Comparison of several control standard endotoxins to the National Reference Standard Endotoxin--an HIMA collaborative study.

A collaborative study, initiated under the auspices of the Health Industry Manufacturers Association (HIMA), was designed to establish the relationship of Escherichia coli O55:B5 endotoxin (the control standard endotoxin of HIMA and the Food and Drug Administration's Office of Medical Devices) to the U.S. National Reference Standard Endotoxin and to two internationally used control standard endotoxins. By using two Limulus amoebocyte lysate test systems, it was established that the E. coli O55:B5 endotoxin lot originally used by HIMA and the Office of Medical Devices to establish Limulus amoebocyte lysate release test criteria for pyrogen testing of medical devices contains approximately 4.5 endotoxin units (EU) per ng. Thus, the 1.0-ng/kg endotoxin dose limit currently established for medical devices is approximately the same as the 5.0-EU/kg endotoxin limit (on an activity basis) established by several other Food and Drug Administration agencies for human and animal parenteral drugs and biological products.     

A collaborative study to establish the 1st national standard of prekallikrein activator in Korea

The aim of this study was to establish the first national standard for prekallikrein activator (PKA) calibrating to the first international standard PKA. A collaborative study among five laboratories, including three manufacturers and two national control laboratories, was carried out to evaluate the suitability of a candidate to serve as a national standard of PKA. The candidate was manufactured in GMP facility following approved human serum albumin fractionation procedure and freeze-dried 5% albumin solution containing PKA. Participants were provided with sufficient samples and asked to use lab-made prekallikrein substrate prepared in accordance with European Pharmacopeia and also to use a commercial prekallikrein provided as part of the study. The PKA concentration of the candidate was 61.8 IU per vial using lab-made prekallikrein. However, the concentration was 54.2 IU per vial using commercial prekallikrein. The variability obtained at each laboratory ranged from 1.9% to 5.1% for within-a-day and from 5.6% to 9.0% for day-to-day. The candidate showed excellent stability from accelerated degradation study and real-time stability study. As a conclusion, the candidate preparation was suitable to serve as a Korean National Standard for PKA.     

A collaborative study to establish an International Standard Rabies immunoglobulin of human origin

Because the supply of the International Standard for Anti-rabies Serum was very low, the WHO initiated a search for a replacement product. The US Food and Drug Administration agreed to undertake a collaborative study using a human rabies immunoglobulin previously purchased for use as a US standard. The potency of this product was determined, in International Units (IU) per millilitre using the rapid fluorescent focus inhibition test for measuring rabies antibody. The mean potency value was found to be 59 IU per ampoule. In June 1984 this preparation was accepted by WHO as the International Standard for Rabies Immunoglobulin.     

Report of a collaborative study to calibrate the Second International Standard for parvovirus B19 antibody

A collaborative study was undertaken to assess the suitability of a replacement for the First International Standard for parvovirus B19 IgG, human serum and to calibrate it in IU. The proposed standard, which is a pool of sera from 16 US blood donors, was assayed along with the First International Standard, a coded duplicate of the proposed standard and a plasma sample from a single blood donor. Nine laboratories from eight countries participated in the studies and five different assay kits were used. Two kits contained VP1+VP2, one kit contained VP1 only and two kits, one of which was used by five participants contained VP2 only. Differences in detection of the proposed standard and the individual plasma were observed with assay kits containing different antigens, VP1, VP2 or VP1+VP2. However, since VP1 is a minor capsid protein and on its own does not assemble into virus like particles and the dominant response in individuals appears to be against VP2, it was considered reasonable to utilize only the data from kits containing VP2 antigen for the calibration of the proposed standard. The results of this study demonstrated that the proposed standard coded 01/602 was suitable to serve as the replacement International Standard for parvovirus B19, serum IgG, and this preparation was established as the Second International Standard for parvovirus B19 antibody, plasma human, with an assigned unitage of 77 IU per ampoule by the Expert Committee on Biological Standardisation of the World Health Organisation in February 2003.     

A collaborative study to establish the International Reference Reagent for hepatitis B vaccine containing plasma-derived hepatitis B surface antigen

A collaborative study was conducted to establish a suitable international reference reagent for hepatitis B vaccine for use in immunogenicity assays. The limiting dilution required to induce antibodies in 50% of the test animals was determined for the proposed international reference reagent and three other plasma-derived hepatitis B vaccines. The minimum antigenic dose of these preparations varied widely (100-fold range) between laboratories. However, the expression of potencies of vaccines relative to the proposed International Reference Reagent reduced the variation between laboratories to within a 10-fold range. The reference reagent is intended for use in assays of hepatitis B vaccines in mouse (or guinea-pig) immunogenicity studies. For products made by different procedures, clinical trials in humans are necessary to establish a correlation between the immunogenic potency in animals and man.     

