血清酶测定结果可溯源性研究

目的　通过对酶标准品的测定 ,探讨血清酶测定结果的可溯源性。方法　向参加研究的实验室 (50家 )各发放酶校准品 1支 ,各实验室用常规方法测定校准品 ,记录检验结果 ;计算校准品测定结果总体均值与标示值间的偏倚。结果　丙氨酸氨基转移酶 (ALT)、天门冬氨酸氨基转移酶(AST)、肌酸激酶 (CK)和乳酸脱氢酶 (LD)的测定结果均值与标准品标示值的相对偏差≤± 10 % ,分别为 6 3 % ,5 5% ,-5 9%和 -5 0 % ;碱性磷酸酶 (ALP)的实验室检测结果与标准品标示值间的相对偏倚为 -3 6 6% ,淀粉酶测定结果的实验室间CV为 2 9 2 %。结论　我国临床实验室对血清酶的测定结果有向国际标准溯源的基础     

Calibration standards and phantoms for fluorescence optical measurements

We report on the development and production of tissue-like optical phantoms for detection and characterization of fluorescent objects close to the tissue surface. The phantoms mimic bulk optical properties such as light scattering and absorption of female breast tissue and human skin. Fluorescence sources are embedded for a given well-defined localization and volume to simulate pathologic lesions that are marked by specific molecular substances labeled with a fluorescent dye. The phantoms were made of epoxy resin with added titanium dioxide and black epoxy color paste for adjustment of the tissue-like scattering and absorbing properties. Indocyanine green was used as a fluorescent dye. The phantoms were evaluated with an experimental set-up for a new method for in vivo laser fluorescence tomography in the frequency domain.     

酶测定中试剂和校准物对溯源的影响

目的研究试剂和校准物对酶测定溯源的影响。方法用美国德灵公司生产的DIMENSION RXL全自动生化分析仪及配套的试剂、校准物作为标准测试系统,朗道试剂和德灵校准物作为测试系统1,朗道试剂及配套校准物作为测试系统2,分别校准后测定新鲜临床标本,同时进行2个水平的质控。结果测试系统2和标准测试系统产生的回归关系更密切。结论配套的试剂和校准物能建立更好的溯源性,非配套的试剂和校准物对测试结果的溯源性会产生负面影响。     

Isoamylase measurements with monoclonal antibody test strips

A test strip with fixed monoclonal antibodies binding human salivary amylase (5) was used for the measurement of pancreatic amylase in human sera. The amount of salivary isoamylase adsorbed by the test strip increased with the amount of enzyme in the test solution, reaching a maximum absorption capacity of 620 mU at a test quantity of 900 mU. At about 160 U/l of pancreatic isoamylase (in a mixture containing 214 U/l of total alpha-amylase, both enzymes with 4-nitrophenyl-alpha-D-maltoheptaoside at 25 degrees C) imprecision within-run (and day-to-day) was 2.8% (and 6.8%) at this level of clinical importance. The preliminary 95%-reference interval from 33 healthy adults was calculated to be 16.6-44.2 U/l. The method proved valuable for the interpretation of slightly elevated alpha-amylase levels (up to twice the upper reference limit) and unusual lipase/amylase ratios.     

Use of the enzyme method for antibody identification

Enzyme methods for red blood cell antibody testing may have two goals: detection of weak antibodies by increasing the strength of the reactions and differentiation of the antibodies in an antibody mixture by abolishing the reaction of antibodies against enzyme-labile antigens. We analyzed the phenotype listing sheets of all lots of one year (expiration date in 2002) of 8 products (5 manufacturers) (together 130 worksheets). The aim was to find out how often some antibodies could only be detected after enzyme treatment, when there is an additional antibody against one of the following enzyme-labile antigens in the patient's serum: Fya, Fyb, M, N, S and s. If there is one of these antibodies against an enzyme-labile antigen, an additional antibody against one of the following enzyme-stable antigens D, C, E, c, e, CW, K, Kpa, Jsa, Jka, Jkb, Lea, Leb, P1, Lua cannot be detected on average in 20% of the panels. Moreover, in a further 37% of the panels there is no red blood cell suspension carrying the Kpa, in 44% none carrying the Jsa and in 19% no one carrying the Lua antigen. On the other hand, an antibody against one of the high-incidence antigens k, Kpb, Jsb or Lub can be detected in each of the 130 panels, also if there is an additional antibody directed against one of the above-mentioned enzyme-labile antigens. As also nowadays in some antibody identification panels antibodies against some enzyme-stable antigens might be covered by antibodies against enzyme-labile antigens, the enzyme method can be a helpful completion in antibody differentiation in some sera with multiple antibodies.     

