Volume 41, Issue 1

Elastase is a protease released by polymorphonuclear neutrophils (PMN) during the inflammatory process. Since 1987, seminal elastase-inhibitor complex (Ela/α 1 -PI) has been proposed as a marker of male silent genital tract inflammation. Measured by immunoassay in seminal plasma, Ela/α 1 -PI at a cut-off level of ≥230 μg/l, is useful in the detection of genital tract inflammation. The prevalence of increased seminal Ela/α 1 -PI in infertile men is significantly higher than that observed in fertile men. The Ela/α 1 -PI level is positively correlated with other seminal fluid markers of male genital tract inflammation: reduced semen volume, citric acid, fructose, and increased albumin, complement component C3, caeruloplasmin, immunoglobulins IgG and IgA, and cytokines interleukins-8 and -6. A higher seminal Ela/α 1 -PI level is significantly associated with tubal damage in female partners. After antibiotic therapy, a decrease of Ela/α 1 -PI level is observed. The presence of tubal damage in the partner may negatively affect the response to antibiotic treatment. A higher seminal Ela/α 1 -PI is associated with lower percentage of sperm with single-stranded deoxyribonucleic acid (DNA) and better fertilization rate in in vitro fertilization. Besides infertility, the determination of Ela/α 1 -PI is useful to confirm the presence of prostate and other male accessory gland bacterial inflammation. Screening for PMN Ela/α 1 -PI is easy to perform and reproducible and is a reliable quantitative test for diagnosis and prognosis of silent genital tract inflammation of couples. Moreover, sequential determinations allow the follow-up of inflammation during and after therapy.

The objective of this work was to evaluate the capability of both seminal plasma and sperm cells to scavenge different forms of oxyradicals and the possible correlation with sperm motility parameters. A total of 14 individuals were analyzed by computer-assisted sperm analysis (CASA) and the results integrated with the measurement of total oxyradical scavenging capacity (TOSC) toward peroxyl radicals, hydroxyl radicals and peroxynitrite in seminal plasma and spermatozoa. TOSC values revealed some significant correlation with kinetic sperm cell parameters, including curvilinear velocity (VCL), straight-line velocity (VSL) and linearity (LIN). A lower antioxidant capacity toward hydroxyl radical was found in the seminal fluid of men with reduced sperm motility. Such correlations were not found with peroxyl radicals and peroxynitrite, neither when TOSC values were analyzed in spermatozoa. The TOSC assay is a useful tool for studying the relationship between oxyradical toxicity and abnormal sperm cell motility. Although further investigations are needed, the data clearly establish different role for various forms of oxyradicals, i.e. , hydroxyl radicals, in altering sperm motility. Measurement of TOSC is suggested as a useful means of indicating relationship between reactive oxygen species and sperm cell kinetics in clinical trials and antioxidant-based treatments.

We identified four novel polymorphisms in the CACNA1S gene that encodes the α 1 -subunit of the dihydropyridine receptor. Mutations in this gene are associated with two genetic diseases: malignant hyperthermia and hypokalemic periodic paralysis. The nucleotide substitutions c2403T→C and c5398T→C did not result in amino acid replacement, the nucleotide substitution c4475C→A caused the replacement of the Ala1492 with an Asp residue and an A insertion was identified in intron 36. By using methods based on digestion with restriction enzymes we calculated the frequencies of these novel polymorphisms, as well as heterozygosity, in normal subjects from southern Italy.

The effect of peroxynitrite on the development of atherosclerosis is one of the major foci of recent studies. Here, the cytotoxic effect of peroxynitrite was investigated by quantitatively measuring nitrated tyrosine, 3-nitrotyrosine (3-NT) levels in atherosclerotic blood vessels. Atherosclerotic vessels were obtained from the patients who underwent either coronary artery or peripheric artery bypass surgery. Internal thoracic arteries of the patients were treated as non-atherosclerotic control vessels. 3-NT was measured by reverse-phase HPLC and plasma nitrite-nitrate levels were measured by spectrophotometry. 3-NT levels were significantly elevated in atherosclerotic vessels (46.6±23.3 nmol/mg protein, n = 15; p < 0.001) in comparison to control vessels (15.8±2.5 nmol/mg protein, n = 10). Vessel 3-NT correlated weakly with plasma nitrate levels (r = 0.38). Thus, atherosclerotic arteries have higher 3-NT levels than non-atherosclerotic blood vessels.