An international collaborative study on the First International Standard of bovine luteinizing hormone for immunoassay

An ampouled freeze-dried preparation of bovine pituitary luteinizing hormone (bLH), coded EHC-bLH-1, has been evaluated in an international collaborative study and shown to be suitable and sufficiently stable to serve as a standard for bLH. Eight laboratories provided immunoassay data, one laboratory provided receptor assay data, and bioassay data were obtained from 4 laboratories. The geometric mean potency estimate obtained by immunoassays, expressed as milliunits of the USDA bLH-B-5 preparation per ampoule, was 25.6, which is consistent with the result obtained by in-vivo bioassays. The geometric mean estimate obtained by receptor assays or by in-vitro bioassays was lower, i.e. 13.2 milliunits per ampoule. The reason for this discrepancy is currently under investigation. With the authorization of the Expert Committee on Biological Standardization of the World Health Organization this preparation was established in 1985 as the International Standard for Luteinizing Hormone, Bovine, for Immunoassay with a unitage of 25 mi.u. per ampoule.     

Reference values of selenium in plasma in population from Barcelona. Comparison with several pathologies

Plasma selenium reference values from healthy donors in the metropolitan area of Barcelona are determined. A random sample from 156 healthy adults (control group) is analysed by using electrothermal atomic absorption spectrometry with Zeeman effect background correction.

The relationship between several pathologies and Se content is also evaluated. Se content from 64 samples from subjects with chronic renal failure and 54 from subjects suffering from several malignancies are determined and the results are compared to the reference values. Moreover, Se contents are determined and compared in two groups of children, healthy (19 samples) and children of mothers infected with HIV-1 (16 samples).

In the control group, Se plasma concentration ranges between 50 and 145 μg · L⁻¹ (82.2 ± 17.5 μg · L⁻¹). Significantly lower values are found in the two pathologies studied (malignancy and chronic renal failure), compared to the control group. However, no significant differences in Se content are found between the two groups studied regarding malignancy and chronic renal failure.

In children of mothers infected with HIV-1, Se status is significantly lower than that of healthy children.     

International collaborative study by in vitro bioassays of the first international standard for porcine inhibin.

A lyophilized preparation of inhibin from porcine ovarian follicular fluid, ampoule code 86/690, was made internationally available as a research standard for in vitro bioassays in 1987. A study involving ten participants in eight countries assessed the stability and suitability of this research standard to serve as an international standard. Each of the participants used in vitro assays, the majority of which depended upon the inhibition of release of follicle-stimulating hormone from dispersed rat anterior pituitary cells. The research standard 86/690 was compared with coded ampoules of 86/690 stored under conditions of accelerated thermal degradation and with inhibins from different species. Intra- and interlaboratory variation for estimates of potency of a coded duplicate ampoule of the research standard provided the basis for comparisons of non-identical inhibins, but the fourfold variability of potency estimates for identical ampoules was such that no conclusions about the differences seen for non-identical inhibins could be made. Predictions of stability from consensus estimates of potency of ampoules that have undergone accelerated thermal degradation indicated that the research standard had satisfactory stability. On the basis of this study, the research standard 86/690 was deemed sufficiently stable and suitable to serve as a standard for in vitro bioassays and was established by the World Health Organization Expert Committee on Biological Standardization as the First International Standard for Porcine Inhibin. The possible presence, in biological extracts (standard or sample), of other bioactive proteins, such as activin and follistatin, complicates the quantitative interpretation of bioassay data. A standard of highly purified human inhibin is now required as a standard for immunoassays used for clinical research purposes; sufficient quantities of recombinant human inhibins have recently been donated for ampouling and evaluation by bio- and immunoassay in the subsequent phase of the standardization of inhibins.     

A collaborative assay of the proposed third British Reference Preparation for Pertussis Vaccine and of the relative potencies of the second International Standard and the second British Reference Preparation for Pertussis Vaccine

A collaborative assay has been carried out to estimate the mouse protective potency of a freeze-dried preparation of Bordetella pertussis (88/522) intended to serve as the third British Reference Preparation for Pertussis Vaccine (third BRP). The opportunity was also taken of reassessing the relationship between the second International Standard for Pertussis Vaccine and the second British Reference Preparation for Pertussis Vaccine (second BRP). Workers in nine laboratories took part in the study and together completed 19 assays which were considered to be statistically valid. Based on the results of the study it is proposed that ampouled preparation code number 88/522 be established as the third BRP with an assigned potency of 50 IU per ampoule. The evidence of this study also suggests that the relationship between the second International Standard for Pertussis Vaccine and the second BRP has not changed significantly since they were originally established.     