The diagnostic value of urinary enzyme measurements in hypertension

N-Acetyl-beta-D-glucosaminidase (NAG), beta-D-galactosidase, alkaline phosphatase (ALP) and leucine aminopeptidase (LAP) were assayed in the urine of 100 normal and 112 hypertensive subjects. Age-related urinary activities for these enzymes in the normotensive control subjects are presented. A new procedure for the assay of urinary ALP using 2-methoxy-4-(2'-nitrovinyl)phenyl (MNP) phosphate is described. Thirty-five of the hypertensive patients were considered to have primary renal disease. The urinary activity of NAG was increased in 27 (77%) of these patients and the detection of primary renal disease was not enhanced by measurements of the other urinary enzymes. Testing the urine both for NAG activity and protein, led to the detection of 91% of these patients. The assay procedures described are simple to perform and can be carried out in outpatient clinics. The measurement of urinary NAG activity is a cheap and reliable method for detecting renal disease in hypertensive patients but maximum diagnostic yield is achieved when proteinuria is determined as well.     

Repeatability of fibre type and enzyme activity measurements in human skeletal muscle

Summary. In order to assess the variability in repeated determination of human muscle fibre type distribution, fibre area and enzyme activity measurements, two biopsies were taken within 10 days in the same vastus lateralis for 12 females and 13 males, and in the right and in the left muscles for 25 other subjects (13 females and 12 males). Within muscle, intraclass reliability coefficients were 0·88, 0·82 and 0·56 for type I, IIa and IIb per cent fibres, respectively, and ranged from 0·74 to 0·82 for fibre areas and from 0·71 to 0·90 for enzyme markers of different metabolic pathways. Correlations between right and left muscle measurements were also high for fibre areas (from 0·85 to 0·91) and enzyme activities (from 0·71 to 0·87), except for phosphofructokinase (r=0·63). In contrast, the right and left thigh muscle correlation reached 0·67, 0·40 and 0·64 for type I, IIa and IIb fibre distribution, respectively. Thus, the variation in muscle sampling and technical procedures reached about 15% of the total variation (i.e. total differences between subjects) for the proportion of fibre type I and IIa and about 20–25% for fibre areas and enzyme activities. On the other hand, the technical error for the proportion of fibre type I and IIa is about 6–7%. This implies that differences brought about by any experimental treatment on these skeletal muscle characteristics in human studies have to be of a relatively large magnitude before being detectable. On the other hand, fibre areas and enzyme activities measured in single needle biopsy sample, from one of the vastus lateralis muscles, are quite representative of the other vastus lateralis. Similarity in fibre type proportion between right and left vastus lateralis cannot be postulated, however, without investigating both muscles.     

Solid phase measurements of antibody and lectin binding to xenogenic carbohydrate antigens

OBJECTIVES: In future pig-to-man xenotransplantation it is important to master tools that identify potentially xenogenic alphagalactose (Galalpha) antigens in the doner tissue. DESIGN AND METHODS: We have measured the binding potentials of Galalpha detecting lectins and antibodies, including a naturally occurring subfraction from human serum, to Galalpha containing neoglycoproteins and mouse laminin that were immobilized on microtiter plates. RESULTS: Galalpha reactive antibodies with similar monosaccharide specificity have distinct structural preference for sugar ligands. Laminin and neoglycoproteins were treated with alpha-galactosidase and subsequently incubated with antibodies and lectins. The enzyme treatment was more deleterious on antibody binding than on lectin binding. CONCLUSION: Antibodies and lectins may bind to different galactose determinants on the glycoproteins. Two anti-Galalpha1 antibodies that both have been raised against glycans on rabbit red blood cells may recognize Galalpha-antigens with varying specificities. Binding results obtained after digestion with alpha-galactosidase indicate that some xenoreactive Galalpha groups are not directly accessible for removal by the enzyme.     

