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Determination of percentage of RoundUp Ready soya in soya flour using real-time polymerase chain reaction: interlaboratory study.

An interlaboratory study was conducted to evaluate a method for determination of the percentage of RoundUp Ready (RR) soya in soya flour using Taqman technology. The method included DNA extraction from the test portion with cetyltrimethylammonium bromide buffer followed by chloroform extraction and Wizard resin cleanup steps. The DNA was then assayed with primer and probe sets specific for lectin as the endogenous control and the RR insert as the target. The percentage of RR soya in the soya fraction of the sample was calculated by using a matrix-matched standard curve. Ten samples of split-level blind duplicates were sent to 22 laboratories in 12 countries worldwide. Test portions contained 0, 0.5, 0.7,1.6, 2, and 3.9% (w/w) RR soya prepared gravimetrically from commercially available RR standard reference materials. Based on the results for test materials, the relative standard deviation for repeatability (RSDr) for the method ranged from 9.3 to 19.3% and, for reproducibility (RSDR), ranged from 20.3 to 33.7%.

DNA, Plant↗

Age-associated changes in quantitative ultrasonometry (QUS) of the os calcis in Lebanese women-assessment of a Lebanese reference population.

OBJECTIVE: This study was aimed to assess age changes in quantitative ultrasonometry (QUS) in a large sample of Lebanese women to determine a Lebanese reference population. DESIGN: Cross-sectional study. SUBJECTS AND METHODS: Broadband ultrasound attenuation (BUA) and speed of sound (SOS) and the stiffness index (SI) of the os calcaneus was measured in 4,320 women with a mean age of 52.5 years (age range 20 to 79 years) using three identical Achilles Express (GE/Lunar) and one Achilles Plus (GE/Lunar) ultrasonometry devices. Women were randomly selected and asked to participate in a nationwide screening program using the media, conferences, telephone calls etc. Measurements were performed at Red Cross centers located all over the country. No inclusion or exclusion criteria were used. RESULTS: There was an overall decline of 19.2% for BUA, 3.1% for SOS and 30.3% for SI between late adolescence and old age. In premenopausal women, BUA decreased only slightly by 3%, while postmenopausal women showed a significant decline of 16.2%. In contrast, SOS continuously decreased from the age of 42; there was a decline of 0.8% from adolescence to the menopause; postmenopausal women showed a larger decline of 2.4%. The SI of premenopausal women decreased by 6%, while postmenopausal women showed a significantly larger decline of 24.3%. SI value for the female Lebanese young adult reference is 8% lower than that of the American and European women (92 SI units compared to 100). At the age of 42, SI value for the Lebanese women is 10.4% lower than the American women and 7.5% lower than the European women (86 SI units compared to 96 and 93, respectively). At the age of 75, SI values for the Lebanese women is 4.4% lower than the American women and the European women (65 SI units compared to 68). The decline in stiffness index for the Lebanese women between age 20 and 75 years is about 30.3% compared to 32% for the American or European reference curves. The rate of decrease for the Lebanese women was 0.2 SI units per year for the premenopausal period, and 0.7 SI units per year for the postmenopausal period. CONCLUSION: The age-related female, Lebanese reference curve was significantly different from the American and the European reference curves used by the manufacturer. Therefore, the use of our standardized reference data instead of the proposed US or European database reduces the risk of overestimating osteoporosis in the Lebanese population. The impact of our results on the prevalence of osteoporotic fracture in Lebanon has to be evaluated later on.

Journal Article↗

Differential expression of c-myc protein in B and T lymphocytes.

Overexpression of c-myc may play a role in the multistep pathogenesis of B- and T-cell malignancies. To determine whether this expression is inappropriate requires information on the normal cellular counterparts. There is no agreement in the literature on the levels of expression of c-myc mRNA and protein in normal peripheral blood lymphocytes and there are no reports on the differential expression in different lymphocyte populations. The aim of this study was to assess the state of c-myc expression in normal peripheral blood lymphocytes at the single cell level by immunocytochemistry and flow cytometry. Two monoclonal antibodies against c-myc and specific peptide inhibition controls were tested in mononuclear cells from nine healthy volunteers and the HL60 cell line. The expression of c-myc in B- and T-lymphocyte subsets was studied by two-colour immunocytochemistry and flow cytometry. Using calibrated reference standards, we quantified the c-myc protein and results were referred as molecules of equivalent soluble fluorochrome. Almost all lymphocytes express c-myc by both techniques. Two patterns of nuclear staining (weak and strong) were found by immunocytochemistry and this was confirmed by two peaks of fluorescence intensity by flow cytometry. Double immunostaining showed that the stronger pattern of c-myc staining corresponds to B lymphocytes and the weak one to T cells. Quantification confirmed these results which demonstrated a statistically significant difference in the expression of c-myc in these two lymphocyte populations (p < 0.005). Our results demonstrate for the first time that normal circulating B cells express higher levels of c-myc protein than T lymphocytes.

