Multicentre evaluation of the Boehringer Mannheim/Hitachi 917 analysis system.

The new selective access analysis system BM/Hitachi 917 was evaluated in an international multicentre study, mainly according to the ECCLS protocol for the evaluation of analysers in clinical chemistry. Forty-three different analytes, covering 56 different methods--enzymes, substrates, electrolytes, specific proteins, drugs and urine applications--were tested in seven European clinical chemistry laboratories. Additionally, the practicability of the BM/ Hitachi 917 was tested according to a standardized questionnaire. Within-run CVs (median of 3 days) for enzymes, substrates and electrolytes were <2% except for creatine-kinase MB isoform and lipase at low concentration. For proteins, drugs and urine analytes the within-run CVs were < 4% except for digoxin and albumin in urine. Between-day median CVs were generally < 3% for enzymes, substrates and electrolytes, and < 6% for proteins, drugs and urine analytes, except for lipase, creatine kinase and MB isoform, D-dimer, glycosylated haemoglobin, rheumatoid factors, digoxin, digitoxin, theophylline and albumin in urine in some materials. Linearity was found according to the test specifications or better and there were no relevant effects seen in drift and carry-over testing. The interference results clearly show that also for the BM/Hitachi 917 interference exists sometimes, as could be expected because of the chemistries applied. It is a situation that can be found in equivalent analysers as well. The accuracy is acceptable regarding a 95-105% recovery in standard reference material, with the exception of the creatinine Jaffé method. Most of the 160 method comparisons showed acceptable agreement according to our criteria: enzymes, substrates, urine analytes deviation of slope +/- 5%, electrolytes +/- 3%, and proteins and drugs +/- 10%. The assessment of practicability for 14 groups of attributes resulted in a grading of one-three scores better for the BM/Hitachi 917 than the present laboratory situation. In conclusion, the results of the study showed good analytical performance and confirmed the usefulness of the system as a consolidated workstation in medium-sized to large clinical chemistry laboratories.

Multicentre evaluation of KONE Optima analysis system.

Optima from KONE Instruments Corporation is a new selective laboratory analyzer for turbidimetric, colorimetric and ion selective electrode measurements. Overall analytical performance of Optima and reagents provided by KONE was evaluated according to ECCLS guidelines, in a multicentre study involving four different laboratories, including substrates (cholesterol, high-density lipoprotein-cholesterol, creatinine), specific proteins (transferrin, IgG), enzyme activities (gamma-glutamyltransferase, alanine aminotransferase) and electrolytes (sodium, potassium, chloride). The results obtained attest good precision of the system for all the analytes tested: the range of within-run CV is 0.5 %-4.3 %, and range of between-day CV % is 0.8 %-7.9 % (median of four laboratories). Except for total cholesterol (5 % overestimation compared to the reference method) accuracy of measurement is adequate. Creatinine and uric acid assays were subjects of interference (defined as deviation > 10 % from target value) by bilirubin and haemoglobin respectively.

Relation between left ventricular function and oxidative stress in patients undergoing bypass surgery.

OBJECTIVE: To determine whether preoperative left ventricular ejection fraction (LVEF) is related to the degree of myocardial oxidative stress during bypass surgery in man. DESIGN: Observational study. SETTING: Tertiary care centre. PATIENTS AND INTERVENTIONS: 31 patients (LVEF range was 20% to 68%) undergoing elective coronary bypass surgery with blood cardioplegic reperfusion were studied. Arterial and coronary sinus blood was collected before aortic cross clamping (T0) and at 0 (T1), 15 (T2), and 30 (T3) minutes after unclamping. Transmural left ventricular biopsies were also obtained from 15 patients at T0 and at T1. MAIN OUTCOME MEASURES: Glutathione and adenine nucleotides were measured in myocardial biopsies, while coronary sinus-artery differences for glutathione, nucleotides, and products of lipid peroxidation were calculated from blood specimens. Creatine kinase (myocardial band; CK-MB) was measured in plasma at four and 12 hours after operation. RESULTS: Myocardial glutathione and adenine nucleotides were correlated (p < 0.02) with preoperative LVEF both at T0 (r = 0.909 and 0.672) and T1 (r = 0.603 and 0.605). Oxidised glutathione released from the heart during reperfusion was inversely correlated with LVEF (r = -0.448, -0.466, and -0461 at T1, T2, and T3, p < 0.01), while reduced glutathione (r = 0.519 and 0.640 at T1 and T2) and glutathione redox ratio (r = 0.647, 0.714, 0.645, and 0.702 at T0, T1, T2, and T3) showed a direct correlation (p < 0.01). Lipid peroxidation at T1 was negatively related to LVEF (r = -0.492). CK-MB was also negatively related to LVEF (r = -0.440 at 4 h and -0.462 at 12 h). CONCLUSIONS: The capacity to counterbalance oxidative burst following ischaemia and reperfusion appears to be related to the functional ability of the heart.

