf putting task. However, considering the still existing problems of movement artifacts during EEG measurements, eligible sports and exercises are limited to those being relatively motionless during execution. Further studies are needed to confirm these pilot results.     

Barnacle reattachment: a tool for studying barnacle adhesion

Standard approaches for measuring adhesion strength of fouling organisms use barnacles, tubeworms or oysters settled and grown in the field or laboratory, to a measurable size. These approaches suffer from the vagaries of larval supply, settlement behavior, predation, disturbance and environmental stress. Procedures for reattaching barnacles to experimental surfaces are reported. When procedures are followed, adhesion strength measurements on silicone substrata after 2 weeks are comparable to those obtained using standard methods. Hydrophilic surfaces require reattachment for 2-4 weeks. The adhesion strength of barnacles in reattachment assays was positively correlated to results obtained from field testing a series of experimental polysiloxane fouling-release coatings (r = 0.89). The reattachment method allows for precise barnacle orientation, enabling the use of small surfaces and the potential for automation. The method enables down-selection of coatings from combinatorial approaches to manageable levels for definitive field testing. Reattachment can be used with coatings that combine antifouling and fouling-release technologies.     

Water balance in Monodelphis domestica (Didelphidae) from the semiarid Caatinga of Brazil

1. Water conservation and energy metabolism in a semiarid-area, neotropical marsupial (Monodelphis domestica) were studied in the laboratory. 2. The rate of energy consumption in this species is low, corresponding with results reported for Australian and other neotropical marsupials. 3. Evaporative water loss rate is low and comparable to that of several desert small rodents, when body size differences are taken into account; however, the ratio of metabolic water production-to-evaporative water loss is lower than in the rodents. 4. Urine osmotic, urea, and chloride concentrations in water-stressed animals are within the range of values reported for some arid-area granivorous rodents, for insectivorous/carnivorous Australian marsupials, and for a North American insectivorous desert rodent. 5. Water-balance computations indicate that this species should be able to maintain water balance at 25 degrees C on a high-protein diet with a water content of about 60% without access to drinking water; this water requirement is comparable to that of ecologically similar marsupials and placentas in arid areas of other continents.     

Learning outcomes and student-perceived value of clay modeling and cat dissection in undergraduate human anatomy and physiology

Alternatives and/or supplements to animal dissection are being explored by educators of human anatomy at different academic levels. Clay modeling is one such alternative that provides a kinesthetic, three-dimensional, constructive, and sensory approach to learning human anatomy. The present study compared two laboratory techniques, clay modeling of human anatomy and dissection of preserved cat specimens, in the instruction of muscles, peripheral nerves, and blood vessels. Specifically, we examined the effect of each technique on student performance on low-order and high-order questions related to each body system as well as the student-perceived value of each technique. Students who modeled anatomic structures in clay scored significantly higher on low-order questions related to peripheral nerves; scores were comparable between groups for high-order questions on peripheral nerves and for questions on muscles and blood vessels. Likert-scale surveys were used to measure student responses to statements about each laboratory technique. A significantly greater percentage of students in the clay modeling group "agreed" or "strongly agreed" with positive statements about their respective technique. These results indicate that clay modeling and cat dissection are equally effective in achieving student learning outcomes for certain systems in undergraduate human anatomy. Furthermore, clay modeling appears to be the preferred technique based on students' subjective perceptions of value to their learning experience.     

Qualitative and quantitative differences in normal vaginal flora of conventionally reared mice, rats, hamsters, rabbits, and dogs

We examined quantitatively the vaginal flora of conventionally reared mice, rats, hamsters, rabbits and dogs, species that are widely used as laboratory animals. Vaginal specimens were examined according to the method of analyzing intestinal flora (Mitsuoka's procedure). The total number of bacteria (aerobes and anaerobes) and the prevalence of specific bacteria were determined. The total number of bacteria was highest during estrus and lowest during diestrus or anestrus in mice, rats, hamsters, and dogs. The most predominant bacteria during estrus were streptococci in mice; gram-negative rods (GNR), streptococci, and members of the family Bacteroidaceae in rats; GNR, Bacteroidaceae and gram-positive anaerobic cocci in hamsters, and Bacteroidaceae in dogs. The increase in the total number of bacteria during estrus was caused by an increase of predominant bacteria in the vagina. Aerobes were more predominant than anaerobes in mice, and number of aerobes was comparable to that of anaerobes in rats and dogs. On the other hand, in hamsters, anaerobes were more predominant than aerobes and the total number of bacteria was highest among the laboratory animals (mice, rats, hamsters, rabbits, and dogs). However, in rabbits, bacteria were not isolated from about 90% of the vaginal specimens. Rabbits do not have cyclic reproductive stages and are usually in precoital status in the laboratory. In precoital rabbits, vaginal epithelium manifests few signs of secretion. Therefore, we suspect that the vaginal environment in precoital rabbits is comparable to that during diestrus or anestrus in mice, rats, hamsters, and dogs. These results suggest that the vaginal flora of laboratory animals is influenced by the estrous cycle, and probably by mucous secretion. Our data imply that vaginal flora differ among laboratory animals species, and researchers need to take into consideration the estrous cycle of laboratory animals when studying their vaginal flora.     

