Factors affecting test reproducibility among laboratories.

Reproducibility is the ability of an assay to provide consistent results (when testing the same samples) in different laboratories. The validation of a new diagnostic assay should include specific assessment of assay reproducibility to determine the degree to which results are unaffected by minor changes in experimental conditions. Ideally, assessment of reproducibility involves the testing of identical samples in multiple laboratories by multiple analysts using the same method, reagents and controls, albeit with different equipment. Such an assessment will provide estimates of the precision and accuracy of an assay across laboratories. In reality, although the reproducibility of an assay is often assessed by multiple laboratories testing identical samples, the reagents, controls and testing platforms used, while similar, are usually not the same. Thus, reproducibility testing permits the assessment of variability resulting from different testing platforms, reagent supplies and operators. The determination of minor versus major variations in test conditions that may be anticipated in multi-laboratory use is part of the assessment at this stage of validation. Once validated, there are ongoing monitoring requirements to assess the performance characteristics and ensure they are consistently maintained. The use of quality assurance programmes is required, as this offers continued monitoring of assay performance by measuring the precision and accuracy of results for well-characterised samples and controls. Tests recommended by the World Organisation for Animal Health as fit for purpose are widely used internationally and need to have satisfactory reproducibility.

La reproductibilité d'un test désigne son aptitude à fournir des résultats constants (en testant les mêmes échantillons) lors d'analyses effectuées par différents laboratoires. Toute validation d'un nouvel essai diagnostique doit inclure une étape spécifique d'évaluation de la reproductibilité de l'essai, visant à vérifier jusqu'à quel point les résultats du test demeurent inchangés en cas de légères variations dans les conditions de réalisation de l'essai. Idéalement, l'évaluation de la reproductibilité consiste à soumettre des échantillons identiques à une même méthode d'essai réalisée dans plusieurs laboratoires par des analystes à chaque fois différents et en utilisant les mêmes réactifs et contrôles, mais avec des équipements différents. Une telle évaluation fournit une estimation de la précision et de l'exactitude d'un essai conduit par plusieurs laboratoires. Dans la pratique, si la reproductibilité d'un essai est souvent évaluée par des laboratoires différents à partir d'échantillons similaires, les réactifs, les contrôles et les plateformes d'essai ne sont généralement pas les mêmes d'un laboratoire à l'autre, bien qu'étant similaires. Les essais de reproductibilité permettent ainsi d'évaluer la variabilité induite par l'utilisation de plateformes de test et de réactifs différents par des opérateurs eux-mêmes différents. La détermination du caractère mineur ou substantiel des variations des conditions d'essai susceptibles d'être anticipées dans un cadre d'utilisation impliquant de nombreux laboratoires fait partie de l'évaluation à ce stade de la validation. Une fois la validation faite, des exigences de contrôle continu sont requises afin d'évaluer les caractéristiques de performances et de veiller à leur maintien dans le temps. Les laboratoires doivent se doter d'un programme d'assurance qualité qui garantisse le suivi continu des performances des essais à travers une mesure de la précision et de l'exactitude des résultats à partir d'échantillons et de contrôles bien caractérisés. Les tests dont l'aptitude à l'emploi selon l'objectif prévu a été établie et qui de ce fait sont recommandés par l'Organisation mondiale de la santé animale ont vocation à être utilisés dans le monde entier ; il est donc important qu'ils soient suffisamment reproductibles.