Role of leucocytes in free radical production during myocardial revascularisation.

OBJECTIVE: To evaluate the role of leucocytes in free radical production in patients with depressed or normal ejection fraction undergoing coronary bypass. DESIGN: Two randomised control trials. SETTING: Tertiary care centre. PATIENTS AND INTERVENTIONS: In the first study, 22 patients with ejection fractions of < or = 40% received blood cardioplegic reperfusion with (n = 11) or without (n = 11) leucocyte depletion. In the second study, 22 patients with ejection fractions > or = 45% received either leucocyte depleted (n = 11) or blood cardioplegia (n = 11). MAIN OUTCOME MEASURES: Glutathione, hypoxanthine, and lipid peroxidation products were measured in coronary sinus blood and plasma before aortic cross clamping and at 0, 15, and 30 minutes after unclamping. Haemodynamic variables and creatine kinase MB isoenzymes were monitored on the first postoperative day. Comparison between treatments was performed on difference (delta) between measurements at time 0 and at baseline, and on slopes obtained by fitting measurements after unclamping with a linear regression model. RESULTS: At unclamping no difference in delta for plasma glutathione redox ratio (oxidised/total glutathione, %) was observed between treated and control groups with low ejection fraction (delta = 16 (SD 8.39) and 24 (7.0) redox ratio %, respectively). Baseline value recovery rate (redox ratio %/min) was significantly faster in treated v control patients (slope -0.912 (0.380) v -0.158 (0.200), P < 0.005, respectively). Cardiac index showed a trend to greater improvement in the treated group (slope 0.04 (0.03) v 0.003 (0.002) 1/min/m2/h, P < 0.02, treated v controls, respectively). In patients with normal ejection fraction, leucocyte depletion did not result in significant improvement v controls. CONCLUSIONS: Leucocyte depletion seems to provide benefit only in patients with left ventricular dysfunction.

Ceftazidime kinetics of diffusion in inoculated agar plates.

The ceftazidime concentration in agar plates inoculated with Pseudomonas aeruginosa was determined by high-performance liquid chromatography at fixed points (3, 6, 9, 12, and 15 mm) from the disk center and at fixed times (2, 4, 6, 16, and 24 h) to study the antibiotic kinetics of diffusion. A statistical difference between the concentrations determined in the presence of microorganisms and in uninoculated plates after 16 and 24 h was evidenced and was probably ascribable to the drug hydrolysis carried out by the induced beta-lactamase.

Detection of HIV-1 proviral sequences in lymphocytes using a qualitative polymerase chain reaction assay.

The performance and clinical relevance of a qualitative PCR-based assay for the detection of HIV-1 DNA sequences in peripheral blood mononuclear cells (PBMCs) was evaluated by two different laboratories. Four hundred and one samples were obtained from 397 individuals from different risk populations. All blood donors tested had negative results; positive signals were obtained from all infected patients. HIV-1 DNA was detected in 3 of 17 infants born to seropositive mothers; Western blot indeterminate blood donors and exposed health-care workers had negative results. Our results demonstrate that this PCR assay provides both sensitive and specific results and is suitable for testing large numbers of samples and for rapid identification of HIV-1 infection.

Open-chain peptides obtained by acidic hydrolytic cleavage of cyclosporin A.

Hydrolysis of cyclosporin A (CsA) was studied in order to clarify the still undefined point of attack of the acidic degradation. Among ether extractable and water-soluble products formed from CsA in HCl, two open-chain peptides were isolated by high-performance liquid chromatography which were identified as the deca- and nonapeptides deriving from CsA through the hydrolytic cleavage of amino acid residue 11 and both residues 11 and 10, respectively. Identification was carried out by fast atom bombardment tandem mass spectrometry.

High-performance liquid chromatographic method for measuring hydroxylysine glycosides and their ratio in urine as a possible marker of human bone collagen breakdown.

