Development of national secondary reference materials of urea and creatinine in frozen human serum / 中华检验医学杂志

Objective:To develop a national secondary reference material of Urea and Creatinine in frozen human serum as a standard for metrological traceability.Methods:According to JJF1343-2012 "General and Statistical Principles for Characterization of Reference Materials" and JJF 1006-1994 " Technical Norm of Primary Reference Material ", the homogeneity, stability, and commutability were evaluated;Using the JCTLM recommended methods, the value of the reference materials was assigned through collaboration with 6 accredited reference laboratories from Guangdong Provincial Hospital of Chinese Medicine, Beijing Aerospace General Hospital, Shenzhen Mindray Bio-Medical Electronics, Maccura Biotechnology, Beijing Leadman Biochemistry, and Zhejiang MedicalSystem Biotechnology. Uncertainty components including inhomogeneity, stability and value assignment were evaluated.Results:The results of one-way analysis of variance of homogeneity for the reference materials showed P>0.05, and the stability evaluation was less than the critical value of the t-test. The measured values were in the 95% confidence interval in the four conventional detection systems for commutability, and the certified values and expanded uncertainties were urea:(14.7±0.3) mmol/L ( k=2),Cr:(313.9±14.5) μmol/L ( k=2). Conclusion:The prepared secondary reference materials of urea and creatinine had promising homogeneity, stability, and commutable, the values of urea and creatinine concentration in reference materials were accurate and reliable.

Research Progress on Biological Matrix Reference Materials in Forensic Toxicology / 法医学杂志

Biological matrix reference material is a reference material that combines the target material with the biological matrix. The biological matrix reference material has higher consistency with the authentic specimens in forensic toxicology, and its application has a positive effect on improving the accuracy of test results. This paper reviews the research on the matrix reference materials corresponding to three common biological test materials (blood, urine and hair). In order to provide reference for the development and application of biological matrix reference materials in forensic toxicology, this paper mainly introduces the research progress of preparation technology of biological matrix reference materials and some existing products and their parameters evaluation.

Method and application of high efficient preparation of procalcitonin standby reference material based on protein recombination technology / 中华检验医学杂志

Objective:To construct a prokaryotic expression vector for human retinol binding protein 4 (hRBP4) that allows technicians to obtain hRBP4 purified protein with low cost, high efficiency, high concentration and high purity.Methods:The hRBP4 coding sequence provided by National Center for Biotechnology Information was optimized by E. coli codons, and a synthetic DNA fragment was cloned into the PET-28A (+) prokaryotic expression vector to construct a recombinant hRBP4 expression plasmid. The recombinant protein was transformed into E. coli BL21, and the induced expression conditions (temperature, rotate speed and isopropyl β-d-thiogalactoside concentration) were optimized. The recombinant protein was purified by His fusion tag. Results:The recombinant hRBP4 prokaryotic expression plasmid was successfully constructed, and the expression concentration and induction temperature of the recombinant protein were optimized. The results of sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that a band with a relative molecular weight of 26 000 daltons was clearly visible in the purified product. The purified hRBP4 protein could be detected clinically, and there was a good linear relationship between the dilution ratio and the detection concentration.Conclusions:The recombinant hRBP4 protein has high purity, high concentration, and short production cycle. It has the potential to become a candidate for reference materials for laboratory quality evaluations.

Elaboration of reference material for cadmium and arsenic in hydrobiological products, for the purpose of being used in a laboratory intercomparation program / Elaboración de un material de referencia para cadmio y arsénico en productos hidrobiológicos, para propósito de ser utilizado en un programa de intercomparación de laboratorios

