External Quality Assessment Schemes: need for recognised requirements.

Programs for Accreditation of clinical laboratories consider participation in External Quality Assessment Schemes (EQAS) a key element in the evaluation of testing procedures and improving them. One of the main functions of EQAS is to assess whether laboratories perform tests competently. It is therefore of utmost importance for laboratories to participate in EQAS that are in line with formally recognised requirements. Specific proposals have been made on how to design and execute EQAS by International Working Groups, but there seems to be no consensus on the best strategies to use and quality specifications to set out. The Clinical Pathology Accreditation (CPA) Program for EQA Scheme Accreditation (CPA-EQA) is the only program in Europe to provide a formal recognition of the quality of EQAS activities. The present paper reports on the experience of the Centre of Biomedical Research which is following an accreditation process for their own schemes in line with the CPA-EQA program and a proposal to set requirements that Italian schemes must follow to be recognised as valid and effective.

Assessment of package inserts for diagnostic kits.

To assure the quality of service in laboratory medicine, it is necessary to implement a quality system which comprises the entire testing process. The use of quality reagents is an important aspect of the process. Despite the fact that it is the responsibility of the laboratory to ensure the quality of the analytical system (including reagents) and since it is impossible to evaluate all commercial diagnostic kits, the laboratory often depends on statements issued by the manufacturer to select the most appropriate diagnostics for a particular laboratory. In this study we report the results of the analysis of information provided in 887 package inserts enclosed in the more widely used commercial diagnostic kits, following the Standard for the labelling of clinical laboratory materials of the European Committee for Clinical Laboratory Standards (ECCLS). Only a third of these were in agreement with the guidelines of ECCLS Standard, reporting complete and correct information. We believe that it is necessary to implement a constructive cooperation between manufacturers of diagnostic materials and clinical laboratories to produce a more uniform approach to improvements in laboratory quality assurance.

Troponin T, creatine kinase MB mass, and creatine kinase MB isoform ratio in the detection of myocardial damage during non-surgical coronary revascularization.

The presence of myocardial injury during non-surgical coronary revascularization has been evaluated by means of highly specific and sensitive biochemical markers. Troponin T, creatine kinase-MB isoenzyme mass concentration, and creatine kinase MB2/MB1 isoform ratio have been determined in 80 patients who underwent coronary revascularization with percutaneous transluminal coronary angioplasty (PTCA). Forty-five patients underwent balloon angioplasty, 15 rotational atherectomy, 10 directional atherectomy, and 10 elective coronary stenting. Serum concentration of the evaluated markers did not increase significantly after 57 uncomplicated revascularization procedures, including 15 rotablation procedures, nor after 8 PTCAs complicated by localized coronary type B and C dissections. Significant elevation of all markers above the upper limits of the reference interval (P < 0.05) was detected after occlusion of small side branches (< 0.5 mm diameter) in 5 patients. Creatine kinase MB2/MB1 isoform ratio was the earliest marker to increase. After recanalization of occluded vessels in 8/10 patients with 6-60 days old myocardial infarction only troponin T concentrations increased from a baseline of 0.28 microgram/l to a median peak of 0.80 microgram/l. This increase was statistically not significant (P = 0.12). In conclusion, myocardial damage was not detected following uncomplicated non-surgical revascularization obtained with different techniques. Markers of myocardial injury provide high sensitivity after small side branch occlusion.

New and traditional serum markers of bone metabolism in the detection of skeletal metastases.

OBJECTIVES: The evaluation of "new" and "traditional" markers of osteoblastic and osteoclastic activity, in patients with bone metastases. DESIGN AND METHODS: Our series consist of 40 patients with clinical, radiological, and scintigraphic evidence of bone metastases, and 40 age-matched healthy subjects. In all samples, traditional markers were evaluated by measuring total alkaline phosphatase (ALP), tartrate-resistant acid phosphatase (TrACP) activity, and osteocalcin (BGP) concentration. To assess new biochemical bone markers, bone isoenzyme of alkaline phosphatase (ALP-B) activity, carboxyterminal propeptide of type I procollagen (PICP), and carboxyterminal telopeptide of type I collagen (ICTP) concentrations were measured. RESULTS: Our findings showed that the best diagnostic efficiency is provided by ICTP (0.94) followed by total ALP (0.90), ALP-B (0.80), and TrACP (0.76). The efficiency of BGP and PICP was, instead, very low (0.64 and 0.60, respectively). CONCLUSION: Our results confirm the utility of the new serum markers such as ALP-B and ICTP assays in detecting bone metastases.