Glucosyl-galactosyl-hydroxylysine (GGHYL) and galactosyl-hydroxylysine (GHYL) are constituents of collagen protein. The ratio of the two hydroxylysine glycosides varies with the collagen type and, moreover, for a given collagen type, it also varies according to the connective tissue. For example, in type I collagen (the most abundant in the body), the GGHYL/GHYL ratio tends to be greater in soft connective tissues and lower in bone. The hydroxylysine glycosides are not recycled during collagen turnover and are excreted in the urine. Therefore, the urinary GGHYL/GHYL ratio, which reflects the proportion of the two metabolites in the various collagens, may indicate the type of connective tissue affected by pathological turnover, and may thus be a promising marker of bone metabolism. In this paper a method is described for the measurement of urinary hydroxylysine glycosides by reversed-phase liquid chromatography after purification of the sample by solid-phase extraction. The method presented is analytically reliable and suitable for routine use in a clinical laboratory.

Determination of serum glucose by isotope dilution mass spectrometry: candidate definitive method.

We report a rather simple method to determine glucose concentration in serum, using isotope dilution mass spectrometry and [13C6]glucose as internal standard. The procedure involves a single step of sample purification and the conversion of the analyte into its aldononitrile pentaacetate. The between-day and within-day contribution to total variance for a single measurement was determined by assaying Standard Reference Material (SRM) 909 serum. The method was then applied to measurement of glucose concentration in three lyophilized sera: SRM 909 and two other commercially available sera. In the two studies, the concentration of SRM 909 serum was found to be 0.8% above and 0.3% below the reported value (6.25 mmol/L), respectively; the overall coefficient of variation for determinations in all sera ranged from 0.37% to 0.56%. The precision and the accuracy of the method satisfy the requirements for a Definitive Method.

Total urinary hydroxyproline determined with rapid and simple high-performance liquid chromatography.

A precolumn derivatization method was optimized for rapid and specific analysis of total urinary hydroxyproline by HPLC. After an overnight hydrolysis, urine samples dried and reconstituted with the internal standard cysteic acid (in sodium hydrogen carbonate, pH 9.3) were derivatized with N,N-diethyl-2,4-dinitro-5-fluoroaniline (FDNDEA) at 100 degrees C for 20 min. The DNDEA-hydroxyproline adduct was separated on an Ultrasphere ODS column with a mobile phase of acetate buffer (containing triethylamine, 6 mL/L, pH 4.3) and acetonitrile (80/20, by vol), and was detected at 360 nm. A single run took 18 min with a hydroxyproline retention time of 7.3 min. The assay showed a linear response to hydroxyproline concentrations from 5 to 100 mg/L with a detection limit of 0.8 ng injected, corresponding to 2 mg/L in urine. Mean (SD) analytical recovery was 94.2 (13)% and 104 (9)% at 10 and 50 mg/L, respectively. Within-run and between-run CVs (n = 10) were 3.74% and 4.33%, respectively, for 25 mg/L. Results for samples (n = 50) analyzed by HPLC (y) vs ion-exchange chromatography with postcolumn ninhydrin reaction (x) correlated well: y = 0.98x + 1.02 (r = 0.985, Sxy = 3.13). In another comparison, involving 173 samples, a colorimetric procedure (Hypronosticon, x) gave slightly higher values than the HPLC method (y): y = 0.83x + 2.21 (r = 0.937, Sxy = 4.6).

Tumor-associated trypsin inhibitor as a possible marker in male infertility.

The concentrations of tumor-associated trypsin inhibitor (TATI) in the seminal plasma of infertile males was studied. The TATI levels in seminal plasma were not correlated with either sperm count or ejaculate volume. High levels were observed in some men with unexplained infertility and high or normal sperm counts, whereas normal levels were observed in males with antisperm antibodies. The concentrations in seminal plasma were stable in the same subjects. These results suggest that TATI may be an important marker of reproductive pathology in men.

Tumor-associated trypsin inhibitor in induced and acquired immunodeficiency. Studies on transplanted and HIV-infected patients.

A new tumor marker, tumor-associated trypsin inhibitor (TATI), was studied in 5 patients who received successful kidney or pancreas grafts and in 30 subjects with antibodies against human immunodeficiency virus. Serum TATI concentrations were very high during the four first days after transplantation. Thereafter the serum levels decreased when the peptide was eliminated through the kidney. Consequently, the urine values were very high. The TATI concentrations of HIV positive subjects were compared with serum levels of HIV antigen and antibody, by Western blotting and determination of peripheral T-lymphocyte subpopulations. The occurrence of high concentrations of TATI in some HIV positive subjects and especially in AIDS patients, suggests that TATI could be useful in exploring physiopathological aspects of severe immunodeficiencies even if TATI levels were not correlated with the commonly used markers of the immune system status. The increased levels of TATI in immunological disorders suggests its possible use in assessing the immune response against cancer.