Antibodies, Monoclonal↗

Simultaneous quantitation of Krebs cycle and related acids by mass fragmentography.

Methods are described for simultaneous quantitation of Krebs cycle and related acids by gas chromatography--mass spectrometry using deuterium-labelled acids and n-butyl-d9-esters of the organic acids as internal standards. Using sulphuric acid as esterification catalyst, only lactic, succinic, fumaric, malic, maleic and citric acids were found to be stable to hydrogen exchange and could be used as reference standards in the deuterated form. In contrast, pyruvic, oxalacetic, alpha-ketoglutaric and malonic acids were found to exchange their deuterium readily and could not be employed for this purpose. All the acids could be quantitated using n-butyl-d9-esters of reference organic acids as internal standards, following a separate preparation of the n-butyl derivatives of the unknown acids. The method is suitable for routine analysis of organic acids at the picogram level in perchloric acid extracts of tissues.

Adipates↗

Wavelength and alignment tests for confocal spectral imaging systems.

Confocal spectral imaging (CSI) microscope systems now on the market delineate multiple fluorescent proteins, labels, or dyes within biological specimens by performing spectral characterizations. However, we find that some CSI present inconsistent spectral profiles of reference spectra within a particular system as well as between related and unrelated instruments. We also find evidence of instability that, if not diagnosed, could lead to inconsistent data. This variability confirms the need for diagnostic tools to provide a standardized, objective means of characterizing instability, evidence of misalignment, as well as performing calibration and validation functions. Our protocol uses an inexpensive multi-ion discharge lamp (MIDL) that contains Hg+, Ar+, and inorganic fluorophores that emit distinct, stable spectral features, in place of a sample. An MIDL characterization verifies the accuracy and consistency of a CSI system and validates acquisitions of biological samples. We examined a total of 10 CSI systems, all of which displayed spectral inconsistencies, enabling us to identify malfunctioning subsystems. Only one of the 10 instruments met its optimal performance expectations. We have found that using a primary light source that emits an absolute standard "reference spectrum" enabled us to diagnose instrument errors and measure accuracy and reproducibility under normalized conditions. Using this information, a CSI operator can determine whether a CSI system is working optimally and make objective comparisons with the performance of other CSI systems. It is evident that if CSI systems of a similar make and model were standardized to reveal the same spectral profile from a standard light source, then researchers could be confident that real-life experimental findings would be repeatable on any similar system.

Animals↗

Standardization of flow microfluorometers using glutaraldehyde fixed lymphocytes.

The preparation of glutaraldehyde fixed lymphocytes as a reference standard in flow microfluorometry is described. The ease of preparation, the stability of the cells over time, their ability to stain reproducibly in the range expected of fresh human peripheral blood and bone marrow cells and, most importantly, the fact that they may be used to resolve optimally and situate correctly the G1 peak of normal mammalian cells, make them a reliable tool for the calibration of flow microfluorometers and for detection of aneuploid populations.

Fluorometry↗

Candidate reference methods for determining target values for cholesterol, creatinine, uric acid, and glucose in external quality assessment and internal accuracy control. I. Method setup.

In Germany, the target values for External Quality Assessment (EQA) and internal accuracy control are determined by Reference Methods for several analytes, including cholesterol, creatinine, uric acid, and glucose. We present candidate Reference Methods for these compounds, based on isotope dilution-gas chromatography--mass spectrometry methods that have been developed at INSTAND, one of the two official Germany EQA reference institutions. Each Reference Method target value is calculated from six independent measurements performed on three different days. The mean method CVs ranged from 0.66% for glucose to 0.96% for creatinine. The inaccuracy (bias) of the methods is < 0.7%, as compared with the Standard Reference Material 909 of the National Institute of Standards and Technology. The maximum total error of a Reference Method value, including the 95% confidence interval and systematic errors, is < 2.3%. The presented candidate Reference Methods have been successfully used to set target values in the German EQA scheme and the internal accuracy control of routine laboratories.