Rabies vaccine standardization: International collaborative study for the characterization of the fifth international standard for rabies vaccine

A collaborative study was carried out to establish a replacement for the International Standard for Rabies Vaccine, the stocks of which are exhausted. Three rabies vaccines for human use derived from different rabies virus strains and prepared on different cell culture substrates were compared with the International Standard for Rabies Vaccine using in vivo and in vitro assay methods in a collaborative study involving 14 participants. The proposed fifth International Standard (PISRAV) which was derived from the same virus strain as the present international standard preparation, the Pitman Moore (PM) strain, was found to be approximately twice as potent relative to the International Standard in immunogenicity assays as in antigenicity assays. On the other hand another vaccine, derived from the LEP strain, was considerably more potent in antigenicity assays than in immunogenicity assays. The glycoprotein of the proposed replacement standard measured in antigenicity assays appeared to be stable at +37 degrees C for 245 days, whereas the immunogenicity of the proposed replacement vaccine was sensitive to this heat treatment and the vaccine lost 66% of its immunogenic potency. The results of this study indicate that the NIH protection test should continue to form the primary basis for potency assay of rabies vaccine as glycoprotein content does not appear to correlate with immunogenic potency for different types of vaccine. The vaccine coded PISRAV has been established as the fifth International Standard for Rabies Vaccine and a potency of 16 International Units of Rabies Vaccine (based on the immunogenicity assays) assigned to the contents of each ampoule. Each ampoule has also been assigned a unitage of 10 IU of PM Rabies Virus Glycoprotein and 135 IU of PM Rabies Virus Ribonucleoprotein.     

Development of a Common Oligonucleotide Reference Standard for Microarray Data Normalization and Comparison across Different Microbial Communities

High-density functional gene arrays have become a powerful tool for environmental microbial detection and characterization. However, microarray data normalization and comparison for this type of microarray remain a challenge in environmental microbiology studies because some commonly used normalization methods (e.g., genomic DNA) for the study of pure cultures are not applicable. In this study, we developed a common oligonucleotide reference standard (CORS) method to address this problem. A unique 50-mer reference oligonucleotide probe was selected to co-spot with gene probes for each array feature. The complementary sequence was synthesized and labeled for use as the reference target, which was then spiked and cohybridized with each sample. The signal intensity of this reference target was used for microarray data normalization and comparison. The optimal amount or concentration were determined to be ca. 0.5 to 2.5% of a gene probe for the reference probe and ca. 0.25 to 1.25 fmol/μl for the reference target based on our evaluation with a pilot array. The CORS method was then compared to dye swap and genomic DNA normalization methods using the Desulfovibrio vulgaris whole-genome microarray, and significant linear correlations were observed. This method was then applied to a functional gene array to analyze soil microbial communities, and the results demonstrated that the variation of signal intensities among replicates based on the CORS method was significantly lower than the total intensity normalization method. The developed CORS provides a useful approach for microarray data normalization and comparison for studies of complex microbial communities.Microarray-based technology has become a robust genomic tool to detect, track, and profile hundreds to thousands of different microbial populations simultaneously in complex environments such as soils and sediments. For example, GeoChip, a comprehensive functional gene array, has been developed for investigating biogeochemical, ecological, and environmental processes (12, 18, 23, 27, 29, 32). Although a massive amount of microarray data can be generated rapidly, one of the bottlenecks in using microarrays for environmental microbial community studies is the lack of an appropriate standard for data comparison and normalization (6). Currently, it is difficult to compare microarray data across different sites, experiments, laboratories, and/or time periods (10). This limits the power of the technology to address ecological and environmental questions.In pure culture-based functional genomics studies, genomic DNAs (gDNAs) have been used as a common reference for hybridizations in which the same amount of gDNAs are used to cohybridize with each target cDNA sample and then to normalize different target cDNAs based on the gDNA standard (4, 5, 8, 9, 19, 21, 23). Several normalization methods such as scale normalization, quantile normalization, and Lowess normalization have been used for gene expression studies (2). Using the gDNA standard method can minimize or eliminate differences in target cDNA quantity, spot morphology, uneven hybridization, labeling, and sequence-specific hybridization behaviors (5), and this allows the comparison of microarray data across different sites, laboratories, experiments, and/or times. The main rationale for gDNA as a common reference is that it provides complete coverage for all genes represented on the array because the DNA composition from a particular organism should be identical across different treatment samples even though RNA expression is different (8). However, this approach is not applicable to microbial community studies because not all communities have identical DNA compositions. Pooling of equal amounts of gDNA or RNA from every target sample to make a common sample could be used as an alternative reference for cohybridization (1, 22). However, the disadvantage of the sample pooling approach is that samples do not provide large amounts of DNA or RNA in a reliable and reproducible way. For example, groundwater samples usually have a very low biomass and thus would not provide enough DNA for pooling. In addition, the sample pool itself is uncharacterized, and gene abundance may be diluted out so that insufficient DNA is present to result in a positive signal some array features, especially for those genes in low abundance. Moreover, a new sample pool would be required for every new experiment, making comparison across experiments difficult. Thus, other approaches need to be developed for microbial community studies.Dudley et al. (7) used a 25-mer oligonucleotide that matched a small portion of the parental EST clone vector contained in every PCR product printed on the array for normalization of pure culture RNA expression. Although the oligonucleotide generated a stable hybridization signal on every array feature, this method requires a universal sequence tag as a “capture” sequence, limiting its general use in microbial community studies. Thus, in the present study, we developed a common oligonucleotide reference standard (CORS) approach by co-spotting a common oligonucleotide with each array feature to improve the accuracy and comparability of microarray data for microbial community studies. This method was evaluated by using a pilot array, a whole-genome array, and a functional gene array, and all results demonstrate that the developed CORS is a reliable and reproducible method for microarray data normalization and comparison for microbial community studies.     

