Volume 51, Issue 2

Recently, different bacteriological laboratory interventions that decrease reporting time have been developed. These promising new broad-based techniques have merit, based on their ability to identify rapidly many bacteria, organisms difficult to grow or newly emerging strains, as well as their capacity to track disease transmission. The benefit of rapid reporting of identification and/or resistance of bacteria can greatly impact patient outcomes, with an improvement in the use of antibiotics, in the reduction of the emergence of multidrug resistant bacteria and in mortality rates. Different techniques revolve around mass spectrometry (MS) technology: matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), PCR combined with electrospray ionization-mass spectrometry (PCR/ESI-MS), iPLEX MassArray system and other new evolutions combining different techniques. This report emphasizes the (r)evolution of these technologies in clinical microbiology.

Chagas disease, caused by Trypanosoma cruzi infection, is ranked as the most serious parasitic disease in Latin America and has huge potential to become a worldwide problem, due to increasing migration, and international tourism, as well as infectant transfer by blood contact and transfusion, intrauterine transfer, and organ transplantation. Nearly 30% of chronically-infected patients become symptomatic, often with a latency of 10–30 years, developing life-threatening complications. Of those, nearly 90% develop Chagas heart disease, while the others manifest gastrointestinal disease and neuronal disorders. Besides interrupting the infection cycle and chemotherapeutic infectant elimination, starting therapy early in symptomatic patients is important for counteracting the disease. This would be essentially supported by optimized patient management, involving risk assessment, early diagnosis and monitoring of the disease and its treatment. From economic and logistic viewpoints, the tools of laboratory medicine should be especially able to guarantee this. After summarizing the basics of chronic Chagas disease, such as the epidemiological data, the pathogenetic mechanisms thought to drive symptomatic Chagas disease and also treatment options, we present tools of laboratory medicine that address patient diagnosis, risk assessment for becoming symptomatic and guidance, focusing on autoantibody estimation for risk assessment and heart marker measurement for patient guidance. In addition, increases in levels of inflammation and oxidative stress markers in chronic Chagas disease are discussed.

Background: The increasing need to reduce the costs of providing diagnostic laboratory services has prompted initiatives based on the centralization and consolidation of laboratory facilities. However, the majority of papers and experiences reported in literature focus on “cost per test” thus overlooking the real value of a laboratory service, which requires more complex economic evaluations, such as cost-benefit, cost-effectiveness, and cost-utility analysis. It is important to perform cost analysis, which is no mean feat, by taking into consideration all variables affecting the final and true cost per test. Methods: The present study was conducted in order to evaluate the costs of delivering laboratory services in 20 Italian clinical laboratories using a widely accepted methodology, the so-called “activity-based costing analysis”. Results: The finding of a trend towards a decrease in total costs – due to an increase in test volumes – attained statistical significance only for quantities of up to about 1,100,00 tests per year. For 1,800,00 tests and more, the cost per test appeared to range from 1.5 to 2.0 € irrespective of the different volumes. Regarding the relationship between volumes and number of staff, there is an evident linear relationship between the number of senior staff and volumes, whereas this trend is not observed in the case of medical technologists, the degree and type of automation strongly affecting this variable. Conclusions: The findings made in the present study confirm that the relationship between volumes and costs is not linear; since it is complex, numerous variables should be taken into account.

Background: Venous blood specimen collection is a common health care practice that has to follow strict guidelines, non-compliance among sampling staff may compromise patient safety. We evaluated a large-scale 2 h educational intervention that emphasised guideline adherence to assess possible improvements of venous blood specimen collection practices. Methods: Blood specimen haemolysis is usually caused by inadequate venous blood specimen collection and handling, reflecting overall pre-analytical handling. We monitored haemolysis of serum samples with haemolysis index corresponding to ≥150 mg/L of free haemoglobin for specimens sent from 11 primary health care centres and analysed on a Vitros 5,1 clinical chemistry analyser before (2008, n=6652 samples) and after (2010, n=6121 samples) the intervention. Results: The total percentage of haemolysed specimens was 11.8% compared to 10.5% (p=0.022) before the intervention. As groups, rural primary health care centres demonstrated a significant reduction [Odds ratios (OR)=0.744] of haemolysed specimens after intervention, whereas urban primary health care centres demonstrated a significant increase (OR=1.451) of haemolysis. Conclusions: A large-scale 2 h educational intervention to make venous blood specimen collection staff comply with guideline practices had minor effects on collection practices. Educational interventions may be effective in wards/care centres demonstrating venous blood specimen collection practices with larger deviations from guidelines.