Different organization of von Willebrand factor oligomers in type-2A and -2B von Willebrand disease variants: effects of DDAVP infusion and protease inhibitors.

Plasma von Willebrand factor (vWf) displays a complex pattern of repeating multimers, whose heterogeneous size distribution seems to depend on the proteolytic cleavage of the constituent vWf subunit. Smaller vWf multimers are thought to derive by proteolytic cleavage of the larger forms. To clarify the relationship between large multimer representation and the structure of small vWf oligomers, DDAVP was infused in patients with type-2A and -2B von Willebrand disease (vWd) variants which lack circulating high vWf forms. Before infusion, high-resolution multimer analysis demonstrated a more pronounced representation of the satellite bands of each oligomer, mainly concerning fast-moving components, especially in type 2B vWd. After DDAVP, in type-2A vWd each oligomer displayed a different organization depending on whether restoration of large vWf multimers occurred. The lack of large vWf multimer restoration, as shown in citrated samples, was associated with the fast band being significantly more represented than the slower, and almost similar to the central component. In contrast, when the high-molecular-weight vWf forms were restored, as occurred in the samples collected in the presence of protease inhibitors, the relative representation of the fast- and slow-moving bands was similar to that of normal samples. In type-2B vWd, regardless of the anticoagulant used, DDAVP infusion did not restore large vWf multimers, and each oligomer displayed a significant increase in both the central band and fast-moving satellite, the fast being even more highly represented. These findings suggest that, regardless of the origin, the disappearance of large circulating multimers in type-2A and -2B vWd induces an increased representation of the fast-moving satellite of the low-molecular-weight multimers. Moreover, the time course of large and low/intermediate multimer decrease and increase provides a further demonstration that low vWf multimers derive from the larger ones, and that mainly the fast-moving band of the oligomer is involved.

Serum bone alkaline phosphatase in the follow-up of skeletal metastases.

The efficacy of bone alkaline phosphatase (ALP) isoenzyme measurement, using a lectin precipitation method, in confirming metastatic sites was assessed in 65 patients with cancer and skeletal (n = 44), hepatic (n = 15) or lymph node (n = 6) metastases; the control group consisted of 33 healthy adults. In all subjects, total ALP activity and osteocalcin were also assayed. Our results confirm that isoenzyme analysis is more specific than total enzymatic activity measurement in the identification of bone metastases: the mean for total ALP values was increased in all patients, while significantly high mean values of bone fraction (p < 0.05 by ANOVA) were observed only in patients with bone secondaries. In the serial monitoring of 9 patients with skeletal metastases, bone ALP values correlate with pain symptomatology: a progressive decrease in bone isoenzyme activity was observed in patients with a complete remission of pain after radiotherapy, while a progressive increase in activity was observed in the presence of increased bone pain. The measurement of bone isoenzyme activity is useful in screening for skeletal metastases; levels appear to correlate with the course of bone symptomatology, thus providing useful objective evidence of response to treatment.

Evaluation of a new automated system for the determination of CK-MB isoforms.

We evaluated a new analyzer (Cardio REP) specifically designed for cardiac CK-MB isoenzyme and isoforms activity, with a performance time of 24 minutes. Ten AMI patients, with times elapsed between the onset of chest pain and admission to hospital ranging from 30 minutes to 4 hours, were monitored every 3-4 hours until the 16th hour of hospitalization. In each serum sample, in addition to total CK-MB and CK-MB isoforms measured by the Cardio REP analyzer, we also assayed total CK activity, CK-MB activity by immunoinhibition method, CK-MB mass concentration, CK-MB isoforms by REP method, troponin T, and myoglobin. The precision study demonstrated acceptable within assay and between assay CVs% for total CK-MB (8.1 and 10.4), MB1 (9.1 and 14.2), and MB2 (9.1 and 8.2) isoforms. The method was found to be linear up to 371 U/L for MB2 isoform fraction and up to 516 U/L for total CK-MB. Results for CK-MB obtained with the Cardio REP correlated well with those for CK-MB activity obtained with the immunoinhibition method (r = 0.869) and those of CK-MB mass concentration (r = 0.923). The sensitivity of the Cardio REP CK isoforms method was found to be greater than that of the REP CK isoforms method. Time to first increased value of MB2/MB1 ratio and MB2 isoform was earlier in comparison to that for CK-MB mass concentration and similar to that for myoglobin, a marker that, however, lacks specificity. The diagnostic efficiency of CK-MB isoforms and the availability of a real-time, fully automated method for their measurement suggest that utilization of this biochemical marker in emergency for the early diagnosis of AMI.