We compared the analytical accuracy, times, costs and the detection rate of four procedures for the molecular analysis of cystic fibrosis (CF). DNA from 127 subjects bearing different genotypes was tested by denaturating gradient gel electrophoresis followed by sequencing (reference procedure) and, for comparison, by ASO dot-blot (in-house), reverse dot-blot (Innogenetics), ARMS (Zeneca Diagnostics) and OLA-PCR (PE Applied Biosystem). To assess inter-observer variability, all samples were tested twice. To evaluate intra- and between-series variability, two samples were tested twice in each series. All the procedures yielded reproducible results and assigned the correct genotype to each sample. ASO dot-blot is the cheapest procedure but has the longest analytical time (>24 h) and uses radioactive labeling. It can be adapted to analyze peculiar mutations in specific ethnic groups. ARMS from Zeneca Diagnostics is rapid (4 h), easy to perform, but, except for the ∆F508 mutation, does not distinguish the homozygote from the heterozygote genotype. It could be used for carrier analysis in families with known mutations. OLA-PCR has the highest detection rate in most ethnic groups, is automated for capillary electrophoresis but requires a high level of operator expertise: it is suitable when collected series of samples are analyzed from large patient cohorts. Reverse dot-blot is easy to perform and can be semiautomated: it can be used as first-line screening test. Given the heterogeneity of CF mutations, commercial kits should be developed to analyze mutations peculiar to specific ethnic groups.

The sugar absorption test is the usual test for measurement of intestinal permeability. After intestinal absorption of probe sugars the subsequently excreted sugars are measured in urine. We have developed four enzymatic methods for the measurement of the urinary concentration of the probe sugars mannitol, raffinose, lactose and sucrose. Mannitol, lactose and sucrose are directly measured on Hitachi 917 using mannitol dehydrogenase, β-galactosidase and invertase, respectively, as enzyme reagents. Raffinose measurement needs a three hours preincubation with α-galactosidase, after which the liberated sucrose is measured. The analytical performances such as within- and between-run precision, linearity, lowest detection limit, interference of other sugars and comparison with a gas chromatographic method are described for the four methods. These methods are accurate an can easily be performed in any clinical laboratory.

We describe a model to illustrate different chemical interactions that can occur in urine following ingestion of individual and combined health supplements. Two types of interactions are defined: synergism and addition. The model was applied to eight healthy males who participated in a study to investigate the chemical interactions between calcium carbonate, magnesium oxide and sodium citrate-bicarbonate health supplements on calcium oxalate urinary stone risk factors. Subjects ingested these components individually and in combination for 7 days. Twenty-four-hour urines were collected at baseline and during the final day of supplementation. These were analysed using standard laboratory techniques. Three different chemical interactions, all involving citrate, were identified: magnesium and citrate exerted a synergistic effect on lowering the relative superaturation (RS) of brushite; the same two components produced a synergistic effect on raising pH; finally, calcium and citrate exerted an additive effect on lowering the RS of uric acid. We propose that the novel approach described in this paper allows for the evaluation of individual, additive and synergistic interactions in the assessment of the efficacy of stone-risk reducing preparations.

Recent recommendations in the National Cholesterol Education Program Expert Panel on Detection, Evaluation and Treatment of High Blood Cholesterol in Adults (ATPIII) are expected to increase the number of triglyceride (TG) determinations and consequently the risk of misinterpretation of “non-blanked” results with co-determination of free glycerol. Glycerol-kinase deficiency (GKD) is one cause of falsely elevated TG results. The natural history of isolated GKD with symptom-free cases and cases with e.g. severe episodes of hypoglycemia and/or ketoacidosis challenges the laboratories to identify cases of GKD and family members at risk. “Blanked” methods reporting both glycerol and TG concentration are therefore desirable. Molecular studies of the glycerol kinase ( GK ) and DAX1 genes were performed on four cases of “persistent hypertriglyceridemia” found in an Italian population and on two pediatric cases with high serum glycerol concentration. Two new missense mutations were found (C358Y, T96I). Molecular modeling on GK from E. coli , indicate that these mutations are located in parts of the enzyme important for enzyme formation or activity. One splice-site mutation, (IVS9A-1G>A), was found in two brothers. Splice-junction analysis indicates that it destroys the splice site and results in a mixture of mRNA. Deletion of the GK and DAX1 genes was found in one child with symptoms of adrenal failure. A female with glycerolemia and glyceroluria had normal GK activity but possibly slightly decreased ability to oxidize glycerol.

