The dehydroalanine effect in the fragmentation of ions derived from polypeptides.

Tandem mass spectrometry is a powerful approach for the analysis of peptides and proteins due to the primary structural information inherent in the observed products. The fragmentation of peptides and proteins depends heavily on the sequence and ion type of the species of interest. In this perspective special feature article, Scott McLuckey and co-authors show that peptides and proteins containing dehydroalanine, a nonproteinogenic amino acid with an unsaturated side-chain, undergo enhanced cleavage of the N-Cα bond of the dehydroalanine residue to generate c- and z-ions. Since these fragment ion types are not commonly observed upon activation of positively charged even-electron species, they can be used to identify dehydroalanine residues and localize them within the peptide or protein chain. Scott McLuckey is Professor of Chemistry at Purdue University (West Lafayette, IN). His research interests are centered on gas-phase ion chemistry and instrumentation for organic and biological mass spectrometry.

Is the low tri-iodothyronine state a crucial factor in determining the outcome of coronary artery bypass patients? Evidence from a clinical pilot study.

The cardiovascular system is an important target for thyroid hormones. The present study evaluates the changes affecting thyroid hormone metabolism during and 6 days after coronary artery bypass and their relationship with the post-operative outcome of the patients. Thirty-three patients were enrolled in the study; their thyroid hormone profiles were determined at 13 sampling points during surgery and for 6 days afterwards. Serum total tri-iodothyronine (T3) and free T3 (FT3) concentrations decreased significantly after surgery (P<0.001) and they remained significantly low until the end of the study. Free thyroxine (FT4) and T4 declined significantly immediately after surgery (P<0.05 for FT4, P<0.001 for T4) but they returned to baseline values (24 h and 96 h post-surgery respectively). Serum reverse T3 increased remarkably 36 h after surgery (P<0.001) and remained significantly higher than the baseline value throughout the study. A relevant finding was that the days of post-operative hospitalization (10+/-3 days, means+/-S.D.) was inversely correlated with the slope of the recovery of T3 concentration (P<0.001) or with the area under the plasma curves of T3 (P=0.024, time range 72-144 h) and the FT3/FT4 ratio (P=0.037, time range 72-144 h) during the post-operative period. Our data suggest a prolonged reduction of T4 to T3 conversion in patients undergoing cardiac surgery and indicate that the recovery period is the most critical in the evaluation of a possibly successful approach for T3 substitutive therapy.

Performance of immunoassays for ca 19-9, ca 15-3 and ca 125 tumour markers evaluated from an international quality assessment survey.

Data collected in the 1993 and 1994 cycles of an international External Quality Assessment (EQA) programme were cumulatively analysed to evaluate the analytical performance of the methods currently in use for routine assay of mucinous tumour markers CA 19-9, CA 15-3 and CA 125. On average the between-laboratory variability was 14.7 and 15.8 CV% for CA 15-3 and CA 125 respectively. For CA 19-9, a markedly worse between-laboratory variability (on average 27.2 CV%) was found; the agreement of CA 19-9 results worsened in the last few years when new isotopic techniques became available. The variability component attributable to systematic differences between methods/kits was relatively small for CA 15-3 and CA 125 (17% and 21% of the total variability), while it was markedly larger for CA 19-9 (45% of the total variability). The precision of the methods/kits most often used in the survey ranged from 9.6 to 13.9 CV% for CA 125 and from 10.8 to 14.1 CV% for CA 15-3. For these two tumour markers the precision of the traditional IRMAs does not appear to be different from that of the new fully automated non-isotopic techniques. The precision of CA 19-9 methods was on average worse from (11.9 to 19.2 CV%), even though the precision of the two automated systems was better than that of IRMAs. In conclusion, the results of this study indicate that the between-laboratory agreement for CA 15-3 and CA 125 assays appears satisfactory while the CA 19-9 assay shows larger differences between methods and is affected by poorer precision of kits.

Comparison of immunoassays for tumor markers CA 19-9, CA 15-3 and CA 125: data from an international quality assessment scheme.

