RESUMEN
The low biological variation of myoglobin and creatine kinase isoenzyme MB mass (CK-MBm) requires accurate measurements. In the standardization process, in order to effectively measure and correct intermethod variability, the intermethod behaviour of control materials must be the same of patient sera, i.e. they must be commutable. In this work we checked the commutability of some commercially available control materials in pairs of methods for myoglobin and CK-MBm measurements; we assessed the impact of commutable and non-commutable control materials when used for equalizing patient sera results by two different methods and discussed the problems related to external quality assessment schemes. Myoglobin and CK-MBm were measured in sets of 49 and 56 patient sera and in 13 commercially available control materials with two automatic analytical systems. The non-commutability rate was 8.3% for myoglobin and 23.1% for CK-MBm. Recalculation of serum samples results with a control material as calibrator lowered or increased the bias originally present according to whether the material itself was commutable or not. We conclude that also for myoglobin and CK-MBm assays it is necessary to check the commutability of materials to be used in external quality assessment schemes, or to normalize patient results by different methods.
Asunto(s)
Creatina Quinasa/sangre , Isoenzimas/sangre , Mioglobina/sangre , Forma MB de la Creatina-Quinasa , Humanos , Inmunoensayo/métodos , Estándares de Referencia , Reproducibilidad de los ResultadosRESUMEN
In assessing possible in vitro interference from bilirubin on analytical methods, different results are to be expected from using either unconjugated (uB) or conjugated (cB) bilirubin as test materials, because of their different solubilities. In vitro interference of a synthetic soluble bilirubin derivative (ditauro-bilirubin, dtB) on gamma-glutamyltransferase activity measurement has been studied, in comparison with uB. In three out of five analytical methods/systems for the measurement of the enzyme activity, significantly higher (negative) interference was observed in the presence of the soluble derivative. Whichever the mechanism for the observed effect, the opportuneness of using soluble derivatives in order to assess bilirubin interference is pointed out: pathological serum specimens, submitted for laboratory investigations, are indeed frequently loaded with soluble bilirubin conjugates.
Asunto(s)
Bilirrubina/farmacología , gamma-Glutamiltransferasa/metabolismo , Bilirrubina/análogos & derivados , Pruebas Enzimáticas Clínicas/métodos , Relación Dosis-Respuesta a Droga , Humanos , Técnicas In Vitro , Solubilidad , Taurina/análogos & derivados , Taurina/farmacologíaRESUMEN
By means of gel-filtration of bilirubin/albumin mixtures, it is shown that unconjugated bilirubin remains completely linked to albumin (both human and bovine) in tetraborate buffer (pH 9.3), protein-free bilirubin appearing only when the bilirubin/albumin molar ratio exceeds two. On the other hand, bilirubin is completely set free from its protein link in the caffeine reagent. Additional chromatographic and spectrophotometric evidence is reported indicating the formation of a low-affinity complex between bilirubin and caffeine. These data explain why the spectrophotometric properties of bilirubin/albumin mixtures are matrix-dependent if measured in the tetraborate buffer but are no longer so when measured in the caffeine reagent. The relevance of these findings to the spectrophotometric "direct" assay of bilirubin in serum is discussed with reference to the occurrence of "delta-bilirubin" in pathological sera: this tightly protein-bound bilirubin fraction does not split in the presence of caffeine.
Asunto(s)
Albúminas/análisis , Bilirrubina/análisis , Cafeína , Animales , Boratos , Tampones (Química) , Bovinos , Cromatografía en Gel , Humanos , EspectrofotometríaRESUMEN
Serum bilirubin has been fractionated by means of a gel-filtration technique, on Bio-Gel P-10 columns and with caffeine solution as eluent. When sera containing high concentration of direct-reacting bilirubin were applied to the column, a bilirubin fraction was eluted with the protein. This bilirubin fraction is not set free from protein in the presence of urea, guanidine and sodium dodecyl sulfate; moreover it exhibits peculiar behaviour in the direct and indirect diazo-reaction and in enzymatic oxidation by bilirubin-oxidase as well. On the basis of these data, such bilirubin fraction has been identified with the previously described delta-bilirubin. A method for the quantitative measurement of delta-bilirubin, based on gel-filtration separation followed by diazo-reaction, has been worked out. Results by this method satisfactorily compared with the Kodak Ektachem method.