Monitoring skeletal cancer metastases with the bone isoenzyme of tissue unspecific alkaline phosphatase.

The efficacy of bone alkaline phosphatase (ALP) isoenzyme measurement using lectin precipitation in confirming metastatic bone lesions was compared with total ALP and osteocalcin assay in serum. Sixty-five patients with cancer and metastases to bone (n = 44), liver (n = 15) or lymph nodes (n = 6) as well as 33 healthy adults were studied. Assay of bone ALP is as sensitive but more specific than assay of total ALP in the identification of bone metastases. On the other hand, bone ALP did not correlate with osteocalcin, as is the case in other bone diseases. In the serial monitoring of nine patients with skeletal metastases, bone ALP correlated well with the presence of pain and the progression or regression of metastatic spread.

Troponin T as a marker of ischemic myocardial injury.

A study was undertaken to evaluate the clinical relevance of serum troponin T (TnT) as a marker of ischemic myocardial injury, using a new automated enzyme immunoassay. The reference range for serum TnT was established by measuring serum TnT concentrations in blood obtained from 262 healthy subjects. The serum concentration of TnT was compared to serum creatine kinase activity, creatine kinase MB (mass and activity), myoglobin concentration, and lactate dehydrogenase activity: in 77 patients with myocardial infarction (55 received thrombolytic treatment); in 32 patients with unstable angina; in 30 patients with nonischemic heart diseases; and in 40 patients with skeletal muscle injuries. Our findings showed that: a) 99% of healthy blood donors had TnT concentrations < 0.10 micrograms/L; b) the test had a high clinical efficiency in the diagnosis of acute myocardial infarction, with a sensitivity of 1.0 and a specificity of 0.88 at a decision level of 0.20 micrograms/L; c) serum TnT had a later peak value (8-38 h), but a wider diagnostic window (> 126 h) than the traditional markers considered in the study; d) serum TnT had an excellent sensitivity in the detection of microinfarctions in patients with unstable angina pectoris; e) the release patterns of serum TnT were qualitatively different in perfused versus nonperfused patients. Peak serum TnT values and time to peak values were statistically different (p = 0.0336 and p = 0.0001) in reperfused and nonreperfused AMI patients, respectively; f) a ratio of serum TnT at 16 h to serum TnT at 32 h after chest pain > 1 provided a good indication of reperfusion in thrombolytic treatment (94% efficiency).

Performance of a fluorescence polarization immunoassay system evaluated by therapeutic monitoring of four drugs.

Fluorescence polarization immunoassays (FPIA) for amikacin, gentamicin, quinidine, and theophylline (supplied by Roche Diagnostic Systems, made using a Cobas Fara centrifugal analyzer) were evaluated and compared with widely used monitoring analysis methods. For each drug, the between-assay imprecision was ascertained by calibration on the day of assay and by a stored calibration curve made at the beginning of the study. The precision of the amikacin and theophylline assays was acceptable [total coefficient of variation (CV) less than 7.5%] at all concentrations tested for each calibration mode. Imprecision of quinidine and gentamicin assays was significant at low concentrations (1.9 mg/L): total CV = 9.0% for quinidine assessed with stored calibration curve and total CV greater than 8.5% for gentamicin measured with the two calibration modes. The calibration curves for all four assays had a good stability (greater than 30 days). Linear regression analysis demonstrated close agreement between the FPIA (y) and the following comparative techniques (x): Abbott TDx assay for amikacin and gentamicin (r = 0.988, r = 0.974, respectively); Stratus fluorometric enzyme immunoassay for quinidine (r = 0.979); and EMIT Syva assay for theophylline (r = 0.993). It is concluded that fluorescence polarization immunoassay is a rapid and reliable method for the therapeutic monitoring of the four drugs tested. Moreover, the use of reagents on an instrument that can be implemented for a wide range of chemistries has significant advantages and cost benefits over dedicated instruments.