Se entiende por "reproducibilidad" el conjunto de características que permiten que un ensayo depare resultados uniformes al ser aplicado a las mismas muestras en laboratorios distintos. El proceso de validación de una prueba de diagnóstico debe incluir una evaluación específica de su reproducibilidad, a fin de determinar en qué medida los resultados se mantienen inalterados ante cambios menores de las condiciones experimentales. Lo idóneo para evaluar la reproducibilidad es que, en múltiples laboratorios, múltiples analistas sometan a prueba muestras idénticas empleando idénticos métodos, reactivos y controles, pero distinto equipo de laboratorio. Semejante evaluación permitirá estimar la precisión y exactitud que ofrece un ensayo en diferentes laboratorios. En realidad, aunque en la evaluación de la reproducibilidad de un ensayo intervienen a menudo múltiples laboratorios que analizan muestras idénticas, los reactivos, controles y dispositivos de prueba utilizados, aún siendo parecidos, no suelen los mismos. La realización de pruebas de reproducibilidad permite pues determinar la variabilidad que introducen distintos dispositivos de prueba, suministros de reactivos y técnicos de laboratorio. Así, la determinación de las variaciones menores frente a las variaciones importantes de las condiciones experimentales que cabe prever cuando múltiples laboratorios emplean un ensayo forma parte de la evaluación en esta fase del proceso de validación. Una vez validado un ensayo, hay una serie de requisitos de seguimiento continuo que sirven para evaluar las características de rendimiento y garantizar que se mantengan estables en el tiempo. Para ello es necesario utilizar programas de garantía de calidad, que ofrecen la posibilidad de hacer un seguimiento continuo del rendimiento de un ensayo cuantificando la precisión y exactitud de los resultados obtenidos con muestras y controles bien caracterizados. Las pruebas que la Organización Mundial de Sanidad Animal recomienda por considerarlas adaptadas a su finalidad, de uso muy extendido a escala internacional, deben presentar un nivel satisfactorio de reproducibilidad.

Enhanced surveillance of COVID-19 in Scotland: population-based seroprevalence surveillance for SARS-CoV-2 during the first wave of the epidemic.

OBJECTIVES: The impact of the COVID-19 pandemic in Scotland has been amongst the most severe in Europe. Serological surveillance is critical to determine the overall extent of infection across populations and to inform the public health response. This study aimed to estimate the proportion of people who have antibodies to SARS-CoV-2 ('seroprevalence') in the general population of Scotland and to see if this changes over time. STUDY DESIGN/METHODS: Between International Organization for Standardization (ISO) week 17 (i.e. week commencing 20th April) and ISO week 25 (week commencing 15 June), 4751 residual blood samples were obtained from regional biochemistry laboratories in six participating regional health authority areas covering approximately 75% of the Scottish population. Samples were tested for the presence of anti-SARS-CoV-2 IgG antibodies using the LIAISON®SARS-CoV-2 S1/S2 IgG assay (DiaSorin, Italy). Seroprevalence rates were adjusted for the sensitivity and specificity of the assay using Bayesian methods. RESULTS: The combined adjusted seroprevalence across the study period was 4.3% (95% confidence interval: 4.2%-4.5%). The proportion varied each week between 1.9% and 6.8% with no difference in antibody positivity by age, sex or geographical area. CONCLUSIONS: At the end of the first wave of the COVID-19 pandemic, only a small fraction of the Scottish population had antibodies to SARS-CoV-2. Control of COVID-19 requires the ability to detect asymptomatic and mild infections that would otherwise remain undetected through existing surveillance systems. This is important to determine the true number of infections within the general population which, in turn, can help to understand transmission, inform control measures and provide a denominator for the estimation of severity measures such as the proportion of infected people who have been hospitalised and/or have died.

National population prevalence of antibodies to SARS-CoV-2 in Scotland during the first and second waves of the COVID-19 pandemic.

OBJECTIVES: Studies that measure the prevalence of antibodies to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ('seroprevalence') are essential to understand population exposure to SARS-CoV-2 among symptomatic and asymptomatic individuals. We aimed to measure seroprevalence in the Scottish population over the course of the COVID-19 pandemic - from before the first recorded case in Scotland through to the second pandemic wave. STUDY DESIGN: The study design of this study is serial cross sectional. METHODS: We tested 41,477 residual samples retrieved from primary and antenatal care settings across Scotland for SARS-CoV-2 antibodies over a 12-month period from December 2019-December 2020 (before rollout of COVID-19 vaccination). Five-weekly rolling seroprevalence estimates were adjusted for the sensitivity and specificity of the assays and weighted to reference populations. Temporal trends in seroprevalence estimates and weekly SARS-CoV-2 notifications were compared. RESULTS: Five-weekly rolling seroprevalence rates were 0% until the end of March, when they increased contemporaneously with the first pandemic wave. Seroprevalence rates remained stable through the summer (range: 3%-5%) during a period of social restrictions, after which they increased concurrently with the second wave, reaching 9.6% (95% confidence interval [CI]: 8.4%-10.8%) in the week beginning 28th December in 2020. Seroprevalence rates were lower in rural vs. urban areas (adjusted odds ratio [AOR]: 0.70, 95% CI: 0.61-0.79) and among individuals aged 20-39 years and 60 years and older (AOR: 0.74, 95% CI: 0.64-0.86; AOR: 0.80, 95% CI: 0.69-0.91, respectively) relative to those aged 0-19 years. CONCLUSIONS: After two waves of the COVID-19 pandemic, less than one in ten individuals in the Scottish population had antibodies to SARS-CoV-2. Seroprevalence may underestimate the true population exposure as a result of waning antibodies among individuals who were infected early in the first wave.