Comparison of surface potentials due to several singularity representations of the human heart

In electrocardiography the electrical potentials due to the heart actions can be analyzed by assuming the human body to be a conductor of homogeneous medium and the heart to be a combination of singularities within it. For a spherical conductor the “interior sphere theorem” of G. Ludford, J. Martinek, and G. Yeh (Proc. Cambridge Phil. Soc.,51, 389–93, 1955) renders potential expressions due to any singularity. For a conductor of prolate spheroidal shape the potential expressions due to a source-sink pair and a general dipole have been given by J. R. Wait (Jour. App. Physics,24, 496–97, 1953) and the authors (paper at the Conference on the Electrophysiology of the Heart, Feb. 16–17, 1956, in New York, to appear in theAnn. N. Y. Acad. Sciences) respectively. (A theorem which applies to any singularity inside a prolate or oblate spheroid will be published by the authors soon). This paper presents numerical and graphical results of potentials on the surfaces of a prolate spheroid and a sphere due to source-sink pairs and dipoles of several locations and directions and compares the various representations. A discussion on the judicious choice of heart models concludes the paper. This investigation was supported by The National Heart Institute under a research grant H-2263.     

标准品制备方法对FQ-PCR定量基因表达水平的影响

实时荧光定量PCR (FQ-PCR)标准曲线法准确定量基因表达的关键在于标准品与待检样本的扩增效率是否一致. 为检测DNA标准品与样本cDNA扩增效率的一致性,探讨定量用标准品的最佳制备方法,本研究以脂肪酸结合蛋白5(Fabp5)、过氧化物酶体增殖活化受体α (Ppar-α)及β肌动蛋白（β-Actin）的3个基因为对象,分别采用质粒纯化法、PCR产物直接纯化法、PCR产物凝胶回收法制备DNA标准品,10倍梯度稀释后用FQ PCR制作标准曲线. 并以10倍梯度稀释的样本cDNA标准曲线的参数为对照,进行比较分析. 结果表明,不同方法制备的DNA标准品的扩增效率差异较大,并且与cDNA的扩增效率不一致,不能对cDNA样本进行准确定量. 另外,虽然目的基因在cDNA样本中的拷贝未知,不能对基因表达水平进行绝对定量,但因不同cDNA样本的同一基因的扩增效率一致, 可对基因的表达进行准确的相对定量.     