Precipitation method for separating and quantifying bone and liver alkaline phosphatase isoenzymes.

We evaluated a method for quantifying bone isoenzyme of alkaline phosphatase (ALP) which utilizes wheat-germ lectin to precipitate this fraction. In precision studies, CVs ranged from 3.2 to 11.4% (within-day) and from 3.7 to 11.5% (day-to-day). The assay procedure was linear to 1100 U/L and was easily adapted to automated kinetic measurement. Comparison of the precipitation method with an affinity electrophoretic method, which utilizes cellulose acetate as a support, demonstrated a satisfactory coefficient of correlation (r = 0.886). The reference range was determined in sera from 188 healthy adult subjects. The distribution of bone ALP values was also studied in 73 healthy children and in 30 healthy adolescents. To evaluate the clinical applicability of the method, the bone isoenzyme was determined in samples from several groups of subjects (pregnant women, patients with hepatobiliary diseases, patients with hepatocellular carcinoma without skeletal involvement, and patients with bone, liver or lymph node metastases). We found the method suitable for routine determination of bone alkaline phosphatase and for the screening of bone metastases. Because of its technical simplicity and satisfactory analytical performance, it can be used instead of the heat-inactivation procedure.

Ventricular myosin and creatine-kinase isoenzymes in hypertensive rats treated with captopril.

In the myocardium, myosin and creatine kinase isoforms possess different capacities for using O2 and energy-rich phosphates. We studied electrophoretically the distribution of these isoforms in 19 hypertensive rats (two-kidney, one clip model of hypertension) and in age-matched controls. After 6 weeks of hypertension, seven rats were treated with captopril (2 mg/kg daily) for 4 weeks, six were left hypertensive for another 4 weeks, and the remaining rats were killed under ether anesthesia. In the latter, ventricular mass was significantly higher than in controls; V3 isomyosin was 32.3 +/- 6.8% versus 0%, and both creatine kinase-MB and -BB were increased at the expense of creatine kinase-MM (creatine kinase-MB = 29 +/- 2.8% vs. 14.7 +/- 1.8%, p less than 0.001; creatine kinase-BB = 3.1 +/- 0.6% vs. 1.7 +/- 0.8%, p less than 0.001). After 10 weeks of hypertension, ventricular mass, V3 isomyosin, and both creatine kinase-MB and -BB isoforms were found to be persistently higher than in controls. At the same time, captopril-treated rats showed reduced but not normalized blood pressure levels, normalized ventricular mass, and prevalence of the V1 isomyosin (56.9 +/- 22% vs. 47.9 +/- 23.8% in normotensive controls, p = NS). However, higher levels of creatine kinase-MB and -BB were still found in these rats in comparison with the normotensive controls (creatine kinase-MB = 22.4 +/- 5.4% vs. 15.8 +/- 2.8%, p less than 0.025; creatine kinase-BB = 2.3 +/- 0.1% vs. 1.8 +/- 0.3%, p less than 0.02).(ABSTRACT TRUNCATED AT 250 WORDS)

Assay of pancreatic amylase with use of monoclonal antibodies evaluated.

To evaluate a new method for measuring pancreatic amylase in serum, in which the salivary isoenzyme is inhibited with a specific monoclonal antibody, we determined the activity of pancreatic and salivary amylase in sera from 103 healthy subjects and from 114 hospitalized patients having a wide range of total amylase activities. CVs for the proposed method ranged from 0.8% to 5.1% (within day) and from 2.3% to 6.6% (day to day). Results correlated well with those obtained by the wheat-germ inhibition method (r = 0.998) and by electrophoresis on cellulose acetate. Analytical-recovery studies confirmed the good specificity of the monoclonal antibody for salivary amylase (97%) and its low cross-reactivity (0.6%) toward pancreatic amylase. The assay procedure presents a wide range of linearity (141-1817 U/L) and can easily be adapted to an automated kinetic system. We found the proposed method suitable for routine determinations of pancreatic amylase.

Immunoglobulin A (lambda chains) conjugated with lactate dehydrogenase in serum.

An atypical pattern for lactate dehydrogenase (EC 1.1.1.27) isoenzymes in a patient with sclerosis of the bladder neck was ascribable to complexing between lactate dehydrogenase and IgA. This complex formation was also replicable "in vitro." We determined that the IgA bound to lactate dehydrogenase was of the lambda type, a very unusual occurrence.