Data collected in the 1993 and 1994 cycles of an international external quality assessment (EQA) program and in a national multicenter collaborative study were cumulatively analyzed to evaluate the standardization of the methods currently in use for the assay of mucinous tumor markers CA 19-9, CA 15-3 and CA 125. On average the between-laboratory variability was 15.2 and 16.0 CV% for CA 15-3 and CA 125 respectively; the between-laboratory variability found for CA 19-9 was markedly worse (mean 28.3 CV%). The variability component attributable to systematic differences between different methods/kits was relatively small for CA 15-3 and CA 125 (18% and 24% of the total variability) but markedly larger for CA 19-9 (48% of the total variability). The agreement of CA 19-9 results worsened in the last few years when new nonisotopic techniques became available. The precision of the methods/kits most used in the survey ranged from 9.9 to 13.3 CV% for CA 125 and from 11.6 to 13.9 CV% for CA 15-3. For these two tumor markers the precision of the traditional IRMAs does not appear different from that of the new fully automated nonisotopic techniques. The precision of CA 19-9 methods was on average worse (from 11.7 to 19.6 CV%) although two automated systems exhibited a precision better than that of IRMAs. In conclusion, the results of this study indicate that CA 15-3 and CA 125 are satisfactorily assayed whereas CA 19-9 assay appears affected by larger differences between methods and by poorer precision of laboratories and kits.

'Oncocheck': an international external quality assessment scheme for immunoassays of tumor markers.

Starting from November 1990, an international External Quality Assessment Scheme (EQAS) for immmunoassays of tumor markers has been organized. Presently, 238 laboratories from France, Germany, Italy, Japan and Spain participate in the scheme. In this report the main features of the EQAS and data processing are outlined. Results collected during the 1992-cycle allow evaluation of the state of the art of AFP, CEA, CA 19-9, CA 15-3, CA 125 and PSA immunoassays. According to their analytical performances, the 6 tumor marker immunoassays can be classified into several groups, the first including AFP and CA 15-3 for which both total variability and within-kit agreement are good. For CEA assay, performance can be considered as satisfactory even though further improvements of between-lab agreement would be welcome. For the 3 other tumor markers, the higher total variability indicates an urgent need for a better standardization by improvement of either both within-kit and between-kit agreements (CA 19-9) or between-kit agreement mainly (PSA, CA 125).

Analytical performance of CA 19.9, CA 125 and CA 15.3 assays as observed through an external quality assessment program.

Immunoassays of the tumor markers CA 19.9, CA125 and CA 15.3 are generally acknowledged to be a useful tool in the management of cancer patients. As a consequence, many methods developed by different companies are now commercially available. However, discrepancies have been described in the results of marker determinations even when the same monoclonal antibody was used. An external quality assessment (EQA) was carried out; starting from 1989 about 110 laboratories participated; since December 1991 the program was linked with the interlaboratory program Oncocheck organized by the Service de Radiopharmacie et Radioanalyse, University of Lyon. At present more than 200 laboratories of many European countries are involved: cumulatively 47 quality control samples have been prepared and sent to the participants. This manuscript is a report on data collected for CA 19.9, CA 125, and CA 15.3.

Immunoassay of CEA, CA 19-9, CA 125, and CA 15-3 on the automated systems ES 300 and ES 600: methodological evaluation from a multicentre collaborative study.

The analytical performance of the automated Enzymun Test System ES 300 and ES 600 (developed by Boehringer Mannheim) for the assay of the tumour markers CEA, CA 19-9, CA 125, and CA 15-3, was assessed from data collected in a multicentre collaborative study in which eleven laboratories were involved. Results of the 1990 cycle of the external quality assessment (EQA) scheme for tumour markers, supported by the Italian National Research Council (CNR), were also used in this evaluation. The within-assay and between-assay precision was found to be 2.0 and 4.3 CV% for CEA, 2.9 and 6.8 CV% for CA 19-9, 3.6 and 9.4 CV% for CA 125, 2.9 and 6.0 CV% for CA 15-3. The between-lab variability of the four tumour markers on ES 300 and ES 600 systems was 9.4, 10.6, 11.9, 9.2 CV% for CEA, CA 19-9, CA 125 and CA 15-3 respectively. These values were comparable to or better than those obtained with the most precise manual kits used by laboratories participating in the 1990 EQA cycle. The agreement between the results from the Enzymun Test and those obtained using other method/kits was evaluated by assaying control samples previously circulated either in the CNR EQA or in the German EQA. The regression analysis indicates that for CEA, CA 125 and CA 15-3 assays the results produced by ES 300 and ES 600 are in good agreement with the consensus means of the EQAs; CA 19-9 results exhibit a worse correlation and are generally lower than the consensus mean. The linearity of the assays for the four tumour markers was checked by dilution tests performed by participants in the collaborative study; in all cases the dilution of the sample did not affect the values obtained.

External quality assurance of the carcinoembryonic antigen (CEA) assay: main findings in six years' experience.