Blood Glucose↗

Proteinase activity of human renin preparations: the International Reference Preparation (Renin Standard 68/356).

1. Several commonly used preparations of human renin, including the International Reference Preparation (Renin Standard 68/356), were examined for the presence of contaminating proteinase activity by using a 14C-glycinated bovine haemoglobin substrate assay. 2. All of the human renin preparations tested cleaved haemoglobin even in the presence o di-isopropylfluorophosphate and ethylenediaminetetra-acetate (EDTA). For a given amount of renin activity, varying amounts of proteinase activity were seen. The pH optimum also varied between preparations. 3. Small peptide inhibitors of human renin were not able to inhibit the proteinase activity. Furthermore a diazoacyl reagent and pepstatin, both potent inhibitors of aspartic acid-active site proteinases, were only partially inhibitory. 4. These and other observations suggest that the proteinase activity of the human renin preparations is not due to renin itself, but to contaminating proteinases of different types. Since these enzymes may produce angiotensin I or peptides which may interfere in renin assays, crude preparations of renin which contain proteinase activity, including the International Reference Preparation, should be used with caution or replaced by proteinase-free human renin which can be easily prepared by use of suitable affinity chromatography.

Chromatography, Affinity↗

Local diagnostic reference levels in standard X-ray examinations.

The national diagnostic reference levels (NDRLs) form an efficient, concise and powerful standard for optimising radiation protection of a patient. However, in a large hospital, where many radiological departments are present, it is also possible to calculate and define lower dose values as local diagnostic reference levels (LDRLs). In our hospital there are eight radiological departments; in each of these, the entrance skin dose (ESD) distributions were determined for 10 standard projections (AP Abdomen, PA and LAT Chest, AP and LAT Lumbar Spine, LAT Lumbo-Sacral Joint, AP Pelvis, PA and LAT Skull and AP Urinary tract) and then the ESDs were compared with data previously published and with Italian NDRLs. All ESD values were below the corresponding NDRLs. The maximum/minimum ratio of ESDs ranged from 3.9 (LAT Skull) to 34.3 (AP Abdomen) for individual adult patients and from 2.1 (PA Skull) to 6.5 (Urinary tract) across the mean values of the radiological departments. Finally, it is shown how LDRLs can be proposed to obtain a more fully optimised radiation protection of patients.

Child, Preschool↗

Growth in childhood thyrotoxicosis.

UNLABELLED: Childhood thyrotoxicosis is an uncommon condition. To investigate the effect of thyrotoxicosis on the growth of children and to detect possible influence of the disease on their final height, 105 Chinese children (90 girls; 15 boys) with thyrotoxicosis were studied longitudinally from diagnosis. At presentation their mean age was 11. 57 years. Their height and weight measurements were converted to standard deviation scores (SDS) using normal height and weight-for-height reference standards for Chinese children established in Hong Kong. Their mean height SDS at diagnosis was +0. 73. Bone age assessment at diagnosis was done in 48 girls and 8 boys and their mean +/- S.D. bone development quotient was 1.16 +/- 0.11. A total of 53 girls have reached adult height and their mean height was 161.3 cm, corresponding to a SDS of +0.63. Their final heights significantly exceeded their target heights. CONCLUSION: This study demonstrates that children with thyrotoxicosis were tall for age and their bone ages were advanced at presentation. They continued to be tall for age after starting treatment and they achieved final heights exceeding their target height.

Adolescent↗

Toxicological screening.

Well-defined dose- and time-related toxic effects of chemicals can often be detected using simple tests with small numbers of animals. The strategies for the establishment of toxicological screening tests are discussed. The most important steps are the definition of the targets, the selection of the methods, and the setting of the test criteria. Screening tests must then be validated with standard reference chemicals, and the test criteria must be adjusted so that the standards can be detected regularly. Maximal flexibility is allowed in the design of the tests. For evaluation, the results obtained in treated animals can be compared with those of controls, using conventional concepts of biostatistics. It is also possible to base the evaluation on preset test criteria. Toxicological screening tests do not replace conventional safety studies, but they help in selecting the most promising candidates in a series of related chemicals and in establishing priorities for further testing. As an example, a screening for the hemolytic effects of chemicals is presented.

Drug Evaluation, Preclinical↗

Determination of copper levels in serum of healthy subjects by atomic absorption spectrometry.