Low-density lipoprotein (LDL) oxidation in vivo depends on lipid composition and on plasma antioxidant status. The aim of our study was to investigate the relationship between plasma lipid composition and LDL oxidation and, in particular, to explore whether LDL-cholesterol/triglycerides ratio (LDL-C/TG) and LDL-cholesterol/high-density lipoprotein (HDL)-cholesterol ratio (LDL-C/HDL-C) can be used as predictive parameters of LDL oxidation in vivo . In 87 volunteers over a wide range of age plasma lipids and LDL oxidation were studied. Blood was collected after 12 h overnight fast. LDL oxidation was estimated by the level of conjugated diene (BDC) in the lipid fraction isolated from plasma after gradient ultracentrifugation. The results were expressed as μmol/l (BDC/l) to evaluate the level of oxidized LDL, and as nmol of BDC for mg of LDL-cholesterol (BDC/LDL-C) for the evaluation of LDL oxidation degree. BDC/l correlated significantly with age, total and LDL-C, apolipoprotein B and TG, while BDC/LDL-C negatively correlated with total cholesterol, apolipoprotein B, LDL/TG and LDL/HDL ratios. Age of subjects significantly correlated with total and LDL-C and apolipoprotein B. TG have a significant inverse correlation with HDL-C. Our results support the hypothesis that among the several factors involved in LDL oxidation the most important determinants are LDL/TG. Plasma triglycerides appear to be very important even when circulating cholesterol levels are within normal limits. Moreover, we found that the LDL/HDL ratio is also very important with regard to the putative protective role of HDL against LDL oxidation in vivo . In conclusion, plasma lipid parameters must be evaluated not only for their absolute values but also for their mutual ratios as expression of plasma lipid homeostasis. Both LDL/TG and LDL/HDL ratios can be used as predictive parameters of in vivo LDL oxidation.

Nasal potential difference (NPD) measurements have been proposed to assess defective ion transport in cystic fibrosis (CF). Implementing it routinely is, however, difficult. Therefore, a modified method based on nasal instillation in supine position at reduced flow rate was tested to evaluate its ability to discriminate CF from non-CF subjects. Classical and modified methods were compared in nine healthy subjects and there were no statistical differences. Following the new method, 97 tests were performed on 74 subjects divided in three cohorts: 21 CF patients and two control groups consisting of 19 patients with other pulmonary diseases and 34 healthy subjects. Twenty five children were enrolled in this study. Maximal NPD in CF patients (−44.9±2.5 mV) was significantly different from that obtained in control groups (−18.1±1.6 and −17.2±1.1 mV). Depolarization after amiloride also discriminated CF patients (25.9±1.4 mV) from control groups (10.5±0.9 and 8.1±0.7 mV). Marked repolarization following isoprenaline plus amiloride in low chloride solution was seen in control groups (−15.7±1.1 and −15.3±1.1 mV). We conclude that the modified method represents a simplified and equally effective approach to discriminate CF patients from non-CF subjects. Moreover, this method presents practical advantages for the patients related to hygiene and convenience, favoring its application in small children.

Objective – To improve the accuracy and completeness of reporting of studies of diagnostic accuracy, to allow readers to assess the potential for bias in the study and to evaluate its generalisability. Methods – The Sta ndards for R eporting of D iagnostic Accuracy (STARD) steering committee searched the literature to identify publications on the appropriate conduct and reporting of diagnostic studies and extracted potential items into an extensive list. Researchers, editors, and members of professional organisations shortened this list during a two-day consensus meeting with the goal of developing a checklist and a generic flow diagram for studies of diagnostic accuracy. Results – The search for published guidelines regarding diagnostic research yielded 33 previously published checklists, from which we extracted a list of 75 potential items. At the consensus meeting, participants shortened the list to 25 items, using evidence on bias whenever available. A prototypical flow diagram provides information about the method of patient recruitment, the order of test execution and the numbers of patients undergoing the test under evaluation, the reference standard or both. Conclusions – Evaluation of research depends on complete and accurate reporting. If medical journals adopt the checklist and the flow diagram, the quality of reporting of studies of diagnostic accuracy should improve to the advantage of clinicians, researchers, reviewers, journals, and the public.