Reference materials (RM) are tools used in the comparability and traceability of measurements. They are widely used by laboratories for method validation and quality control of assay. Chile must evaluate theperformance of laboratories that analyzing metals in fishery products, despite RM have high prices and are scarce. For that reason, a RM in a hydrobiological product was developed. Reference values for arsenic and cadmium elements for a fishmeal were assigned. The measurement methods for characterization of the material were Inductively Coupled Plasma Mass Spectrometry, Atomic Absorption Spectrometry and Neutron Activation Analysis. Reference values with their expanded uncertainty (U) were established for arsenic 2.64 ± 0.42 mg/kg (U; k = 2) and for cadmium 0.86 ± 0.16 mg/kg (U; k = 2). Homogeneity and stability of the RM allowed its use in a proficiency test for eleven food control laboratories. Results for median were 2.114 mg/kg for arsenic, and 0.863 mg/kg for cadmium. The performance values of the participants were evaluated with a z score obtaining 60% satisfaction for arsenic and 73% for cadmium.The material demonstrated to be suitable for use in interlaboratory proficiency assay. (AU)

Materiales de referencia (MR) son herramientas utilizadas en la comparabilidad y trazabilidad entre mediciones. Laboratorios los utilizan ampliamente en validación de métodos y control de calidad. Chile debe evaluar el desempeño de los laboratorios que analizan metales en productos pesqueros, a pesar de los altos precios y escasez del MR. Por esa razón, se desarrolló un MR en producto hidrobiológico. Se asignaron valores de referencia para arsénico y cadmio en harina de pescado. Los métodos de medición para la caracterización del material fueron Espectrometría de Masas de Plasma Acoplado Inductivamente, Espectrometría de Absorción Atómica y Análisis de Activación de Neutrones. Se establecieron valores de referencia con su incertidumbre (U) para arsénico 2.64 ± 0.42 mg/kg (U; k = 2) y para cadmio 0.86 ± 0.16 mg/kg (U; k = 2). La homogeneidad y estabilidad del MR permitieron su uso en una prueba de aptitud para once laboratorios de control de alimentos. Las medianas fueron 2,114 mg/kg para arsénico y 0,863 mg/kg para cadmio. Se evaluaron los rendimientos de los participantes con un estadístico de puntaje z satisfactorio del 60% para el arsénico y 73% para el cadmio. El material demostró ser adecuado para uso en ensayo de aptitud de intercomparación. (AU)

Development of Fucosterol Certified Reference Material / 中国实验方剂学杂志

Objective:According to the GB/T 15000.3-2008， to develop a fucosterol certified reference material based on the project approved by Standardization Administration. Method:Fucosterol was isolated from <italic>Laminaria japonica</italic> dried thallus via 95% ethanol extraction， vacuum concentration， repeated column chromatography separation， recrystallization in petroleum ether-ethyl acetate， and residual solvent removal. Its chemical structure was identified by elemental analysis （EA）， infrared spectrum （IR）， mass spectrometry （MS）， nuclear magnetic resonance （NMR） and X-ray diffraction （XRD）. Its homogeneity， stability， and cooperative certification conducted by 8 laboratories were carried out by high performance liquid chromatography with evaporative light scattering detector. Result:For the fucosterol reference material， the certified value of purity was 99.54% with expanded uncertainty of 0.16% in confidence interval of 95%， the stability was good within 24 months storage period at 2-4 ℃， which met the technical requirements of reference material and passed the acceptance organized by Standardization Administration. Conclusion:The national standard materials of fucosterol has been successfully developed， which can be used for the determination of this component， the evaluation of detection methods， and the detection and quality control of related products.

Preparation and purification of Cry1Ah protein candidate reference material / 生物工程学报