Copper levels in serum samples of 84 healthy subjects living in southeastern Spain were determined using the flame atomic absorption spectrometry technique. Mineralization of samples was carried out with an HNO3/HClO4 (4:1) mixture in a thermostated mineralization block. The accuracy of the method was tested by using a standard reference material. A mean recovery percentage of 104.70% was obtained. The relative standard deviation (R.S.D.) as a measurement of the precision of the method was lower than 5% in the concentration range considered. Mean copper concentrations were 1.092 +/- 0.365 mg/l (with the range 0.304-2.000 mg/l) and 1.113 +/- 0.253 mg/l (corresponding to an interval of 0.648-1.760 mg/l) for women and men, respectively. There are no significant differences between the copper levels in serum according to either sex or geography zone (P > 0.05). For example, there were no differences of copper levels in serum of subjects from coastal and mountainous zones. Estimated daily dietary intakes on copper in women and men were 1.38 and 2.10 mg Cu/day respectively.

Copper↗

Ultrasonic fetal weight estimation and tolerance to measurement error: a comparative analysis.

The objective of the present study was to test the hypothesis that volumetric formulae for fetal weight estimation are more tolerant to measurement error than exponential models. A mathematical model of normal fetal growth was constructed, using published British reference standards for biometric variables. Observed measurements were computer-generated by contaminating reference measurements with error terms according to their published coefficients of variation. The error in weight estimation was computed as the percentage difference between weight estimates derived from the observed biometric variables and the true measurements. A total of nine weight estimation formulae were tested. Campbell's formula appeared to be most affected by observational errors, especially before 38 weeks. In this range, they varied up to 9%. The most tolerant was Shepard's formula, with errors of only approximately 2.8%. Other formulae showed errors of approximately 5-6%. With the exception of Campbell's formula, the effect of gestational age was minimal. There was no correlation between percentage error and fetal size. Combining ultrasound biometric variables into a fetal weight estimate does not always exaggerate the errors of the original measurements. There were no significant differences between volumetric and exponential formulae.

Female↗

Multivariate predictive models for group A beta-hemolytic streptococcal pharyngitis in children.

OBJECTIVES: To create predictive models for the clinical diagnosis of group A beta-hemolytic streptococcal (GABHS) pharyngitis in children. METHODS: Patients aged 6 months to 18 years presenting to a pediatric ED with suspected GABHS pharyngitis were prospectively enrolled in the study. Clinicians recorded pertinent clinical information using a standardized form and obtained a throat swab to culture GABHS using a reference standard method. Twelve demographic and clinical features of patients with positive throat cultures were compared with the features of patients with negative throat cultures. Significantly different features were entered in a stepwise logistic regression analysis to create predictive models for the diagnosis. RESULTS: Eighty-five patients (29%) were culture-positive and 212 (71%) were culture-negative for GABHS. Respective mean ages were 6.2 years and 6.1 years in the two groups. Univariate chi-square analysis of the 12 features identified six variables that were significantly associated with GABHS. All significant features were initially included in a stepwise logistic regression analysis. In model I, four independent variables were identified: moderate to severe presentation of tonsillar swelling, moderate to severe tenderness and enlargement of cervical lymph nodes, the presence of scarlatiniform rash, and the absence of moderate to severe coryza, yielding a 95% probability for GABHS. Excluding the rare scarlatiniform rash, the remaining variables were used in the second regression analysis. In model II, three independent variables were identified: moderate to severe tonsillar swelling, moderate to severe tenderness and enlargement of cervical lymph nodes, and absence of moderate to severe coryza, yielding a probability of 65% for the diagnosis. A probability of <15% was observed in the absence of scarlatiniform rash, the absence of moderate to severe tenderness and enlargement of cervical lymph nodes, and the presence of moderate to severe coryza. CONCLUSIONS: In children with moderate to severe presentation of tonsillar swelling, tenderness and enlargement of cervical lymph nodes, and the absence of coryza, the probability of a positive throat culture is >65%. Conversely, in the absence of a moderate to severe presentation of tonsillar swelling, enlargement of cervical nodes, and the presence of coryza, the probability of a positive throat culture is <15%. If prospectively validated, these models could be integrated into a consistent treat, test, and no treatment/no testing approach to the clinical management of childhood pharyngitis.

Adolescent↗

Hypertension in pregnancy and size at birth.