Asunto(s)
Bilirrubina/sangre , Cromatografía en Gel , Bilirrubina/aislamiento & purificación , Fraccionamiento Químico/métodos , HumanosRESUMEN
A two-wavelength spectrophotometric procedure for the simultaneous determination of haemoglobin and haemoglobin cyanide (HiCN) (or of haemoglobin and haemoglobin azide (HiN3)) concentrations in mixtures has been developed and applied to the determination of the stability constants of HiCN and HiN3. The analytically reliable procedure allowed stability constants to be estimated with about 10% (relative standard deviation, coefficient of variation) uncertainty. Values of 1.9 X 10(6) and 2.0 X 10(5) 1 X mol-1 were obtained for HiCN and HiN3, respectively. These results are discussed in relation to the optimal composition of the reagents for blood haemoglobin assay by the two methods.
Asunto(s)
Azidas/análisis , Hemoglobinas/análisis , Estabilidad de Medicamentos , Humanos , Cinética , Espectrofotometría/métodosRESUMEN
Sodium and potassium were measured in sets of 102 to 107 patients sera, and in 31 commercially available control sera. The results from four routine analytical methods/systems (indirect potentiometry: two; direct potentiometry and enzymatic assay: one each) were compared with those from a flame photometry-based reference method. In the assay of patient sera, substantial agreement was observed in some comparisons, clinically relevant bias in others. The inter-assay changes observed for the control sera differed significantly from those shown by the patients sera (i.e. commercial control sera were non-commutable) in about 12% of the comparisons, as a whole. Recalculation of serum sample results with a single control serum as calibrator lowered or increased the bias originally present according to whether the serum itself was commutable or not. Moreover, the inter-method variability in the assay of commercial control sera was lower with commutable sera, higher with non-commutable sera. With the exception of liquid sera stabilized with ethylene glycol, there was no evident link between any specific characteristic of the commercial control sera (matrix and physical state) and their degree of commutability.
Asunto(s)
Potasio/sangre , Sodio/sangre , Animales , Bovinos , Intervalos de Confianza , Activación Enzimática , Caballos , Humanos , Fotometría , Potenciometría/normas , Potenciometría/estadística & datos numéricos , Estándares de Referencia , Valores de Referencia , Reproducibilidad de los ResultadosRESUMEN
The aim of this work was to check the suitability of control materials to normalize intermethod results for the measurement of free triiodothyronine in patient sera. In the main experiment, 108 patient sera and 11 commercially available control materials were assayed by a pair of methods. In a confirmatory experiment, two of the control materials and 142 patient sera were assayed with an alternative pair of methods. In the main experiment, the intermethod variability of 6/11 control materials differed significantly from that of patient sera (i. e. control materials were non-commutable). Recalculation of patient results using control materials as calibrators lowered the intermethod difference only if commutable materials were used. The confirmatory experiment demonstrated that the pattern of commutability changed if a different pair of methods was used. We conclude that in the case of free triiodothyronine the commutability of control materials should be tested if they are to be used to normalize patient results obtained by different methods.
Asunto(s)
Triyodotironina/sangre , Humanos , Estándares de Referencia , Reproducibilidad de los ResultadosRESUMEN
The performance of the Reflotron system (Boehringer Mannheim) for the determination of urate in whole blood and serum was evaluated. Within-run and day-to-day imprecision of the system were comparable with those for a solution-chemistry enzymatic method (overall CVs in the range 2.2-2.5%). Results for 100 individual specimens with urate concentrations ranging from 16 to 134 mg/L agreed well with the comparison method, both for serum and whole blood. We saw no significant interference from lipemia or hemoglobin. Bilirubin interfered at concentrations greater than 100 mg/L. Hematocrit variation between 25% and 55% did not affect results for whole blood; variation of the applied sample volume from 28 microL to 35 microL (stated sample volume requirement: 30 microL) did not significantly influence the measured value. We consider results produced by the system to be of the same analytical quality as those obtained by the more conventional solution-chemistry methods that are currently available.