With the rapid development of transgenic technology, the safety of genetically modified products has received extensive attention. Certified reference materials for the detection of genetically modified organisms play important roles in ensuring comparability and traceability of the qualitative and quantitative detection of genetically modified products. However, the development of protein reference materials is relatively slow, and one of the difficulties is the preparation of protein candidates with high purity. The cry1Ah1 gene of Bacillus thuringiensis has been used for the development of transgenic insect-resistant crops because of its excellent insecticidal activity against lepidopteran pests such as Asian corn borer, and has obtained transgenic lines with good insect resistance traits. In order to develop Cry1Ah protein certified reference material, it is urgent to establish a preparation and purification system. In this study, a system for preparing Cry1Ah protein by Bt expression system was optimized, and a high-purity Cry1Ah protein (size exclusion chromatography purity: 99.6%) was obtained by ion-exchange chromatography and size exclusion chromatography stepwise purification. The results of biological activity assay showed that there was no significant difference in the insecticidal activity of purified Cry1Ah protein and protoxin against diamondback moths (Plutella xylostella). Finally, the amino acid sequence of the activated Cry1Ah protein was determined using Edman degradation and mass spectrometry. In summary, the obtained Cry1Ah pure protein can be used for the development of protein reference materials.

Essential Elements for Establishing Clinical Next-generation Sequencing Testing

Over the past decade, next-generation sequencing (NGS) has evolved at an astonishing pace and has revolutionized clinical medicine as well as genomics research. The rapid advancements in NGS technologies have been accompanied by accumulating evidence of the analytical and clinical validity, and clinical utility of NGS. NGS is used worldwide. This review provides medical technicians and laboratory physicians with the essential elements for establishing clinical NGS testing. Here the authors briefly describe the advantages and drawbacks of currently available NGS platforms, potential sources of error in NGS workflow, and reference materials.

The measurements of urinary albumin and the progress of its standardization / 中华检验医学杂志

Chronic kidney disease caused by diabetes and hypertension has become a global public health problem. Urinary albumin is one of the key indicators for the diagnosis and grading of chronic kidney disease, and it has been widely used in practice. Accurate and comparable results of urinary albumin detection served as an important basis for clinical application. This paper discussed the status of urinary albumin detection and the progress of standardization, in order to help the laboratory correctly interpret the test results and promote the quality of diagnostic products.

Allergen standardization

Allergen immunotherapy (AIT) and diagnostic tests are based on well qualified allergen extracts, which are derived from biologic organisms. The allergenicity of the extracts is markedly affected by the climate, soil, year of production, storage methods, and manufacturing processes. Thus, standardization is a crucial process to guarantee the clinical efficacy and safety of the treatment and diagnostic reagents in allergic diseases. There are 2 different standardization processes, one is In vivo and the other is in vitro standardization. In vivo standardization is done by skin prick or intradermal tests. For in vitro standardization, measurements of weight/volume and protein nitrogen units have been widely used since the early period of AIT. In the 1970s, immunological methods such as radial immunodiffusion, enzyme-linked immunosorbent assay (ELISA) inhibition test and basophil activation test were developed. Allergen potency measured by ELISA inhibition test reflects the potency measured by skin tests and has been widely used for quality control of batch-to-batch variation. Recently, standardizations focused on the major allergen content of extracts have developed. Standardization for major allergens requires reliable reference materials (RMs) made of recombinant allergens and 2-site ELISA kits. However, only a few reliable RM and 2-site ELISA kits are available. For the standardization process, allergen RMs are essential. The Center for Biologics Evaluation and Research of the U.S. Food and Drug Administration provides 19 allergen RMs, and our research team also proved 9 RMs which are important in Korea. In conclusion, allergen standardization is an essential process for the development of reliable treatment and diagnostic reagents, and allergy specialist should be familiar with the concept of allergen standardization.

Evaluation of a fungal collection as certified reference material producer and as a biological resource center

Abstract Considering the absence of standards for culture collections and more specifically for biological resource centers in the world, in addition to the absence of certified biological material in Brazil, this study aimed to evaluate a Fungal Collection from Fiocruz, as a producer of certified reference material and as Biological Resource Center (BRC). For this evaluation, a checklist based on the requirements of ABNT ISO GUIA34:2012 correlated with the ABNT NBR ISO/IEC17025:2005, was designed and applied. Complementing the implementation of the checklist, an internal audit was performed. An evaluation of this Collection as a BRC was also conducted following the requirements of the NIT-DICLA-061, the Brazilian internal standard from Inmetro, based on ABNT NBR ISO/IEC 17025:2005, ABNT ISO GUIA 34:2012 and OECD Best Practice Guidelines for BRCs. This was the first time that the NIT DICLA-061 was applied in a culture collection during an internal audit. The assessments enabled the proposal for the adequacy of this Collection to assure the implementation of the management system for their future accreditation by Inmetro as a certified reference material producer as well as its future accreditation as a Biological Resource Center according to the NIT-DICLA-061.

