RESUMEN
In a Dutch project for harmonization of fibrinogen assays, the commutability of potential calibrators for fibrinogen was assessed by means of a twin-study design, which is, in essence, a multicentre, split-patient sample, between-field-methods protocol. The study consisted of simultaneous analysis of fresh-frozen patient plasmas and three potential calibrators for fibrinogen by 48 Dutch laboratories forming 24 couples. The state-of-the-art intralaboratory standard deviation was used to assess the commutability of the potential calibrators. The potential calibrators were commutable for the Clauss, but not for the prothrombin time (PT)-derived assays. One potential calibrator was used in an attempt to harmonize fibrinogen assay results in a Dutch field study. The interlaboratory coefficient of variation (CV) of three out of four test samples could be reduced significantly using the common calibrator. The average overall CV for the four test samples was 10.3% using the routine measurements and 7.8% using the common calibrator. Despite the reduction in the overall CV, the bias between Clauss and PT-derived assay results in two coumarin test samples could not be eliminated.
Asunto(s)
Calibración/normas , Fibrinógeno/análisis , Técnicas de Laboratorio Clínico/normas , Fibrinógeno/normas , Humanos , Laboratorios/normas , Países Bajos , Garantía de la Calidad de Atención de Salud , Reproducibilidad de los ResultadosRESUMEN
The acceptance of the polymerase chain reaction (PCR) as an amplification method in molecular diagnostics and the rapid development of capillary electrophoresis (CE) as an analysis method of those PCR products was a reason for us to investigate further integration of those two techniques. Using a fused-silica capillary as a pipette we were able to compose a PCR mixture in the CE apparatus. Because a capillary can be thoroughly rinsed and the CE apparatus is a closed system, the risk of contamination and therefore the occurrence of false positive results is minimized. The fact that a CE system can be fully automated contributes to a more reproducible and standardized PCR composition protocol.
Asunto(s)
Autoanálisis , Electroforesis Capilar/métodos , Ácidos Nucleicos/análisis , Reacción en Cadena de la Polimerasa/métodos , ADN/análisis , Contaminación de Medicamentos/prevención & control , Electroforesis en Gel de Agar , Electroforesis Capilar/instrumentación , Etidio , Reacciones Falso Positivas , Genes ras , Indicadores y Reactivos , Oligonucleótidos/análisisRESUMEN
Capillary electrophoresis (CE) is a convenient, fast and non-radioactive method with possibilities for automatization. To analyse single-stranded DNA molecules in a more automated way, we developed a heating device to melt double-stranded DNA fragments in the capillary during electrophoresis. In this study we used this device to obtain single-stranded DNA, necessary for the detection of point mutations in DNA using the single-strand conformation polymorphism technique. Results show that double-stranded DNA molecules can be melted on-line into single-stranded DNA molecules, although not for 100%. In an attempt to find universal electrophoretic conditions for the analysis of single-stranded DNA, we investigated the influence of several parameters on the yield of single-stranded DNA molecules and on the resolution of the single-stranded DNA peaks. We demonstrate that this heating device is a technical adjustment of CE which contributes to more automated analyses of DNA fragments.
Asunto(s)
ADN de Cadena Simple/análisis , ADN/química , Electroforesis Capilar/métodos , ADN/genética , ADN de Cadena Simple/genética , Genes p53 , Genes ras , Mutación Puntual , Reacción en Cadena de la PolimerasaRESUMEN
Recently a point mutation (G1691A) in the coagulation factor V gene was shown to cause resistance for cleavage by activated protein C. The mutation is associated with an increased thrombotic risk and thus-far the most common genetic cause of thrombophilia. Current techniques to investigate the single base pair mutation at the DNA level use an assay based upon the polymerase chain reaction followed by restriction enzyme digestion or Southern blotting and allele specific probing. The method we describe here consists of a single PCR in which two specially designed allele specific primers and two consensus primers were used in one reaction to distinguish between homozygous normal, heterozygous and homozygous mutant individuals. Amplification products were analysed using Capillary Electrophoresis and on line UV monitoring. The Allele Specific Amplification Protocol and subsequent CE analysis (ASAP-CE) is a convenient, fast, automated and highly reproducible method that can be used in a routine laboratory setting.
Asunto(s)
Factor V/genética , Mutación Puntual , Alelos , Secuencia de Bases , Electroforesis Capilar/métodos , Humanos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa/métodos , Trombosis/genéticaRESUMEN
We compared the use of capillary electrophoresis (CE) in a polymer network with the use of slab gel electrophoresis for the quantitative analysis of polymerase chain reaction (PCR)-amplified DNA samples. We quantified residual lymphoma cells carrying a translocation between chromosomes 14 and 18, in consecutive patient peripheral blood samples that were amplified by competitive PCR. For CE analysis we used a 4% linear polyacrylamide network. Results show that the calculated number of translocations in patient samples using both analyses were comparable. We conclude that CE is a sensitive, non-radioactive, fast and accurate method for quantitation of competitive PCR products.
Asunto(s)
ADN/análisis , Autorradiografía , Secuencia de Bases , Cromosomas Humanos Par 14 , Cromosomas Humanos Par 18 , Electroforesis , Humanos , Ganglios Linfáticos/química , Linfoma/química , Datos de Secuencia Molecular , Oligonucleótidos/análisis , Reacción en Cadena de la Polimerasa , Translocación GenéticaRESUMEN
The use of capillary electrophoresis (CE) in a polymer network for single-strand conformation polymorphism (SSCP) is investigated. SSCP is a method to detect DNA point mutations, essential in the diagnosis of several diseases. The PCR (polymerase chain reaction) amplified p53 gene, a tumour suppressor gene known to be frequently mutated in malignant cells, was subjected to CE analysis. Two single-strand DNA fragments of 372 bp in length differing in only one nucleotide could be separated. We conclude that SSCP using CE in a polymer network is a powerful method for the detection of point mutations in DNA sequences.
Asunto(s)
ADN/química , Electroforesis/métodos , Genes p53 , Mutación Puntual , Polímeros , Secuencia de Bases , Acción Capilar , ADN/aislamiento & purificación , Humanos , Datos de Secuencia Molecular , Mieloma Múltiple/genética , Conformación de Ácido Nucleico , Reacción en Cadena de la Polimerasa , Polimorfismo GenéticoRESUMEN
The recent finding that eight out of 10 multiple myeloma cell lines have p53 gene mutations prompted us to examine the p53 tumour suppressor gene in 25 non-related multiple myeloma patients. None of 19 patient bone marrow samples available for Southern blot analysis showed rearrangements in the p53 gene and only one patient showed loss of the p53 locus. DNA encompassing exons 5, 7, and 8, where p53 mutations commonly cluster, was amplified by PCR. Single-strand conformation polymorphisms of the PCR-amplified exon 5 region were detected in two patients. Direct sequencing of the mutant band revealed that one patient had a C to T transition at codon 138 (Ala to Val) and one patient had a G to C transversion at codon 139 (Lys to Asn). p53 mutations in germline cells in hereditary cancer syndromes predispose the family members to the development of malignancies. We therefore searched for p53 germline mutations in exons 5, 7, and 8 in the affected individuals from three families each with two multiple myeloma patients (these patients include three individuals from the non-related group mentioned above). Using Southern blotting, polymerase chain reaction/single-strand conformation polymorphism (PCR-SSCP) analysis and direct sequencing, no germline mutations were found. These results indicate that mutations in exons 5, 7, and 8 of the p53 gene are infrequent in multiple myeloma.