Low birth weight and high placental weight are associated with increased cardiovascular morbidity and mortality. Children born after hypertensive pregnancies have higher blood pressure than children born after normotensive pregnancies. Hypertension in pregnancy is considered a major risk factor for intrauterine growth retardation. The present study describes size at birth and perinatal characteristics in children born after hypertensive pregnancies. During five consecutive years 17,000 deliveries took place in the well defined geographic area of eastern Göteborg. Hypertension in pregnancy complicated 261 pregnancies. For comparison 260 normotensive pregnancies, matched for maternal age and time of delivery, were used. Standard deviation score for birth weight and length were calculated according to reference standards for birth weight (BWSDS) and length (BLSDS) based on all Swedish births 1977-1981 (n = 475,588). Children born after hypertensive pregnancies had lower birth weight, were shorter and had a shorter gestational period as compared with children born after normotensive pregnancies. Head circumference and placental weight did not differ and there were no significant differences in BWSDS. It is concluded that hypertensive pregnancies are characterized by lower birth weight and shorter gestational period. However, intrauterine growth retardation is not a general characteristic of hypertension in pregnancy.

Adolescent↗

Assessment of hypertension based on chronoengineering.

For the study of blood pressure, the use of automatic fully ambulatory recording systems greatly facilitates data collection and, in combination with proper software, the interpretation of results. Hardware for automatic long-term measurement is here wedded to a set of software modules to assess the circadian variability of human blood pressure. To evaluate changes in blood pressure within a day, the authors analyzed 303 series of systolic and diastolic blood pressures and heart rates automatically monitored every 30 minutes for at least 48 hours. Subjects were clinically healthy men and women, 19-23 years of age. Individual circadian rhythm parameters computed for each time series by least-squares rhythmometry were used, first, to assess rhythm characteristics of all subjects and for each gender separately, and, second, to compute a so-called paradesm, a (here 90%) time-specified prediction region for the amplitude-acrophase pair. The limits thus obtained for the circadian amplitude (an index of within-day variability) of blood pressure are exceeded in cases of amplitude-hypertension. Additionally, the original data were also used to compute time-specified tolerance limits for blood pressure and heart rate in clinical health. By the use of these reference standards many false positive and false negative diagnoses can be eliminated. Such reference limits may serve for an objective and positive definition of health, for the screening and diagnosis of disease, and for gauging a subject's response to treatment. The combined hardware-software method for the establishment of time-qualified tolerance limits presented allows the assessment of the extent and timing of rhythmic change and can be used for the interpretation of single values obtained at specified times.

Adult↗

Overview, conclusions, and recommendations of the IPCS collaborative study on complex mixtures.

The International Programme on Chemical Safety (IPCS) sponsored an international collaborative study to examine the variability associated with the extraction and bioassay of standard reference materials (SRMs) that are complex environmental mixtures provided by the U.S. National Institute of Standards and Technology (NIST). The study was also intended to evaluate the feasibility of establishing bioassay reference values and ranges for the SRMs. Twenty laboratories from North America, Europe, and Japan participated in the study. As part of the mandatory core protocol, each laboratory extracted the organic material from two particulate samples and bioassayed these extracts. A coal tar polycyclic aromatic hydrocarbon (PAH) solution and two mutagenic control compounds were also subjected to bioassay without prior extraction by the participating laboratories. The bioassay used was the Salmonella/microsomal plate incorporation assay. For the optional portion of the study, a laboratory was free to use the SRMs for any type of exploratory research. The primary purpose of the required portion of the study was to estimate the intra- and inter-laboratory variability in mutagenic potencies of the test materials and to determine whether or not the NIST mixtures could be used as reference materials by others performing the Salmonella assay. Repeatability (intra-laboratory variance) of the bioassay results ranged from 16% to 88% depending on the SRM and the bioassay conditions (tester strain and metabolic activation), whereas reproducibility (inter-laboratory variance) ranged from 33% to 152%. Between-laboratory variability was the main source of variation accounting for approximately 55-95% of the total variation for the three environmental samples. Variation in the mutagenic potency of the control compounds was comparable, with the exception of 1-nitropyrene for which the reproducibility ranged from 127% to 132%. In summary, NIST SRMs provided useful materials for an international inter-laboratory study of complex mixtures. By establishing both intra- and inter-laboratory variance for the mutagenicity results for these materials, the usefulness of these SRMs as reference materials for the Salmonella bioassay was established, critical procedures within the bioassay protocol were identified, and recommendations for future efforts were delineated.

International Cooperation↗