Estudo colaborativo nacional para o estabelecimento do material de referência de trabalho da vacina contra sarampo, caxumba e rubéola: evolução para a autossuficência na produção nacional da vacina tríplice viral / National collaborative study for establishing the working reference material of the vaccine against measles/mumps and rubella: evolution to the self-sufficiency in the national production of triple viral vaccine

A monografia farmacopeica da vacina tríplice viral (sarampo/caxumba/rubéola) exige a validação de desempenho do ensaio de potência utilizando-se apropriado material de referência (MR). Com o intuito de estabelecer o primeiro MR de trabalho (MRT) nacional para a vacina tríplice viral, foi realizado o estudo colaborativo nacional com a participação de duas únicas instituições que executam o ensaio de potência desta vacina, o Instituto de Tecnologia em Imunobiológicos (Bio-Manguinhos, produtor nacional) e o Instituto Nacional de Controle de Qualidade em Saúde.O material candidato (cMRTBio), preparado pelo produtor, foi avaliado pelos laboratórios participantes utilizando-se as respectivas metodologias in-house de determinação de potência. O cMRTBio foi considerado apropriado como MR in-house por estar em concordância com as especificações recomendadas nas normativas de compêndios, a saber: variações intra- (< 5 %), inter-ensaios (< 10 %) e entre laboratórios (< 10 %) abaixo dos limites aceitáveis; e potência estimada (log10 CCID50/DH) em 3,72 para sarampo, 4,80 para caxumba e 3,70 para rubéola. Este trabalho reflete o compromisso do único produtor nacional da vacina tríplice viral com a saúde pública, descrevendo-se a expansão da tecnologia, o cumprimento às diretrizes internacionais, o cuidado com o controle da qualidade e culminância para a autossuficiência nacional na produção de vacinas.

The pharmacopoeia monograph for the measles/mumps and rubella (MMR) triple vaccine demands to perform the validation of the potency assay by using the suitable reference material (MR). Aiming at establishing the firstwork MR (MRT) for the MMR triple vaccine, a national collaborative study was performed with the participation of the two unique national institutions working on the vaccine potency evaluation test, the Imunobiological Technology Institute (Bio-Manguinhos, national manufacturer) and the National Institute for Quality Control in Health. The candidate product (cMRTBio) prepared by the manufacturer was evaluated by the participant laboratories by employing the respective in-house methodologies for determining the potency. The cMRTBio was considered suitable as in-house MR, according to the specifications based on the normative compendia, being the intra-assay (< 5 %), inter-assay(< 10 %) and between laboratories variations (< 10 %) below the acceptable limits, and the estimate potency(log10 CCID50/DH) in 3.72 for measles, 4.80 for mumps and 3.70 for rubella. This study reflects the commitment of the unique national MMR vaccine producer to the public health, describing the expansion of technology,the compliance with international guidelines and the careful quality control, leading to the national self-sufficiency in the vaccine production.

Development of a new ferulic acid certified reference material for use in clinical chemistry and pharmaceutical analysis

This study compares the results of three certified methods, namely differential scanning calorimetry (DSC), the mass balance (MB) method and coulometric titrimetry (CT), in the purity assessment of ferulic acid certified reference material (CRM). Purity and expanded uncertainty as determined by the three methods were respectively 99.81%, 0.16%; 99.79%, 0.16%; and 99.81%, 0.26% with, in all cases, a coverage factor (k) of 2 (P=95%). The purity results are consistent indicating that the combination of DSC, the MB method and CT provides a confident assessment of the purity of suitable CRMs like ferulic acid.