National population prevalence of antibodies to SARS-CoV-2 among pregnant women in Scotland during the second wave of the COVID-19 pandemic: a prospective national serosurvey.

OBJECTIVES: This study aimed to determine SARS-CoV-2 seroprevalence among pregnant women in the Scottish population during the second wave of the COVID-19 pandemic. STUDY DESIGN: Prospective national serosurvey. METHODS: We tested 13,428 residual samples retrieved from pregnant women participating in the first trimester combined ultrasound and biochemical screening for fetal trisomy across Scotland for SARS-CoV-2 antibodies over a 6-month period from November 2020 to April 2021. Seroprevalence estimates were adjusted for the sensitivity and specificity of the assays and weighted to reference populations. RESULTS: Seroprevalence rates in the antenatal samples significantly increased from 5.5% (95% confidence interval [CI] 4.7%-6.5%) in the 5-week period up to and including International Organization for Standardization (ISO) Week 51 (w/b Monday 14 December 2020) to 11.3% (95% CI 10.1%-12.6%) in the 5-week period up to and including ISO Week 14 (w/b Monday 5 April 2021). Increasing seroprevalence trends across the second wave were observed among all age groups. CONCLUSIONS: By the end of the second wave of the COVID-19 pandemic, approximately one in 10 women tested around the end of the first trimester of pregnancy had antibodies to SARS-CoV-2, suggesting that the vast majority were still susceptible to COVID-19 as they progressed to the later stages of pregnancy, when risks from infection are elevated for both mother and baby.

Recurrent outbreaks of mumps in Lothian and the impact of waning immunity.

Another large outbreak of mumps occurred in Lothian from October 2017, which coincided with the commencement of the higher education term. During this period 324 cases were notified, most of whom were aged 18-22 years old. Although previous outbreaks had a focus in student populations, 43% of current cases reported that they were not a student. There has been increases in private student housing where students from all universities live, which may have contributed to the wide spread of the outbreak and complicated outbreak control. Information on vaccination status was available for 244 cases (75%), of whom the majority (75.8%) reported having two MMR doses. To investigate potential waning vaccine immunity the mean length of time since last mumps containing vaccine was calculated as 14.3 years. The outbreak was declared over in May 2018 after case numbers returned to background levels. This outbreak highlighted that mumps outbreaks occur cyclically coinciding with new cohorts of susceptible students entering the Lothian population. The lessons from this outbreak are to encourage students to have two MMR doses and also be prepared for mumps outbreaks in the near future. In future outbreaks the utility of a third MMR for outbreak control could be examined.

A nonmyeloablative chimeric mouse model accurately defines microglia and macrophage contribution in glioma.

AIMS: Resident and peripherally derived glioma associated microglia/macrophages (GAMM) play a key role in driving tumour progression, angiogenesis, invasion and attenuating host immune responses. Differentiating these cells' origins is challenging and current preclinical models such as irradiation-based adoptive transfer, parabiosis and transgenic mice have limitations. We aimed to develop a novel nonmyeloablative transplantation (NMT) mouse model that permits high levels of peripheral chimerism without blood-brain barrier (BBB) damage or brain infiltration prior to tumour implantation. METHODS: NMT dosing was determined in C57BL/6J or Pep3/CD45.1 mice conditioned with concentrations of busulfan ranging from 25 mg/kg to 125 mg/kg. Donor haematopoietic cells labelled with eGFP or CD45.2 were injected via tail vein. Donor chimerism was measured in peripheral blood, bone marrow and spleen using flow cytometry. BBB integrity was assessed with anti-IgG and anti-fibrinogen antibodies. Immunocompetent chimerised animals were orthotopically implanted with murine glioma GL-261 cells. Central and peripheral cell contributions were assessed using immunohistochemistry and flow cytometry. GAMM subpopulation analysis of peripheral cells was performed using Ly6C/MHCII/MerTK/CD64. RESULTS: NMT achieves >80% haematopoietic chimerism by 12 weeks without BBB damage and normal life span. Bone marrow derived cells (BMDC) and peripheral macrophages accounted for approximately 45% of the GAMM population in GL-261 implanted tumours. Existing markers such as CD45 high/low proved inaccurate to determine central and peripheral populations while Ly6C/MHCII/MerTK/CD64 reliably differentiated GAMM subpopulations in chimerised and unchimerised mice. CONCLUSION: NMT is a powerful method for dissecting tumour microglia and macrophage subpopulations and can guide further investigation of BMDC subsets in glioma and neuro-inflammatory diseases.