Detection, signal processing, and calibration In lmmunoassay systems.

The new trends in immunochemistry related to the replacement of radioisotopic labels with non-radioactive labels are presented. Immunoenzymatic, fluorescent and chemiluminescent techniques are described in terms of their basic principles and their most common applications. The advantages of computer-controlled calibration are also discussed.

Enhanced chemiluminescence in the measurement of proteins and haptens: evaluation of choriogonadotropin (hCG) and free thyroxin.

We evaluated the Amerlite system (Amersham, Bucks, UK) for hCG and FT4. The within-run imprecision (CV%) for hCG was 4.05 at 19.6 U/l (n = 10), 6.28 at 43.45 U/l (n = 10) and 4.62 at 298.57 U/l (n = 10). The between-run imprecision (five replicates for ten days) was 4.8%, 15% and 11%, respectively. The system was linear up to 200 U/l. A good correlation between Amerlite hCG and an IRMA assay (Becton Dickinson, r = 0.91), Delfia (Pharmacia, r = 0.91) and an automated ELISA assay on ES 600 (Boehringer, r = 0.92) was observed on 70 samples. Within-run imprecision for FT4 was 3.8% at 0.7 ng/dl (n = 10), 3.3% at 1 ng/dl (n = 10) and 4.32% at 5.15 ng/dl (n = 10), and between-run was 5.95%, 4.4% and 8.2%, respectively. The comparison with a commercial direct RIA (Becton Dickinson) showed good correlation (r = 0.90, n = 100 samples). The diagnostic value of the association of thyrotropin and FT4, in comparison with the traditional thyroid tests (T3, T4, thyrotropin, FT4, FT3) has been assessed in various thyroid diseases.

Determination of creatinine in serum and urine by a rapid liquid-chromatographic method.

We describe an HPLC ion-pair procedure for rapid and specific evaluation of creatinine in serum and urine. We used a 15 cm X 4.6 mm ODS column with a 50/50 (by vol) mixture of sodium decanesulfonic acid (10 mmol/L, pH 3.2) and methanol and measured absorbance at 236 nm. Serum (100 microL) or 30-fold-diluted urine (100 microL) was added to 400 microL of acetone. After centrifugation, the supernates (300 microL) were dried, reconstituted with the mobile phase, and injected into the HPLC. Assay precision was tested for concentrations of 10, 29, and 130 mg/L and yielded, respectively, 3.1%, 2.1%, and 1.1% for within-day CV and 2.8%, 2.1%, and 2.2% for total CV. Analytical recovery was 102 (+/- 6.7%). Linearity was demonstrated in the 0-200 mg/L range for serum and 0-3.5 g/L range for urine (r greater than or equal to 0.999). The detection limit for creatinine (signal-to-noise ratio = 3) was 0.5 mg/L. We used cimetidine for internal standardization. Correlation was good between this procedure and the Jaffé kinetic, the enzymatic (creatinine amidohydrolase), and the Fuller's earth alkaline picrate methods.

Antibody against EBV in CSF from HIV-1 and HTLV-I infected patients.

We have measured the levels of antibody (ab) against different Epstein-Barr virus (EBV) antigens in 10 AIDS patients, 7 HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP) patients and 20 control subjects. We found comparable serum levels of anti-EBV ab between AIDS patients, HAM/TSP patients and control subjects. By contrast, anti-EBV ab were present in the large majority of CSF from AIDS patients (70%) and HAM/TSP patients (60%) but only in 15% of control group. Our results support a synergistic role of EBV in retroviral infections of the central nervous system.

Detection of a case of pseudolymphocytosis due to cryoglobulins.

A new type of interference of cryoglobulins on hemocytometric tests is described. The precipitation of temperature-dependent proteins produced a pseudolymphocytosis on a three-part differential leukocyte count of Coulter S-Plus VI, whereas unaffected results, identical to the microscopical count, were obtained using the cytometer Coulter VCS. The laboratory detection of cryoglobulin interference on hematological data is very important in patients with underlying diseases, where the accuracy of absolute and differential leukocyte counts is critical for follow-up. Histograms from the Coulter S-Plus VI can help detect these cases.