Urinary markers of bone collagen degradation such as N-terminal telopeptide, deoxypyridinoline, hydroxyproline and proline-hydroxyproline dipeptide as a novel marker were measured in both morning and daily urine samples collected from 40 postmenopausal women on 10 consecutive days. In addition, osteocalcin, bone alkaline phosphatase and procollagen C-terminal propeptide were analyzed in one serum sample from each of the women. DXA densitometry was employed to differentiate between osteopenic and osteoporotic women. Spearman rank correlation, factor analysis and receiver-operating characteristics were computed to evaluate the results. The highest correlation with a single common factor was found between telopeptide assayed in daily urine, followed by the dipeptide and hydroxyproline assayed in morning urine. These markers also showed high sensitivity for the diagnosis of osteoporosis, while osteocalcin had a high sensitivity for the diagnosis of bone disease. The concentrations of telopeptide, dipeptide and osteocalcin differed significantly between the osteopenic and osteoporotic subjects. Because of the ease of non-hydrolyzed urine processing, the speed of chromatographic analysis and the negligible costs, the dipeptide assay opens new possibilities in the field.

We evaluated the diagnostic utility of urinary α 1 -microglobulin, α 2 -macroglobulin and albumin in the diagnosis of acute prostatitis. We studied 133 men (43±17 years) with, and a reference population (n=36, 41±16 years) without, urinary tract infection. Prostatectomy samples were used to study the potential interference between prostatic proteins and protein analysis. Urinary α 2 -macroglobulin/albumin ratio was significantly lower in prostatitis compared to the reference population, cystitis or acute pyelonephritis (p <0.0001). Low α 2 -macroglobulin concentrations in prostatitis are due to inhibition (p = 0.0001) of the immune reaction between α 2 -macroglobulin in presence of polyclonal rabbit antibodies (used for immunonephelometry) by soluble prostatic proteins (± 60 kDa) which appear in urine in acute prostatitis. The urinary α 1 -microglobulin/creatinine ratio diagnoses acute pyelonephritis (sensitivity 100% and specificity 87%) and the urinary α 2 -macroglobulin/albumin ratio diagnoses acute prostatitis (sensitivity 100% and specificity of 90%). Stepwise multinomial logistic regression analysis reveals that urinary α 1 -microglobulin, α 2 -macroglobulin, albumin and creatinine provide optimal differentiation between acute pyelonephritis and acute prostatitis (pseudo R 2 =0.83; Loglikelihood −30.55, p <0.000001). In conclusion, the combination of hematuria and absence of urinary alpha-2-macroglobulin is diagnostic for acute prostatitis. Even without hematuria, α 2 -macroglobulin remains lower compared to patients without prostatitis.

Proper sample handling is important for the accurate and prompt diagnosis of cryoglobulinaemia as well as for obtaining correct results of other laboratory tests. In this case report, we present two patients for whom improper sampling delayed the diagnosis of cryoglobulinaemia. The routine sampling procedure was not sufficient for detecting these cryoglobulins and we demonstrated that the temperature dropped below 37 °C at different steps during the sample preparation. Adjusting the pre-analytical procedure revealed that both patients possessed a cryoglobulin with high thermal insolubility. We conclude that in cryoglobulinaemia strict adherence to guidelines to keep the temperature >37 °C is crucial for sample collection and must be strongly recommended.