Imported case of measles in a university setting leading to an outbreak of measles in Edinburgh, Scotland from September to December 2016.

In September 2016, an imported case of measles in Edinburgh in a university student resulted in a further 17 confirmed cases during October and November 2016. All cases were genotype D8 and were associated with a virus strain most commonly seen in South East Asia. Twelve of the 18 cases were staff or students at a university in Edinburgh and 17 cases had incomplete or unknown measles mumps rubella (MMR) vaccination status. The public health response included mass follow-up of all identified contacts, widespread communications throughout universities in Edinburgh and prompt vaccination clinics at affected campuses. Imported cases of measles pose a significant risk to university student cohorts who may be undervaccinated, include a large number of international students and have a highly mobile population. Public health departments should work closely with universities to promote MMR uptake and put in place mass vaccination plans to prevent rapidly spreading measles outbreaks in higher educational settings in future.

Impact of rest duration on Achilles tendon structure and function following isometric training.

Intervention programs are often sought to strengthen the Achilles tendon (AT) due to its high injury rate. Long rest periods between loading cycles have been found to increase collagen synthesis by tenocytes, suggesting rest duration may be important for tendon adaptation in vivo; however, exercise programs comparing long and short rest duration have not been directly compared. Fourteen adults completed a 12-week progressive training intervention; training sessions consisted of 5×10 isometric plantarflexion contractions each of 3-s duration performed at 90% of MVC three times weekly. Each leg was randomly allocated to long (LRT, 10-s rest) or short rest training (SRT, 3-s rest). We hypothesized that the leg allocated to LRT would demonstrate superior AT collagen organization compared to the leg receiving SRT, which would be related to improved biomechanical function. AT collagen organization and morphology were measured using ultrasound tissue characterization. AT properties were assessed before and after the intervention using a combination of dynamometry, ultrasound imaging, EMG, and motion capture. Contrary to our hypothesis, collagen organization did not improve following either training protocol; conversely, an unexpected decrease in echotype I proportion was seen after SRT (P<.001) but not LRT (P=.58), indicating an apparent protective effect of rest on collagen organization during isometric training. In contrast, AT adaptation was not appreciably enhanced by increasing intercycle rest duration; both protocols were equally effective at inducing significant strength gains and AT mechanical and material adaptation (P≤.001). Further research is necessary to identify optimal loading characteristics for injury prevention and rehabilitation.

An epidemiological study of A. cantonensis in Jamaica subsequent to an outbreak of human cases of eosinophilic meningitis in 2000.

The infection status of angiostrongylosis in Jamaica was assessed in wild rats and molluscs in the 5 years following the major outbreak of eosinophilic meningitis (EM) in 2000. Parasitological analyses of 297 Rattus rattus and 140 Rattus norvegicus, and 777 terrestrial molluscs from all 14 Parishes on the island revealed Angiostrongylus cantonensis in 32·0% of the rats and in 12·5% of the molluscs. Multivariate analyses confirmed that A. cantonensis occurred significantly more frequently in R. rattus (Odds Ratio [OR] = 1·76), while mean infection intensity in R. rattus was also significantly higher (16·8) than R. norvegicus (11·3) (Mann-Whitney U-test: P = 0·01). Third-stage larvae of A. cantonensis were detected in 29% of 86 Pleurodonte spp.; in 20% of five Poteria spp.; in 18·7% of 369 Thelidomus asper; in 11% of 18 Sagda spp.; and in 6% of 24 veronicellid slugs. Most rodent infections occurred in Northeastern Jamaica (OR = 11·66), a region where infected molluscs were also abundant. Given the prevalence of A. cantonensis infection in rats has significantly increased since the 2000 outbreak, and that a survey of human infections revealed at least ten autochthonous cases in the last 15 years, angiostrongylosis persists as an important zoonosis in Jamaica.