Asunto(s)
Ácido Úrico/sangre , Bilirrubina/sangre , Colesterol/sangre , Reacciones Falso Positivas , Hematócrito , Humanos , Peroxidasa , Control de Calidad , Estadística como Asunto , Triglicéridos/sangre , Urato OxidasaRESUMEN
BACKGROUND: To effectively assess and correct for intermethod variability, calibration and control materials (CCMs) must show the same intermethod behavior as patient sera, i.e., they must be commutable. We describe the commutability of selected CCMs for lipase assays, the impact of noncommutability of CCMs in normalizing patient results, and characteristics of reagents that affect assay specificity and commutability. METHODS: Lipase was measured in 98 patient sera and in 29 commercial CCMs, with 2 commercial methods using different substrates and with 4 experimental methods using 1,2-o-dilauryl-rac-glycero-3-glutaric acid-(6'-methylresorufin) ester as substrate and colipase as cofactor, but differing in the stabilizing proteins used and in the size of the substrate micelles. RESULTS: The noncommutability rate, i.e., the frequency of aberrant intermethod behavior of CCMs in comparison with patient sera, was 27% for liquid CCMs and 47% for lyophilized CCMs. The normalized residuals, measuring the degree of noncommutability, were -2.3 to 2.4 for CCMs with "normal" lipase activity, and -3.5 to 21.7 for CCMs with higher lipase activity. Recalculation of patient results with CCMs as calibrators decreased or increased the original bias according to whether the CCMs were commutable. CONCLUSIONS: For the lipase methods in this study, the frequency of noncommutability of CCMs is affected by assay-specific characteristics, including size of substrate micelles and the presence or absence of added proteins.
Asunto(s)
Lipasa/normas , Calibración , Humanos , Indicadores y Reactivos , Modelos Lineales , Lipasa/sangre , Estándares de Referencia , Sensibilidad y EspecificidadRESUMEN
In assessing interference from bilirubin, the use of a synthetic soluble derivative (ditaurobilirubin, DTB) is recommended as a surrogate for the natural conjugates (Bc). We compared the interference effect of unconjugated bilirubin (Bu), Bc, and DTB, using six mechanized methods for serum creatinine measurement. No significant interference was noted in methods that include removal of proteins or in an enzymatic method involving NADH oxidation. Heavy (negative) interference was observed in an alkaline picrate method, and in direct enzymatic methods based on hydrogen peroxide measurement: interference was always more pronounced in the presence of the two soluble derivatives (Bc and DTB), whose interference was of the same magnitude. These results point out the utility of testing for bilirubin interference by using soluble derivatives, in addition to Bu, and suggest the feasibility of using DTB as a surrogate for Bc for this purpose.
Asunto(s)
Bilirrubina/análogos & derivados , Creatinina/sangre , Taurina/análogos & derivados , Humanos , Indicadores y ReactivosRESUMEN
We evaluated myocardial release of cardiac troponin I (cTnI) in patients treated with conventional coronary artery bypass grafting (CABG), which employs extracorporeal circulation, and different kinds of minimal invasive coronary artery bypass grafting (MICABG), a surgical technique where the operation is performed without extra-corporeal circulation. Furthermore, we evaluated the usefulness of serum cTnI measurement to detect perioperative myocardial infarction (PMI) after coronary artery bypass surgery. Thirty-one patients were included: sixteen underwent CABG, fifteen underwent different MICABG and five patients had PMI. Blood specimens for cTnI measurements were collected up to 72 hours after opening the graft. Aortic cross-clamping time was a minor determinant of myocardial damage; on the other side, the trauma during surgery correlated with the number of involved arteries and with the manoeuvre employed to obtain heart dislocation, and appeared a more important determinant of myocardial damage. In patients with PMI, the cumulative release of cTnI was higher than in patients free from PMI; however, only after 24-72 hours we observed significant differences in serum cTnI values, because the increased perioperative values of cTnI complicated the interpretation of the myocardial status and a single cut-off could not be used to exclude PMI.
Asunto(s)
Puente de Arteria Coronaria , Infarto del Miocardio/sangre , Infarto del Miocardio/cirugía , Troponina I/sangre , Humanos , Procedimientos Quirúrgicos Mínimamente Invasivos , Reproducibilidad de los ResultadosRESUMEN
Some aspects of the measurement of alkaline phosphatase activity concentration in human serum, using N-methyl-D-glucamine as a buffer, were evaluated with a view to the possible routine use of the method. The evaluated characteristics included: the temperature-dependence of the pH of the buffer; the effect of adding the magnesium/zinc ions buffer; the effect of the serum volume fraction; the substrate-starter versus the serum-starter mode; the effect of modifying the formulation of reagents; the within-run and the between laboratory imprecision; the correlation with an alternative routine method. Also, sex- and age-related reference values were produced, based on 2968 values from selected reference sample groups in 7 laboratories. In general, the results demonstrate the robustness of the method, its adaptability to a variety of mechanized analysers, and hence its feasibility as a routine measurement method.