Nonequimolar-response assays for prostate-specific antigen (PSA) are criticized for overestimating total PSA in some men without prostate cancer (PCA), and underestimating total PSA in some men with PCA. We recently studied three nonequimolar-response PSA assays that had undergone modifications. While two of the studied assays achieved equimolar-response characteristics with improved areas under receiver operating characteristic (ROC) curves (AUC), the modification of the Chiron ACS PSA assay (ACS PSA2, Chiron) failed to achieve this. Recently, the ACS assay underwent another modification (ACS PSA, Bayer), which we investigated. Sera from 305 men (155 without and 150 with PCA, PSA ≥2 and ≤30 μg/l, Tandem-E) were measured using both modifications of the ACS assay and equimolar-response reference methods (Tandem-R free and Tandem- E, Hybritech). Molar response relative to the reference method and clinical performance (comparison of AUCs) between the previous and new ACS assay modifications were studied. The new modification of the ACS assay (ACS PSA, Bayer) achieved equimolar-response characteristics but reported lower values (average 10%) than the Tandem-E assay. Compared to the previous modification (ACS PSA2, Chiron), a 3% improvement in AUC (p = 0.01) was found. Using results of the redesigned equimolar-response assay (ACS PSA, Bayer), we calculated that 6 of 155 men without PCA in this sample set could be spared unnecessary biopsy compared with the previous nonequimolar-response assay (ACS PSA2, Chiron) without missing additional PCA (90% sensitivity). These data provide additional evidence for clinical advantages of equimolar-response over nonequimolar-response PSA assay formats.

This international multicenter study was designed to evaluate the technical performance of the new double-monoclonal, single-step Elecsys neuron-specific enolase (NSE) enzyme immunoassay (EIA) and to assess its utility as a sensitive and specific test for the diagnosis of small-cell lung cancer (SCLC). Intra- and inter-assay coefficients of variation, determined in five control or serum specimens in six laboratories, ranged from 0.7 to 5.3 (inter-laboratory median: 1.3%) and from 1.3 to 8.5 (inter-laboratory median: 3.4%), respectively. Laboratory-to-laboratory comparability was excellent with respect to recovery and inter-assay coefficients of variation. The test was linear between 0.0 and 320 ng/ml (highest measured concentration). There was a significant correlation between NSE concentrations measured using the Elecsys NSE and the established Cobas Core NSE EIA II in all subjects (n = 723) and in patients with lung cancer (n = 333). However, NSE concentrations were systematically lower (approximately 9%) with the Elecsys NSE than with the comparison test. Based on a specificity of 95% in comparison with the group suffering from benign lung diseases (n = 183), the cut-off value for the discrimination between malignant and benign conditions was set at 21.6 ng/ml. NSE was raised in 73.4% of SCLC patients (n = 188) and was significantly higher (p < 0.01) in extensive (87.8%) as opposed to limited disease (56.7%). NSE was also elevated in 16.0% of the cases with nonsmall cell lung cancer (NSCLC, n = 374). It is concluded that the Elecsys NSE EIA is a reliable and accurate diagnostic procedure for the measurement of NSE in serum samples. The special merits of this new assay are the wide measuring range (according to manufacturer's declaration up to 370 ng/ml) and a short incubation time of 18 min.

Between 1997 and 2000 we investigated in a prospective study the voided urine samples of all consecutive patients undergoing cystoscopy independent from their clinical background (n = 705) with the BTA-TRAK™ assay (Bard Diagnostics, Redmont, USA) detecting a complement factor H-related protein (CFHrP) and the NMP22 assay (Matritech, Newton, USA) measuring a nuclear matrix protein, which is supposed to be specific for bladder cancer. The individuals were divided into three groups concerning the clinical background: 233 patients had urological diseases, 268 patients had urinary bladder cancer and 150 patients had other urological malignancies. Based on the clinical findings we compared our results with well established diagnostic methods for urinary bladder cancer such as cytology and the detection of hematuria. In addition, we investigated urine samples from 30 healthy individuals and 24 patients with urinary tract infection without performing cystoscopy. Following the recommendations of the European Group on Tumor Markers we used 95% specificity for benign urological diseases and urinary tract infections, which resulted in a sensitivity of 17% for active bladder cancer for the BTA-TRAK™ assay and 31% for NMP22. We compared these results with the detection of hematuria (specificity: 72%) and cytology, which had a sensitivity of 64% and 89%, respectively. Subsequently, we calculated sensitivity and specificity for the detection of relapse of the disease. Again using 95% specificity, in this case for patients with no evidence of disease (NED), in patients with recurrent disease the BTA-TRAK™ assay showed 8% sensitivity as compared to 12% for the NMP22 assay. Due to an insufficient specificity and sensitivity, both tests can neither be clinically useful in screening of high risk patients, nor in primary diagnosis of bladder cancer. They cannot replace neither cystoscopy nor cytology. In the follow-up care more investigations may be necessary to prove the benefit of existing diagnostic strategies for the discrimination between active and inactive bladder cancer.