In vivo biological response to extracorporeal shockwave therapy in human tendinopathy.

Extracorporeal shock wave therapy (ESWT) is a non-invasive treatment for chronic tendinopathies, however little is known about the in-vivo biological mechanisms of ESWT. Using microdialysis, we examined the real-time biological response of healthy and pathological tendons to ESWT. A single session of ESWT was administered to the mid-portion of the Achilles tendon in thirteen healthy individuals (aged 25.7 ± 7.0 years) and patellar or Achilles tendon of six patients with tendinopathies (aged 39.0 ± 14.9 years). Dialysate samples from the surrounding peri-tendon were collected before and immediately after ESWT. Interleukins (IL)-1ß, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-17A, vascular endothelial growth factor and interferon-γ were quantified using a cytometric bead array while gelatinase activity (MMP-2 and -9) was examined using zymography. There were no statistical differences between the biological tissue response to ESWT in healthy and pathological tendons. IL-1ß, IL-2, IL-6 and IL-8 were the cytokines predominantly detected in the tendon dialysate. IL-1ß and IL-2 did not change significantly with ESWT. IL-6 and IL-8 concentrations were elevated immediately after ESWT and remained significantly elevated for four hours post-ESWT (p < 0.001). Pro-forms of MMP-2 and -9 also increased after ESWT (p < 0.003), whereas there were no significant changes in active MMP forms. In addition, the biological response to ESWT treatment could be differentiated between possible responders and non-responders based on a minimum 5-fold increase in any inflammatory marker or MMP from pre- to post-ESWT. Our findings provide novel evidence of the biological mechanisms underpinning ESWT in humans in vivo. They suggest that the mechanical stimulus provided by ESWT might aid tendon remodelling in tendinopathy by promoting the inflammatory and catabolic processes that are associated with removing damaged matrix constituents. The non-response of some individuals may help to explain why ESWT does not improve symptoms in all patients and provides a potential focus for future research.

Plantarflexor stretch training increases reciprocal inhibition measured during voluntary dorsiflexion.

Agonist-mediated reciprocal inhibition (RI) in distal skeletal muscles is an important neurophysiological phenomenon leading to improved movement coordination and efficiency. It has been shown to be reduced in aged and clinical populations, so the development of interventions augmenting RI is an important research goal. We examined the efficacy of using chronic passive muscle stretching to augment RI. The influence of 3 wk of plantarflexor stretching (4 × 30 s, two times/day) on RI of soleus and gastrocnemius initiated by tonic, voluntary dorsiflexion contractions [20% of maximum voluntary contraction (MVC)] was examined in 11 healthy men who performed stretch training and in nine nontraining controls. Hoffmann's reflexes (H-reflexes) were elicited by tibial nerve stimulation during both weak isometric (2% MVC) plantarflexions and dorsiflexion contractions at 20% MVC. Changes were examined at three joint angles, normalized to each subject's range of motion (ROM; plantarflexed = 10 ± 0°, neutral = -3.3 ± 2.9°, dorsiflexed = -16.5 ± 5.6°). No changes were detected in controls. A 20% increase in ROM in the stretch subjects was associated with a significant decrease in maximum H-reflex (H(max)): maximum evoked potential (M(max)), measured during 2% plantarflexion at the plantarflexed and neutral angles in soleus and at the plantarflexed angle in gastrocnemius (P < 0.05-0.01). By contrast, decreases in H(max):M(max) during 20% dorsiflexion contract were also seen at each angle in soleus and at the dorsiflexed angle in gastrocnemius. However, a greater decrease in H(max):M(max) measured during voluntary dorsiflexion rather than during plantarflexion, which indicates a specific change in RI, was detected only at the dorsiflexed angle (-30.7 ± 9.4% and -35.8 ± 6.8% for soleus and gastrocnemius, respectively). These results demonstrate the efficacy of soleus-gastrocnemius stretch training in increasing agonist-mediated RI from tibialis anterior onto soleus-gastrocnemius in young, healthy individuals at dorsiflexed, but not plantarflexed, joint angles.

Age-related changes in mechanical properties of the Achilles tendon.