Preparation and Evaluation of the Reference Materials for Plasma von Willebrand Factor Antigen Testing / 现代检验医学杂志

Objective To prepare and evaluate the reference materials for plasma von Willebrand Factor antigen testing with fresh frozen plasma.Methods The candidates were prepared by low temperature centrifugation in 5 different concentration levels.The homogeneity and stability of the preparation was evaluated according to the ISO Guide35 and CNAS-GL03.The comparability between STAGO and IL system was evaluated according to the WS/T 356-2011.Then the preparations were characterized by six laboratories with the Secondary Coagulation Standard established by NIBSC(SSCLOT4).Results Homogeneity evaluation of the preparation showed that there was no statistically significant difference between the groups (P >0.05),the F values of factor analysis of variance were 0.317~0.844,the uncertainty range was 1.01% ~2.06%.A linear regression based on stability evaluation indicated that the linear trend (within 24 weeks)was insignificant (P >0.05). The uncertainty range of long-term (within 24 weeks)stability was 0.79% ~ 1.20%.The results of the preparations on STAGO and IL system were comparable.The certificated values of the candidates were range from 12.2% to 138.9% with uncertainties were 0.06%~0.09%,respectively.The range of combined standard uncertainty was 0.03% ~ 0.16% while the expanded uncertainty was 2.2%~6.7%.Conclusion The reference materials for von Willebrand Factor antigen testing were stable and homogenous with comparability between STAGO and IL.The method of characterization was accurate and reliable.

Estudo de homogeneidade dos soros utilizados em controle de qualidade interno de testes imunodiagnósticos de HIV/Aids / AidsHomogeneity study of the internal quality control sera for immunodiagnosis of HIV/AIDS

Introduction: The present study reports the data from the first homogeneity assessment of samples composing the serum panels produced at the Immunology Center of Instituto Adolfo Lutz, São Paulo. These samples have been distributed to the public laboratories and those partaking in the Brazilian Unified Health System, and to the participants in the Internal Quality Control Program for human immunodeficiency virus (HIV) antibody (Ab) testing. Objective: To assess the homogeneity of serum samples in panels from different lots for HIV/acquired immunodeficiency syndrome (AIDS) immunodiagnosis by using the statistical method to ensure quality of the reference material. Method: Sera homogeneity was evaluated by means of enzyme-linked immunoassay/enzyme immunoassay (ELISA/EIA) for detection of HIV Ab, and the one-way analysis of variance was employed for analyzing the data. No statistically significant differences were found among the several serum vials. Conclusion: The sera dispensed in the vials were homogeneous in the respective lots...

Introdução: No presente estudo estão descritos os resultados das primeiras análises feitas sobre a avaliação da homogeneidade das amostras componentes de painéis de soros produzidos no Centro de Imunologia do Instituto Adolfo Lutz e distribuídos aos laboratórios públicos e conveniados ao Sistema Único de Saúde e participantes do Programa de Controle de Qualidade Interno para imunodiagnóstico de vírus da imunodeficiência humana/síndrome da imunodeficiência adquirida (HIV/AIDS). Objetivo: Avaliar a homogeneidade das amostras de soro componentes de painéis de diferentes lotes para imunodiagnóstico de HIV/Aids por meio de método estatístico para garantir a qualidade do material de referência. Material e método: A homogeneidade das amostras de soro foi avaliada por meio de enzyme-linked immunoassay/enzyme immunoassay (ELISA/EIA) para detecção de anticorpos anti-HIV, e os resultados foram submetidos à análise de variância fator único. Não foram encontradas diferenças significativas entre os resultados obtidos para os diversos frascos de soro. Conclusão: As amostras distribuídas nos frascos foram homogêneas entre si nos respectivos lotes...