Assessing the health of sediment ecosystems: use of chemical measurements

1. This paper outlines the use of chemical measurements as surrogate indicators of the health of sediment ecosystems. 2. For chemical measurements to be used for this purpose, links must be able to be made between adverse biological effects and the chemical composition of sediments. 3. Benchmark sediment quality guidelines allow decisions to be made about whether or not a sediment ecosystem is likely to be impacted. These guidelines are established using an effects database and a weight-of-evidence approach that gives ranges of chemical concentrations associated with known adverse biological effects. 4. If guidelines are exceeded the measured values are compared to concentrations of that contaminant in a comparable background or reference sediment. Exceedance of these concentrations leads to site-specific estimates of contaminant bioavailability. If a potential problem is indicated, laboratory sediment bioassays are used to assess the likelihood that the sediment will harm biota. 5. Trace metals are used as an example of a sediment contaminant.     

Denaturing RNA electrophoresis in TAE agarose gels

Current methods of analytical RNA electrophoresis are based on the utilization of either complicated laboratory instrumentation or toxic, carcinogenic, or expensive chemicals. We suggest here the use of classical Tris-acetate-ethylenediamine tetraacetic acid (TAE) agarose gels combined with prior denaturation of RNA samples in hot formamide for the electrophoretic separation of RNA species. We present a brief comparison of the proposed TAE/formamide method with the most common 3-(N-morpholino)propanesulfonic acid/formaldehyde agarose gel protocol and show that both methods produce comparable results for size determination of RNA molecules and subsequent Northern blotting of gels. In addition to purified RNA samples, the robustness of the TAE/formamide protocol is demonstrated by its suitability for the analysis of RNA quality in crude yeast cell lysates containing large amounts of proteins, DNA, and other contaminating molecules. We therefore propose the TAE/formamide agarose electrophoresis as a rapid, simple, and cheaper alternative to current methods of RNA electrophoresis. Additionally, another benefit is the reduced exposure of laboratory personnel to hazardous chemicals.     

Evidence for episodic secretion of testosterone in laboratory mice.

The concentration of testosterone (T) in the peripheral plasma of laboratory mice is extremely variable. This variability is already evident at 20--25 days of age and is not eliminated by brief or chronic exposure to male or female mice, or by isolation. The variation in T levels in plasma samples collected from the same animals on different occasions is comparable to the variation between individuals bled on a single occasion. The concentration of T in the testis is as variable as that in the peripheral plasma. It is suggested that in the laboratory mouse T is produced and released in an episodic fashion, that elevations in T levels in peripheral plasma of mice are greater than those observed in other species, and that testicular secretory episodes are interspersed with periods of minimal steroidogenic activity.     

Circadian rhythm does not affect responses to chemical cues in the red swamp crayfish, Procambarus clarkii

Studies of crayfish chemical ecology have been conducted in both day and night conditions. This variation may hinder the comparison of data among studies, if the responses by crayfish to chemical cues are dependent upon the time at which the cues are encountered. We tested the hypothesis that responses to chemical cues are dependent on observation time using the red swamp crayfish, Procambarus clarkii. Procambarus clarkii is known to exhibit a light-regulated circadian rhythm, with nocturnal activity peaks. Habitat use differed significantly between non-stimulated periods and periods of exposure to a food stimulus, but no effects of photoperiod (normal vs. reversed) or laboratory conditions (dark vs. light) were observed. The results suggest that, all else being equal, (1) studies of crayfish chemical ecology can be successfully conducted in a variety of experimental conditions, and (2) previous studies conducted at various times of the day should have comparable results.     

The combined determination of indolyl-3-acetic and abscissic acid in plant materials

A method for isoelectric focusing of antibodies in agarose gels with ampholytes synthesized in the laboratory from pentaethylenehexamine is presented. The ampholytes are easy to prepare, give results comparable to those with commercial ampholytes, and are much less expensive. Substituting agrarose bonded to plastic film for the polyacrylamide gels on glass plates commonly used offers many advantages and enhances the usefulness of isoelectric focusing as a tool for studying antibody molecules.     

The Suitability of Some Algae for Mass Cultivation for Food, with Special Reference to Dunaliella bioculata

Twenty-five strains of freshwater and saltwater algae have beeninvestigated for their suitability for mass cultivation forfood. Under laboratory conditions a strain of Dunaliella bioculatahas been found to give yields comparable with those obtainedfrom Chlorella. It is suggested that only when the growing algalcells are exposed in very thin layers will the yield not belimited by light penetration. The storage polysaccharide ofD. bioculata contains a 1:4-glucosan resembling starch. Allthe essential amino-acids have been shown to be present withthe possible exception of methionine or valine and tryptophane.     

Method repeatability for measuring Enterococcus in southern California beach sands

Aims: A recent study that evaluated 22 methods for enumerating faecal indicator bacteria in sand recommended standardization to a preferred method, but all researchers involved in that study had extensive experience in processing sand samples. The purpose of this study was to evaluate how well the recommended method can be transferred to laboratories without such experience. Methods and Results: Eight southern California laboratories that rarely measure bacteria in sand processed six sand and three water samples in replicates to assess repeatability. Among‐laboratory variability was found to be less than within‐laboratory variability, with no significant differences in results among any of the laboratories. Moreover, within‐laboratory variability was comparable between the sand and water samples, indicating that the elution procedure added little additional method error even when performed by laboratories without prior experience. Conclusions: The simple extraction method for enumerating Enterococcus in beach sands was easily transferable to and repeatable among laboratories with little or no prior experience. Significance and Impact of the Study: The demonstrated success of technology transfer will further demonstrate the success of method standardization and adoption, aiding in understanding of how sands affect surface water quality.