The stiffness of a tendon, which influences muscular force transfer to the skeleton and increases during childhood, is dependent on its material properties and dimensions, both of which are influenced by chronic loading. The aims of this study were to: (i) determine the independent contributions of body mass, force production capabilities and tendon dimensions to tendon stiffness during childhood; and (ii) descriptively document age-related changes in tendon mechanical properties and dimensions. Achilles tendon mechanical and material properties were determined in 52 children (5-12 years) and 19 adults. Tendon stiffness and Young's modulus (YM) were calculated as the slopes of the force-elongation and stress-strain curves, respectively. Relationships between stiffness vs. age, mass and force, and between YM vs. age, mass and stress were determined by means of polynomial fits and multiple regression analyses. Mass was found to be the best predictor of stiffness, whilst stress was best related to YM (< 75 and 51% explained variance, respectively). Combined, mass and force accounted for up to 78% of stiffness variation. Up to 61% of YM variability could be explained using a combination of mass, stress and age. These results demonstrate that age-related increases in tendon stiffness are largely attributable to increased tendon loading from weight-bearing tasks and increased plantarflexor force production, as well as tendon growth. Moreover, our results suggest that chronic increases in tendon loading during childhood result in microstructural changes which increase the tendon's YM. Regarding the second aim, peak stress increased from childhood to adulthood due to greater increases in strength than tendon cross-sectional area. Peak strain remained constant as a result of parallel increases in tendon length and peak elongation. The differences in Achilles tendon properties found between adults and children are likely to influence force production, and ultimately movement characteristics, which should be explicitly examined in future research.

Substantial Achilles adaptation following strength training has no impact on tendon function during walking.

Tendons are responsive to mechanical loading and their properties are often the target of intervention programs. The tendon's mechanical properties, particularly stiffness, also govern its function, therefore changes to these properties could have substantial influence on energy-saving mechanisms during activities utilizing the stretch-shortening cycle. We investigated Achilles tendon (AT) function in vivo during walking with respect to a training intervention that elicited significant increases in AT stiffness. 14 men and women completed 12-weeks of isometric plantarflexor strength training that increased AT stiffness, measured during isometric MVC, by ~31%. Before and after the intervention, participants walked shod at their preferred velocity on a fully-instrumented treadmill. Movement kinematics, kinetics and displacement of the gastrocnemius medialis muscle-tendon junction were captured synchronously using 3D motion capture and ultrasound imaging, respectively. A MANOVA test was used to examine changes in AT force, stress, strain, stiffness, Young's modulus, hysteresis and strain energy, measured during walking, before and following strength training. All were non-significant for a main effect of time, therefore no follow-up statistical tests were conducted. Changes in joint kinematics, tendon strain, velocity, work and power and muscle activity during the stance phase were assessed with 1D statistical parametric mapping, all of which also demonstrated a lack of change in response to the intervention. This in vivo examination of tendon function in walking provides an important foundation for investigating the functional consequences of training adaptations. We found substantial increases in AT stiffness did not impact on tendon function during walking. AT stiffness measured during walking, however, was unchanged with training, which suggests that increases in stiffness may not be evident across the whole force-elongation relation, a finding which may help explain previously mixed intervention results and guide future investigations in the functional implications of tendon adaptation.

Flow cytometric analysis of intercellular adhesion between B-cell precursor acute lymphoblastic leukemic cells and bone marrow stromal cells.

The growth of B-cell precursor acute lymphoblastic leukemic (BCP ALL) cells in vitro is dependent on interactions with bone marrow (BM) stromal cells. We have recently demonstrated that the rate of cell division of BCP ALL cells increases when cultured in direct contact with BM stromal cells. In this study we describe a new method for examining the direct binding of BM stromal cells and BCP ALL cells at a cellular level. For this binding assay, BCP ALL cells from six patient samples were first stained with the lipophilic fluorescent probe PKH 26 GL and mixed with BM stromal cells in suspension. In all cases, aggregates between BCP ALL and BM stromal cells were identified by flow cytometry and isolated. Using this assay we have examined some of the mechanisms involved in this binding process. The pattern of aggregate formation at various leukemic/stromal cell ratios showed that the aggregate formation increased by increasing the number of either cell type and that the binding could not be saturated. This suggests that the interaction between these cells is an equilibrium reaction. Functional studies showed that the majority of BCP ALL-BM stromal cell binding is dependent on the presence of divalent cations and requires active cellular metabolism. Finally, by use of inhibitory monoclonal antibodies (moAbs) directed against cell adhesion molecules including anti-CD29, VCAM and CD18, we have demonstrated that the involvement of these molecules in the direct cellular interactions could be detected by this method. However, the maximum inhibition observed was 36% which suggests either that the avidity is low or that other adhesion molecules are involved. The data show that the use of flow cytometric analysis of aggregate formation (rather than cell binding to intact cell layers) allows the study of cell interactions at the individual cell level which can reveal additional cellular adhesion mechanisms.