Necesidades metrológicas en los laboratorios clínicos / Metrological needs in clinical laboratories / Necessidades metrológicas nos laboratórios clínicos

Los resultados generados por los laboratorios clínicos son elementos claves para el diagnóstico y tratamiento de enfermedades, así como para el seguimiento de los pacientes, por lo tanto se requiere un control metrológico sobre el proceso de medición, el cual determinará el grado de comparabilidad y de confianza requerido sobre los resultados obtenidos. Esta revisión aborda el tema de comparabilidad de las mediciones y de cómo ésta se ve influenciada por factores como la conmutabilidad de los materiales de referencia, los métodos de medición, la disponibilidad de materiales y procedimientos de referencia, además de los intervalos de referencia y límites de decisión para un mensurando en particular. Finalmente se exponen cuáles son los mecanismos adoptados que permitirán garantizar la comparabilidad de las mediciones en esta área.

The results produced by clinical laboratories are key elements in the diagnosis and treatment of diseases and to monitor patients, so, it requires a metrological control over the measurement process, which will determine the degree of comparability and trust required on the obtained measurement results. Therefore, in this paper we discuss about the comparability of measurements and how it is influenced by factors such as commutability of reference materials, measurement methods, availability of materials and reference procedures, in addition to reference intervals and decision limits for a particular measurand. Finally it is exposed what are the mechanisms that allow ensure measurements comparability on this area.

Os resultados gerados pelos laboratórios clínicos são elementos chaves para o diagnóstico e tratamento de doenças, assim como para o seguimento dos pacientes, sendo preciso um controle metrológico sobre o processo de medição, que determina o grau de comparabilidade e confiança requerido sobre os resultados obtidos. Neste documento trata-se o tópico de comparabilidade das medições e de como a mesma se encontra influenciada por fatores como a comutatividade dos materiais de referência, os métodos de medição, a disponibilidade de materiais e procedimentos de referência, além dos intervalos de referência e limites de decisão para um mensurando em particular. Finalmente, são expostos quais são os mecanismos adotados que permitirão garantir a comparabilidade das medições nesta área.

Participating in the evaluation of combined value assignment of Cystatin-C as a national standard material candidate / 国际检验医学杂志

Objective As a collaborator of Beijing Institute of Medical Device Testing for value assignment of state standard ma-terial candidate Cystatin-C ,we have used the internationally accepted reference material to assign value for state standard material candidate Cystatin-C ,and help Beijing Institute of Medical Device Testing get Cystatin-C national standard material certificate . Methods According to the target value and operational procedure of international reference material ERM-DA471 ,We have tested 6 dilutions of standard material candidate Cystatin-C on calibrated Hitachi 7180 immunoassay system .Results The results demon-strate good repeatability and commutability ,and have been accepted in calculating the final value for the candidate standard materi-al ,our data has assisted Beijing Institute of Medical Device Testing in passing the criteria and obtaining Cystatin-C national standard material certificate .Conclusion Compared to the data from all participating collaborators ,our results hit right on the target value , and no significant matrix effects have been observed .

Purity Determination and Uncertainty Evaluation of Ribavirin Purity Reference Material / 医药导报

Objective To develop the ribavirin purity certified reference material( CRM ) which has measurement traceability and high accuracy,and establish an effective method of evaluation. Methods The homogeneity and the stability were checked by differential scanning calorimetry(DSC). High performance liquid chromatography(HPLC)and DSC methods were used for purity determination of ribavirin. Results Ribavirin showed satisfactory homogeneity and stability. The certified value of ribavirin was 99. 5%with an uncertainty of 0. 4%(k=2,P=0. 95). Conclusion Ribavirin purity CRM obtained in this paper has been proven to be a national primary CRM with high accuracy and traceability,which can be used to validate analytical methods,improve the accuracy of measurement data,establish meteorological traceability of analytical results as well as control the quality of ribavirin in the pharmaceutical industry.