Evaluation of an enzyme immunoassay kit for estrogen receptor measurements

Using an enzyme immunoassay (EIA) procedure, we evaluated the ability of the glass beads in the ER-EIA kit of Abbott Laboratories, which were coated with monoclonal anti-estrogen receptor (ER) antibodies, to bind hormone-free and hormone-filled Type I ER in cytosols, buffer washes, and 0.4 mol/L KCI extracts of ultracentrifugal pellets of breast cancer tissue homogenates. The unmodified ER-EIA technique yields higher values than does the dextran-coated charcoal (DCC) method for Type I ER. However, the antibody-coated beads fail to bind hormone-free ER and react with only a certain proportion of ER-[3H]estradiol complexes. The antigen saturation limit of the beads could not be determined because the quantity of antigen bound by the beads was disproportionate and unrelated to the total amount of the antigen available in the cytosols. In buffer extracts of tissue pellets that were ER-negative by the DCC assay, the EIA method detected high quantities of ER. We recommend checking the ability of the monoclonal antibodies to recognize proteins other than Type I ER in the extra-nuclear and nuclear compartments of target cells before using them for immunohistochemical detection of ER.     

Calibration of a distributed SWE model using MODIS snow cover maps and in situ measurements

In this letter, we propose the calibration procedure for a Snow Water Equivalent (SWE) forecasting model, using Moderate-Resolution Imaging Spectroradiometer (MODIS) multi-temporal snow cover maps and in situ measurements. The presented study refers to one of the largest artificial lakes in the Western Europe – the Serre-Ponçon reservoir, on the Durance river, in the region of the French Alps. The SWE model, an integral part of the MORDOR (MOdèle à Réservoirs de Détermination Objective du Ruissellement) hydrological model, provides SWE as a function of local precipitation and temperature, as well as of accumulation and melting correction coefficients. The principal motivation for the proposed calibration method comes from the significant model sensitivity with respect to these two coefficients, which, given that they account for the influences of topology and mountain winds, ought to vary spatially. Three different optimization procedures are compared using the set of in situ measurements acquired by the EDF (Eléctricité de france) cosmic-ray snow sensors for 4 out of 36 ground stations in the regions of interest. The appropriate optimization method is selected and the corresponding representative optimal coefficients are derived for these four stations. Further, by combining the selected optimization algorithm and the continuous activation function, we propose a new method for deriving the spatially varying coefficients characterizing the entire region, using multi-temporal MODIS snow cover binary maps. When analysed with respect to the mean square error (MSE) criterion, the SWE model, calibrated in this manner, appears to be significantly more accurate than the original version (using a priori estimated, spatially fixed coefficients). Furthermore, the calibration procedure based on MODIS data is comparable and, for some ground stations, exhibits even better performances than the one based on the in situ measurements.     

Homogeneous enzyme immunoassay for macromolecular antigens using hybrid antibody

A new homogeneous enzyme immunoassay for the determination of macromolecular antigens has been developed based upon competitive enzyme inhibition using hybrid antibody-containing anti-ligand and anti-enzyme inhibitory antibodies. The hybrid antibody was prepared by the reaction of anti-glucose-6-phosphate dehydrogenase (G6PDH) mouse IgG maleimide with anti-human IgM goat IgG-SH. In the absence of the antigen, the hybrid antibody inhibits the enzyme activity. On the other hand, in the presence of an excess amount of antigen, the hybrid antibody binds to the antigen and is unable to bind with the enzyme. The hybrid antibody inhibited 80% of the original activity of enzyme and saturated the inhibition of G6PDH within 10 min. The enzyme activity was proportional to the concentration of human IgM. The measurable range for IgM was 78 μg/ml to 1 mg/ml. Human IgG present in the serum did not affect this method.     

Qualitative membrane enzyme immunoassay for detection of cytomegalovirus antibody.

A new qualitative membrane immunoassay for use in detecting IgG antibodies to cytomegalovirus (CMV) was compared with a standard microplate enzyme immunoassay (EIA). A total of 179 patients were tested by the EIA for IgG- and IgM-specific antibodies to CMV and then compared with the new membrane immunoassay. Among the 179 sera, five specimens were repeatably invalid. Among the 174 evaluable sera, 128 (73.6%) were positive for IgG antibody to CMV by the EIA method. Of these 128 sera, the Transtat-CMV was positive in 124 (96.9%). The remaining 46 sera were negative for IgG antibodies by both methods. The Transtat-CMV membrane immunoassay appears to be a reliable and easily used method not requiring additional equipment or extended incubation times. As a qualitative-only assay, it cannot be used for diagnosis.     