A method for in situ absolute DD yield calibration of neutron time-of-flight detectors on OMEGA using CR-39-based proton detectors.

Neutron time of flight (nTOF) detectors are used routinely to measure the absolute DD neutron yield at OMEGA. To check the DD yield calibration of these detectors, originally calibrated using indium activation systems, which in turn were cross-calibrated to NOVA nTOF detectors in the early 1990s, a direct in situ calibration method using CR-39 range filter proton detectors has been successfully developed. By measuring DD neutron and proton yields from a series of exploding pusher implosions at OMEGA, a yield calibration coefficient of 1.09 ± 0.02 (relative to the previous coefficient) was determined for the 3m nTOF detector. In addition, comparison of these and other shots indicates that significant reduction in charged particle flux anisotropies is achieved when bang time occurs significantly (on the order of 500 ps) after the trailing edge of the laser pulse. This is an important observation as the main source of the yield calibration error is due to particle anisotropies caused by field effects. The results indicate that the CR-39-nTOF in situ calibration method can serve as a valuable technique for calibrating and reducing the uncertainty in the DD absolute yield calibration of nTOF detector systems on OMEGA, the National Ignition Facility, and laser megajoule.

A novel approach to the measurement of different in vitro leukaemic cell growth parameters: the use of PKH GL fluorescent probes.

The application of the fluorescent cell membrane probes PKH2 and PKH 26 GL in the measurement of leukaemic cell growth was examined on four cell lines K562, NALM-6, ACV (a pre-B cell line) and HL-60 using flow cytometry. As the amount of probe per cell reduces at each cell division, the fluorescence can be used to measure cell proliferation. By measuring the mean fluorescence intensity of the cells at the beginning of culture and at various time points, and by combining this information with a viable cell count, it was possible to determine: (1) the number of viable cells; (2) their rate of proliferation; (3) their number of cell divisions; and (4) the maintenance of cells in a viable state over a period of time. It was demonstrated that these parameters could be reliably established using the red fluorescent probe PKH26 GL. In contrast, the green fluorescent probe PKH2 GL showed dye transfer resulting in an underestimation of the number of cell divisions and an overestimation of the maintenance of cells in a viable state. The potential advantages of the use of PKH26 GL over conventional assays for the measurement of leukaemic cell growth parameters are discussed.

Posture determines the nature of the interaction between angiotensin converting enzyme inhibitors and loop diuretics in patients with chronic cardiac failure.

The effects of inhibition of the renin angiotensin aldosterone system on the natriuretic and diuretic actions of an intravenous dose of frusemide 40 mg in patients with chronic cardiac failure maintained on oral diuretics were studied in the supine and erect positions. In the patients studied in the supine position the total 4 hour diuresis was decreased from 995 (92) ml to 668 (66) ml and the total 4 hour natriuresis fell from 105 (14) mmol to 67 (14) mmol following the administration of captopril. Creatinine clearance fell from 87 (8) ml/minute to 52 (15) ml/minute. In the patients studied in the erect position the total 4 hour diuresis was 596 (87) ml without captopril and 562 (83) ml with captopril. Total 4 hour natriuresis was 71 (13) mmol without captopril and 65 (9) mmol with captopril. Creatinine clearance was reduced by captopril from 82 (7) ml/minute to 47 (12) ml/minute. The reduction in the diuretic and natriuretic response to frusemide caused by captopril in the supine position is mediated through a fall in glomerular filtration rate. However, in the erect position, which is associated with even further increases in activity of the renin angiotensin aldosterone system, the reduction in diuresis and natriuresis that a fall in glomerular filtration rate would cause is offset by abolition of the rise in sodium retaining hormones, angiotensin II and aldosterone that mediate the antinatriuretic effect of the erect position.

Detoxification enzyme activities (CYP1A1 and GST) in the skin of humpback whales as a function of organochlorine burdens and migration status.