Estimación de la incertidumbre para el análisis de potencia de insulina humana isofana ADN recombinante por cromatografía liquida de alta resolución fase reversa según USP34 NF29 2011 / Uncertainty estimation for the USP34 NF29 2011 analysis of potency recombinant DNA isophane human insulin from PR-HPLC

El propósito de una medición es determinar el valor de una magnitud, llamada el mensurando. La imperfección natural de las mediciones, hace imposible conocer con certeza absoluta el valor verdadero de una magnitud, ya que toda medición lleva implícita una incertidumbre, el cual es un parámetro no negativo que caracteriza la dispersión de los valores atribuidos a un mensurando. En la presente investigación se empleo una estrategia diferente al procedimiento “bottom-up” propuesto por la ISO, el cual nos permitió estimar de forma global la incertidumbre, mediante la agrupación de valores determinados durante la verificación del método analítico. El valor obtenido en nuestro estudio fue de 3.46 UI/mL con un 90% de confianza. Observándose como fuente de mayor contribución al cálculo, el efecto de la matriz del medicamento, dependiente del tipo de insulina presente, además de la precisión y trazabilidad del patrón de referencia, los cuales contribuyen en la incertidumbre final de forma similar. En este sentido, nuestros esfuerzos deben dirigirse a la optimización de los procesos de homogeneización, adquisición del patrón de referencia tipo primario, con la finalidad de garantizar a lo largo del tiempo, resultados reproducibles, trazables y confiables.

The purpose of measurement is to determine the value of a quantity, called the measurand. The natural imperfection of determining measurements, makes impossible to know with absolute certainty the true value of a magnitude, since all measurement implies an uncertainty, which is a non-negative parameter that characterize the dispersion of the values attributed to a measurand. In this research, we employed a different strategy to the "bottom-up" procedure, proposed by the ISO, which allowed us to calculate globally the uncertainty, by grouping values, determined during the verification of the analytical method. The obtained value in our study was 3.46 IU/mL with 90% confidence. The matrix effect of the drug, depending on the insulin type present inside, was observed as the bigger contribution source to the calculation, as well as the accuracy and traceability of the reference pattern, which contribute in the final uncertainty similarly. Thus, our efforts should be directed to the optimization of the homogenization processes and the acquisition of the primary type reference pattern, in order to ensure over time, reproducible, traceable and reliable results.

Desenvolvimento de material de referência para microbiologia de alimentos contendo Listeria monocytogenes em matriz queijo / Development of reference material for food microbiology containing Listeria monocytogenes in matrix

A NBR ISO/IEC 17025:2005, uma das normas mais utilizadas em laboratórios de ensaio, descreve como um de seus critérios para a garantia da qualidade dos resultados analíticos a participação periódica dos laboratórios em ensaios de proficiência (EP). Os analitos, utilizados nos EP são materiais de referência (MR) provenientes de um mesmo lote, e devem apresentar características de homogeneidade e estabilidade. O objetivo deste estudo foi produzir um MR qualitativo destinado ao ensaio de pesquisa de Listeria monocytogenes em matriz queijo pela técnica de liofilização. Para a produção do MR, foi utilizado como matriz o queijo Minas frescal (QMF) ultrafiltrado. A matriz foi distribuída em frascos, contaminada com a bactéria alvo e submetida à liofilização, tendo a sacarose como crioprotetor. O MR produzido foi considerado homogêneo e estável na temperatura de ≤-70°C durante todo o período estudado (10 meses). O material apresentou estabilidade a 4, 25, 30 e 35°C por quatro dias e a -20°C por 48 dias, e os resultados estatísticos indicam tendência à estabilidade. Conclui-se que o material apresentou todos os requisitos necessários de um MR de qualidade e poderia ser transportado aos laboratórios participantes de um EP à temperatura máxima de 35°C por até quatro dias, uma vez que os resultados indicaram a manutenção da concentração celular nesse período. Esse foi o primeiro trabalho a descrever uma metodologia de produção de MR contendo L. monocytogenes em matriz queijo.