Commutability and traceability in EQA programs

ObjectivesThe concept of commutability of samples has focused laboratories on the importance of traceability. However, the critical role of External Quality Assurance (EQA) in achieving the primary role of traceability (i.e. facilitating comparable patient results in different laboratories) has largely been lost. The aim of this paper is to review the role of EQA in achieving traceable/commutable results.Design and methodsThe role of commutability and traceability in EQA and Internal Quality Control (IQC) are discussed. Examples of commutable EQA samples are given to highlight the problem of assuming EQA material does not behave like patient samples.ResultsWe provide the conventional traceability chain (top down) and the role of EQA in a “bottom up” model using conventional EQA samples.ConclusionsThe quest for commutable samples has compromised the value of EQA without an understanding that some EQA materials are commutable for some measurands.EQA plays a key role in performance improvement, but laboratories need to understand the importance of using a range of values appropriate to the assay to identify areas of quality need. Traceability and EQA using conventional samples are not mutually exclusive concepts.     

Metrologic traceability of total thyroxine measurements in human serum: efforts to establish a network of reference measurement laboratories

BACKGROUND: Assuring/demonstrating metrologic traceability of in vitro diagnostics necessitates the availability of measurand-specific reference measurement systems (RMSs) and the possibility for industry to work with competent reference measurement laboratories (RMLs). Here we report the results of a European project to investigate the feasibility of developing a RMS for serum total thyroxine. METHODS: Four candidate RMLs (cRMLs) developed/implemented variants of a candidate reference measurement procedure (cRMP) based on isotope dilution-liquid chromatography-mass spectrometry. The sole constraint implemented was calibration with a common thyroxine primary calibrator. The RMPs were externally validated and assessed for comparability in round-robin trials using common samples, i.e., 5 lyophilized and 33 frozen native sera. At the same time, the performance of the cRMLs organized in a network was assessed. For uniform external quality assessment, common performance specifications were agreed on. RESULTS: All cRMLs performed the cRMPs with fulfillment of the predefined specifications: total and between-laboratory CVs < or =2.0% and 2.5%, respectively, and a systematic deviation < or =0.9%, estimated with a target assigned from the mean of means obtained by the cRMLs. The mean expanded uncertainty for value assignment to the native sera was 2.1%. CONCLUSIONS: A network of cRMLs, with externally conformed competence to properly perform RMPs, has been established. Performance specifications were defined and will form the basis for admittance of new network members. A serum panel, successfully targeted during the validation process, is available for split-sample measurements with commercial routine measurement procedures. The model can now be used for other measurands for which traceability to the Systeme International d'Unites is needed.     

Noninterchangeability of specific radioimmunoassay and monoclonal antibody fluorescent polarization immunoassay in cyclosporine measurements

Summary— This study compares cyclosporine (CsA) concentrations in whole blood from patients receiving bone marrow ( n = 10), renal ( n = 48), heart ( n = 50) or liver ( n = 50) transplants, as measured by monoclonal antibody flurorescence polarization immunoassay (FPIA) and specific ¹²⁵I-radioimmunoassay (RIA). The FPIA overestimated CsA by an average of 25%. Results were higher for all indications: FPIA/RIA ratios were 1.17 for bone marrow, 1.23 for renal and 1.27 for both heart and liver transplants, and these values were significantly different from 1.0. The percentage of overestimation was higher at low CsA concentrations (≤ 100 μg/L) than at high CsA concentrations (≥ 400 μg/L). In all indications, results by both methods correlated well ( r > 0.96) but slopes and intercepts were different from 1.0 and 0.0, respectively, and these parameters varied greatly between the grafted populations. These findings obtained with the two methods could not be attributed to matrix effect because the mean FPIA/RIA ratio for spiked control samples was 1.0. The discrepancy between the FPIA and RIA could be explained by the lower specificity of the monoclonal antibody contained in the FPIA kit. These results suggest that FPIA is not as accurate as RIA and that the two methods are not interchangeable in CsA level measurement.