The activities of glutathione-s-transferase (GST) and cytochrome P-450 1A1 (CYP1A1) enzymes were measured in freshly extracted epidermis of live-biopsied, migrating, southern hemisphere humpback whales (Megaptera novaeangliae). The two quantified enzyme activities did not correlate strongly with each other. Similarly, neither correlated strongly with any of the organochlorine compound groups previously measured in the superficial blubber of the sample biopsy core, likely reflecting the anticipated low levels of typical aryl-hydrocarbon receptor ligands. GST activity did not differ significantly between genders or between northward (early migration) or southward (late migration) migrating cohorts. Indeed, the inter-individual variability in GST measurements was relatively low. This observation raises the possibility that measured activities were basal activities and that GST function was inherently impacted by the fasting state of the sampled animals, as seen in other species. These results do not support the implementation of CYP1A1 or GST as effective biomarkers of organochlorine contaminant burdens in southern hemisphere populations of humpback whales as advocated for other cetacean species. Further investigation of GST activity in feeding versus fasting cohorts may, however, provide some insight into the fasting metabolism of these behaviourally adapted populations.

Effects of resistance training on tendon mechanical properties and rapid force production in prepubertal children.

Children develop lower levels of muscle force, and at slower rates, than adults. Although strength training in children is expected to reduce this differential, a synchronous adaptation in the tendon must be achieved to ensure forces continue to be transmitted to the skeleton with efficiency while minimizing the risk of strain-related tendon injury. We hypothesized that resistance training (RT) would alter tendon mechanical properties in children concomitantly with changes in force production characteristics. Twenty prepubertal children (age 8.9 ± 0.3 yr) were equally divided into control (nontraining) and experimental (training) groups. The training group completed a 10-wk RT intervention consisting of 2-3 sets of 8-15 plantar flexion contractions performed twice weekly on a recumbent calf-raise machine. Achilles tendon properties (cross-sectional area, elongation, stress, strain, stiffness, and Young's modulus), electromechanical delay (EMD; time between the onset of muscle activity and force), rate of force development (RFD; slope of the force-time curve), and rate of electromyographic (EMG) increase (REI; slope of the EMG time curve) were measured before and after RT. Tendon stiffness and Young's modulus increased significantly after RT in the experimental group only (∼29% and ∼25%, respectively); all other tendon properties were not significantly altered, although there were mean decreases in both peak tendon strain and strain at a given force level (14% and 24%, respectively; not significant) which may have implications for tendon injury risk and muscle fiber mechanics. A decrease of ∼13% in EMD was found after RT for the experimental group, which paralleled the increase in tendon stiffness (r = -0.59); however, RFD and REI were unchanged. The present data show that the Achilles tendon adapts to RT in prepubertal children and is paralleled by a change in EMD, although the magnitude of this change did not appear to be sufficient to influence RFD. These findings are of importance within the context of the efficiency and execution of movement.

Range of motion, neuromechanical, and architectural adaptations to plantar flexor stretch training in humans.

The neuromuscular adaptations in response to muscle stretch training have not been clearly described. In the present study, changes in muscle (at fascicular and whole muscle levels) and tendon mechanics, muscle activity, and spinal motoneuron excitability were examined during standardized plantar flexor stretches after 3 wk of twice daily stretch training (4 × 30 s). No changes were observed in a nonexercising control group (n = 9), however stretch training elicited a 19.9% increase in dorsiflexion range of motion (ROM) and a 28% increase in passive joint moment at end ROM (n = 12). Only a trend toward a decrease in passive plantar flexor moment during stretch (-9.9%; P = 0.15) was observed, and no changes in electromyographic amplitudes during ROM or at end ROM were detected. Decreases in H(max):M(max) (tibial nerve stimulation) were observed at plantar flexed (gastrocnemius medialis and soleus) and neutral (soleus only) joint angles, but not with the ankle dorsiflexed. Muscle and fascicle strain increased (12 vs. 23%) along with a decrease in muscle stiffness (-18%) during stretch to a constant target joint angle. Muscle length at end ROM increased (13%) without a change in fascicle length, fascicle rotation, tendon elongation, or tendon stiffness following training. A lack of change in maximum voluntary contraction moment and rate of force development at any joint angle was taken to indicate a lack of change in series compliance of the muscle-tendon unit. Thus, increases in end ROM were underpinned by increases in maximum tolerable passive joint moment (stretch tolerance) and both muscle and fascicle elongation rather than changes in volitional muscle activation or motoneuron pool excitability.