The standard most used in testing laboratories, ISO/IEC 17025:2005, describes the participation of laboratories in periodic proficiency testing (PT), as a criteria for quality assurance of analytical results. The analyses used in PT are reference materials (MR) from the same lot, and must have characteristics of homogeneity and stability. This study aimed to produce a qualitative RM for detection of Listeria monocytogenes assays in cheese matrix. For the production of RM, Minas Frescal cheese (MFQ) was used as matrix. The matrix was distributed in flasks, contaminated with the target bacteria and submitted to freeze-drying. Sucrose was used as cryoprotector. The RM produced was considered homogeneous and stable at -70°C during the entire period of study (10 months). The material showed stability at 4, 25, 30 and 35°C for 4 days and at -20°C the RM showed stability for 48 days, and the statistical results indicate a tendency to maintain stability. It was concluded that the material showed all the requirements of an RM quality and could be transported to the laboratory participants of a PT at 35°C up to 4 days, since the results indicate the maintenance of cell concentration during this period. This is the first study to describe a methodology for producing MR containing L. monocytogenes in cheese matrix.

Estabilidad de un suero control multienzimático utilizado como material de referencia en los laboratorios clínicos / Stability of a multienzyme control serum used as reference material in clinical laboratories

Una de las vías fundamentales para garantizar la calidad de los ensayos realizados en los laboratorios clínicos es mediante el uso de materiales de referencia. Una problemática a la que nos enfrentamos es la escasez de estos productos en el mercado nacional dado su alto costo. Objetivo: evaluar la estabilidad de un suero bovino adulto enriquecido con las enzimas alanina aminotransferasa (ALAT/TGP), aspartato aminotransferasa (ASAT/TGP), fosfatasa alcalina (FA) y amilasa. Métodos: se evaluó la estabilidad a tiempo real de la matriz enriquecida con las diferentes enzimas durante 12 meses a 2 temperaturas (refrigeración y congelación). Se evaluó el efecto del glicerol sobre la actividad enzimática de los extractos, así como el efecto de los preservantes propilenglicol y etilenglicol en la estabilidad de las enzimas. Resultados: los extractos enzimáticos obtenidos comenzaron a perder la actividad biológica a partir de los 15 días, independientemente de la temperatura de almacenamiento y de la presencia o no de glicerol. Los resultados del ensayo a tiempo real realizados a la matriz enriquecida, mostraron que la estabilidad varió con el tiempo y con el tipo de enzima, independientemente del preservante ensayado, disminuyendo por debajo de los límites aceptables de actividad enzimática luego de 3 meses de almacenamiento del producto a 4 ºC. Conclusiones: se logró un material de referencia multienzimático estable por un período de 3 meses

A fundamental method to assure the quality of clinical laboratory tests is the use of reference materials. A problem we are faced with is the scarcity of these products in the domestic market, due their high cost. Objective: Evaluate the stability of an adult bovine serum enriched with the enzymes alanine aminotransferase (ALT, GPT), aspartate aminotransferase (AST, GPT), alkaline phosphatase (AP) and amylase. Methods: This enzyme-enriched matrix underwent real-time stability assessment during 12 months at two temperatures (refrigerated and frozen). An evaluation was made of the effect of glycerol on the enzymatic activity of extracts, as well as the effect of the preservatives propylene glycol and ethylene glycol on enzymatic stability. Results: The enzyme extracts obtained began to lose their biological activity at 15 days, irrespective of the storage temperature and the presence or absence of glycerol. The real time assessment of the enriched matrix showed that stability varied with time and enzyme type, irrespective of the preservative tested, and fell below acceptable limits of enzymatic activity after three months of storage at 4 ºC. Conclusions: A multienzyme reference material was obtained which was stable for a period of 3 months