Comparison of 5 Thyroid Ultrasound Stratification Systems for Differentiation of Benign and Malignant Nodules and to Avoid Biopsy Using Histology as Reference Standard

ObjectiveWe aimed to compare the thyroid ultrasound risk stratification systems (RSSs) of the American College of Radiology (ACR) Thyroid Imaging Reporting and Data System (TI-RADS), European TI-RADS, Korean TI-RADS, and American Thyroid Association (ATA), American Association of Clinical Endocrinologists, American College of Endocrinology, and Associazione Medici Endocrinologi guidelines to differentiate benign from malignant thyroid nodules and to avoid unnecessary fine needle aspiration (FNA).MethodsThe records of 1143 nodules ≥1 cm that underwent FNA biopsy and thyroidectomy between 2012 and 2020 at our institution were reviewed. Ultrasound categories and FNA recommendation indications of 5 international RSSs were compared with histopathological findings as benign or malignant. The ultrasound categories and recommended FNA indications, the proportion of the avoidable FNA procedures, and false negative rates (FNRs) by different systems were compared with each other.ResultsOf the 1143 nodules, 45% had thyroid malignancy. FNA recommendation and ultrasound risk classification of ATA guidelines had the highest area under curves of 0.619, and 0.715, respectively. ACR TI-RADS, American Association of Clinical Endocrinologists/American College of Endocrinology/Associazione Medici Endocrinologi guidelines, European TI-RADS, ATA guidelines, and Korean TI-RADS would have avoided FNA for 34.7%, 31%, 25.7%, 20%, and 6% of nodules with an FNR of 24%, 28.5%, 22%, 7.2%, and 1.9%, respectively.ConclusionOur findings showed that all RSSs classified the nodules appropriately for malignancy. ATA guidelines had the highest area under curves and a low FNR, whereas ACR TI-RADS would have spared more patients from FNA with a high FNR.     

台湾地区全民健保下分级诊疗制度之借鉴

借鉴我国台湾地区全民健保下分级诊疗制度,落实和完善现行分级诊疗制度以保证医疗资源的合理配置。利用文献分析,对比研究两岸不同卫生体制下分级诊疗制度实施现状,结合专家访谈的形式明确台湾分级诊疗制度的特点。通过建立短期医疗网计划、成立社区医疗群、改革医保补偿机制、构筑医联体等方式优化分级诊疗制度。     

Assessing Diagnostic Tests: How to Correct for the Combined Effects of Interpretation and Reference Standard

We describe a general solution to the problem of determining diagnostic accuracy without the use of a perfect reference standard and in the presence of interpreter variability. The accuracy of a diagnostic test is typically determined by comparing its outcomes with those of an established reference standard. But the accuracy of the standard itself and those of the interpreters strongly influence such assessments. We use our solution to examine the effects of the properties of the standard, the reliability of the interpreters, and the prevalence of abnormality on the measured sensitivity and specificity. Our results provide a method of systematically adjusting the measured sensitivity and specificity in order to estimate their true values. The results are validated by simulations and their detailed application to specific cases are described.     

International collaborative study to assess candidate reference preparations to control the level of anti-D in IVIG for use in Europe and the United States.

Regulatory requirements to control the level of anti-D in intravenous immunoglobulin (IVIG) products with European and United States (US) licences are to be introduced. A reference preparation of IVIG containing anti-D at 0.0475 IU/ml and having a nominal titre of 8 using the proposed direct haemagglutination reference method was deemed suitable to define the anti-D limit. This preparation, code 02/228, and a negative control IVIG preparation, code 02/226, were established by the World Health Organization as International Reference Reagents (IRRs). As stocks of the IRRs are limited, new larger fill stocks of positive and negative reference preparations, codes 04/132 and 04/140, respectively, were produced. The results from an international collaborative study involving 16 laboratories showed that preparations 04/132 and 04/140 are indistinguishable from the corresponding IRRs 02/228 and 02/226, respectively, using the proposed direct haemagglutination reference method. Stocks of 04/132 and 04/140 have been shared with the European Directorate for the Quality of Medicines (re-coded as 23613 and 23614, respectively) and with the Center for Biologics Evaluation and Research of the United States Food and Drug Administration (re-coded as CBER Lots 1B and 1N-b, respectively) for use as European and US Biological Reference Preparations, respectively.     

美国国家生物工程食品信息披露标准法案评析

美国“国家生物工程食品信息披露标准”法案出台的主要目的是统一转基因食品标识立法,避免出现州各自为政、部分州与联邦对立的局面,减少州际食品生产和交易的成本。法案优先于州标识立法,它在要求“强制”的同时,也为经营者提供了多种信息披露方式。披露要求及标准则由农业部在两年内制定规章予以确定。这一法案是美国各方妥协的结果,并未影响原有的生物技术政策和管理原则。在我国,转基因技术相关立法上亦存在矛盾和冲突,转基因食品标识问题尚未有定论。美国立法妥协的艺术值得我国借鉴,各方应当认可国家发展生物技术的目标。我国转基因食品标识立法需要高层次立法的明确授权,设置更多样的标识方式,并进行充分的法律实施评估。