In 1984 we initiated a national external quality assessment (EQA) program (supported by the Italian National Research Council, CNR) for the CEA assay; at present, about 200 Italian laboratories are participating in the program. The laboratories assayed the quality control (QC) samples according to their routine procedures and returned the results together with the name of the method/kit they used. The collected results were computer-processed and reports were sent back to the participants. A significant reduction of the CVt (mean between-laboratory agreement) of the CEA assay was observed throughout the EQA survey (from 35% in 1985 to 20-25% in the last cycles). In order to better clarify the differences in variability observed in the first QC cycles against the last ones, we used the ANOVA technique to evaluate the components of variability. The improvement in between-laboratory agreement was mainly due to the reduction of the between-kit component (from 30.5% to 15.2%), rather than to the smaller decrease observed for the within-kit variability (from 18.4% to 14.0%). The results reported for QC samples from different materials showed differences in the between-lab variability and substantial changes of the kit biases, thus suggesting a different specificity of the antibodies used in the various method/kits against different families of CEA molecules. Considerable uncertainty was also encountered in the clinical classification of low pathological samples, which seems mainly due to the variability in cut-off values used by the laboratories for the clinical assessment of the same analytical results. Our data indicate a progressive increase in the reliability of CEA determination during our study and confirm that EQA has improved the reliability of analysis carried out by the participating laboratories, thus stimulating the kit manufacturers to provide more reliable products.

The CNR external quality assessment program for immunoassays: statistical analysis and reports for participants.

The results collected in the CNR/Tecno-standard external quality assessment (EQA) program for immunoassays of hormones and tumor markers are computer-processed to prepare a "periodic" report and an "end-of-period" report to be sent back to the participants in the survey. The aim of the periodic report is to allow comparison between the result obtained by a laboratory on a single EQA sample with those of all the other laboratories and with the users of the same method/kit; the report contains a histogram of all results and the mean, SD, CV, median and range (computed after trimming of outliers). The same statistics are also reported for data grouped according to the method/kit used. The end of period report provide the participant with a scored estimate of individual analytical performance (average bias and average imprecision) achieved assaying all the EQA samples dispatched in the control cycle (usually a six-month period during which 12-18 samples have been assayed); this cumulative report contains estimates of the performance of those kits more widely used in the survey. Beside helping laboratories to monitor their performance against an external reference, the EQA allows the collection of a large amount of data from which the state of the art and trends in the quality of immunoassays can be soundly evaluated and documented. To achieve this aim, the average total variability is computed from all data collected in the EQA cycle; this index is used for comparing the between-laboratory agreement of different immunoassays and for demonstrating trends of the quality over time.(ABSTRACT TRUNCATED AT 250 WORDS)

External quality assessment of assays for hormones and tumor markers in Italian laboratories.

External quality assessment (EQA) programs run by CNR/Tecnostandard for immunoassays of hormones and tumor markers, started in 1980, presently include as many as 20 analytes; about 300 laboratories are involved in these programs. For all immunoassays submitted to the EQA, the inspection of cumulative results allows the current situation to be documented for total variability and its within-kit and between-kit components (the former accounting for the reproducibility and robustness of the kits and the latter for their systematic differences of estimation). For 13 assays subjected to EQA for longer, the variability trends over time are depicted, and single factors affecting the overall quality of particular assays are identified. Among these, experimental simplification of kit structure, alignment of calibrators with an acknowledged reference material, and adoption of monoclonal-antibody based two-sites assays can be mentioned. On the contrary, neither automation of the procedures nor (more expectedly) increasing use of nonisotopic techniques has proved effective in significantly improving the analytical quality.

A new chemiluminescence immunoassay for triiodothyronine and thyroxine: evaluation using quality control sera assayed in an interlaboratory survey.

A recently developed chemiluminescence immunoassay system (LIA-mat) for triiodothyronine and thyroxine, set up by Byk-Sangtec Diagnostica (Dietzenbach, Germany), has been evaluated and compared with radioimmunoassays and with a chemiluminescence enhanced enzyme immunoassay (Amerlite), using control materials circulated in a national interlaboratory quality control, as well as patient sera. The LIA-mat assays are competitive methods which use coated monoclonal antibodies and triiodothyronine- or thyroxine-ABEI (aminobutylethylisoluminol) conjugate as tracers. The working range of LIA-mat T3 (computed from the within-assay precision profile) extended from 1.4 to 12.3 nmol/l; the between-assay precision was 8.1 - 19.3 CV%. Regression analysis of the LIA-mat T3 results (y) against the consensus means (x) of the participants in the national interlaboratory survey yielded: y = -0.14 + 1.05 x, r = 0.95. The working range of LIA-mat T4 extended from 33 to 515 nmol/l; the between-assay precision was 5.4 - 9.2 CV%. An excellent agreement was found between LIA-mat T4 results (y) and the consensus means (x) of the laboratories participating in the national interlaboratory survey (y = 3.79 + 1.02 x, r = 0.98).

A method for retracing the putative antigen/antibody ratio of immune complexes in biological fluids.

It is well known that immune complex (ICs) diseases are caused by a number of factors which influence the localization, clearance and inflammatory potential of ICs. Several studies suggest that the Ag/Ab ratio is one of the most important of these. Previous studies have clarified that IC detection methods which differ, either in their recognition unit or in the phase used (solid or liquid), show a very poor correlation with each other. This study was developed in order to verify the hypothesis that different methods recognise different kinds of ICs on the basis of their Ag/Ab ratio. We used 3 homogeneous EIAs employing a probe complex enzyme--anti enzyme which competes with circulating ICs for the recognition unit (bovine conglutinin, C1q or monoclonal rheumatoid factor) to detect 10 unrelated in vitro-made ICs at different relative Ag/Ab concentrations (from 8 x Ag excess to 8 x Ab excess). We demonstrated that the 3 recognition units recognised the ICs principally on the basis of their Ag/Ab ratio. These results were then used to set up a mathematical model capable of retracing the Ag/Ab ratio of the ICs present in unknown samples. This was employed to test a panel of sera from patients with systemic lupus erythematosus, essential mixed cryoglobulinemia and rheumatoid arthritis; we obtained very suggestive results but they require further prospective studies to understand the full significance of this parameter.

External quality control survey for alpha-fetoprotein assay.

Starting from 1984 we organized an interlaboratory quality control (QC) survey for the tumor marker AFP in which 100-150 laboratories have been involved. Seven consecutive QC cycles have been carried out during the period 1984-1988; 15-20 samples have been prepared and sent in each cycle. The 11 kits more used in the survey, which use three different immunological methods (3 kits EIA, 3 kits RIA, and 5 kits IRMA) were evaluated. Since the results of AFP assays were expressed as ng/ml of different local standards of the various kits, the participants were asked to convert their results in UI/ml of the 1st IRP 72/225 distributed by WHO. The total variability of AFP assay resulted approximately constant (19.9-24.7 CV%) during the all QC period. The validity of the consensus mean of AFP determinations obtained in the survey and assumed as reference value has been checked by recovery experiments. Regression analysis indicates that the consensus means found in these samples are in very good agreement with the added AFP. The analytical performance achieved by the most popular methods/kits, observed in the last period, are also reported.

Minimizing between-kit variability in immunoassay for carcinoembryonic antigen by use of a common standard.

Between-laboratory agreement of CEA determinations collected in a multicenter study has been substantially improved (CV decreased from 60.4 to 21.6%) by converting the results from mass concentration units (ng/mL) of local kit standards to int. units/L of the international standard first IRP 73/601. Conversion factors for the kits most used were computed, during the same survey, from results of two analytical-recovery experiments in which control samples, supplemented with the international standard, were analyzed by participating laboratories (n = 96).

Components of variance analysis of data produced in a national quality-control survey of radioimmunoassays of thyroxin, triiodothyronine, thyrotropin, prolactin, and progesterone.

Data collected in a collaborative survey for radioimmunoassays have been studied by using analysis of variance to estimate the within-kit (CVw.kit) and the between-kit (CVb.kit) components of the total variability (CVT). This analysis has been applied to the results for triiodothyronine, thyroxin, thyrotropin, prolactin, and progesterone produced by 80-150 laboratories that assayed blind, replicate samples. Total variability was lowest in the thyroxin assay (CVT = 10.9%), associated with a very close between-kit agreement (CVb.kit = 4.0%); in the triiodothyronine assay, on the other hand, the large between-kit component (CVb.kit = 10.1%) increased the total variability to 16.1%. In the prolactin assay the CVT of 19.3% included 17.5% CVw.kit and 8.1% CVb.kit. Assays for thyrotropin and progesterone involve analyses of two pools at different hormone concentrations. The CVb.kit component was very high in the low-concentration pool, both for thyrotropin (25.1%) and progesterone (45.2%); in the higher-concentration pool it decreased to 8.3% for thyrotropin but remained high (21.6%) for progesterone. Applying analysis of variance to the triiodothyronine and thyroxin data obtained by different laboratories using the same kit showed that most kits yielded significantly different measurements when used in different laboratories.