Background: Cerebrospinal fluid (CSF) leaks are potentially life-threatening conditions that can be diagnosed by detection of β 2 -transferrin using protein electrophoresis. Another less commonly available test is β-trace protein quantitation using immunoassay. The objectives of this study were to evaluate a new immunofixation-based β 2 -transferrin test for detection of CSF leaks and to compare it to an existing agarose gel electrophoresis test and β-trace protein immunoassay. Methods: For method comparison, 63 consecutive samples from physician-ordered β 2 -transferrin tests were analyzed using two different electrophoresis methods, agarose gel fractionation followed by acid-violet staining, and high resolution agarose gel electrophoresis followed by β 2 -transferrin immunofixation. A subset of samples (16/63) were analyzed for β-trace protein. Results were compared against patient chart data for the presence of a CSF leak. Additional studies were performed to assess the stability, detection limit, and analytical specificity of the β 2 -transferrin immunofixation test. Results: The β 2 -transferrin immunofixation test had a sensitivity of 100% (40/40) and specificity of 71% (12/17) for detection of CSF leaks. By comparison, the agarose gel test had a sensitivity of 87% (35/40) and specificity of 94% (16/17). β-trace protein had a sensitivity of 100% (10/10) and specificity of 86% (5/6). Serum and saliva could be differentiated from CSF by the β 2 -transferrin immunofixation test based on their migration patterns. However, whole blood samples appeared positive for β 2 -transferrin at a threshold of ∼4 g/L hemoglobin. At a cut-off of 3 mg/L, β-trace protein was increased in 10/10 cases with documented CSF leak and in 1/6 patients without CSF leak. Conclusions: Both the new immunofixation test for β 2 -transferrin and the β-trace protein were effective at detecting CSF leaks. Users of the β 2 -transferrin immunofixation test should be cautioned against interpreting samples with blood contamination.

Background: About 30% of cutaneous psoriasis (PsC) patients develop psoriatic arthritis (PsA) in the joint, which is under-recognized by dermatologists. Biomarkers for PsA are needed so that early referral to a rheumatologist is made. Kallikreins (KLKs) are secreted serine proteases implicated in skin desquamation and inflammation. This study examined KLK potential as serum biomarkers of PsA in cutaneous psoriasis patients. Methods: KLKs were measured by ELISAs in synovial fluids of three PsA patients and three control early osteoarthritis (OA) patients, as well as in a cohort of 152 serum samples collected from age- and sex-matched PsC patients, with (n=76) or without PsA (n=76). KLK expression in psoriatic plaques was examined by immunohistochemistry. Univariate and multivariate logistic regression analyses were conducted to analyze the association between serum KLK levels and disease class (PsC, PsA). Serum KLKs that associated with PsA were correlated with clinical parameters of skin and joint activity. Results: Among the seven KLKs tested, KLK6 and KLK8 were elevated in both PsA synovial fluids and psoriatic plaques, but only serum KLK8 levels were associated with psoriatic disease (odds ratio=2.56, p=0.03). Although significantly elevated in PsC and PsA sera compared to healthy controls, KLK8 did not discriminate PsA from PsC patients. KLK8 correlated positively with the psoriasis area and severity index (PASI) (r=0.43, p=0.001) independent of age, sex and psoriasis duration (β=1.153, p=0.0003) and exhibited no correlations with tender or swollen joint counts. Conclusions: Increased KLK8 serum level in PsA patients reflects disease activity in the skin but not in the joints. Serum KLK levels are not useful for screening psoriasis patients for PsA.

Background: Soluble urokinase plasminogen activator receptor (suPAR) is a biomarker increasingly used for the assessment of systemic inflammation. We aimed to evaluate suPAR for the assessment of inflammatory activity in rheumatoid arthritis (RA) patients in remission. Methods: In our cross-sectional study we measured plasma suPAR and C-reactive protein (CRP) levels as well as erythrocyte sedimentation rate (ESR) in 120 RA patients at various stages of disease activity and 29 healthy age-matched controls. Results: suPAR, CRP and ESR values were higher in RA patients compared to healthy individuals. When suPAR levels were analyzed according to DAS28 scores of RA patients, suPAR level in the subgroup with DAS28≤2.6 was lower than in the subgroup with DAS28>2.6, but still higher than in controls [4.45 (3.33–5.56) ng/mL vs. 3.66 (3.10–4.67) ng/mL vs. 2.80 (2.06–3.42) ng/mL, p<0.0001, median (interquartile range)]. In contrast, CRP and ESR values were comparable in the subgroup with DAS28≤2.6 and in healthy individuals. We further analyzed the correlation between the number of tender and/or swollen joints and suPAR levels in RA patients in remission. suPAR values were significantly higher in patients with four tender and/or swollen joints than in patients with 2–3 or 0–1 tender and/or swollen joints. Conclusions: While CRP and ESR values indicate remission of the chronic inflammatory process in RA, suPAR values are still elevated compared to healthy individuals. suPAR might be particularly valuable in the recognition of inflammatory activity in patients who are in remission according to DAS28 scores but have symptoms of tender and/or swollen joints.

Background: Post-menopausal osteoporosis (PO) affecting a large fraction of elderly women, is triggered by the decline in 17β-estradiol (E2) level. Experimental studies in animal models and cell cultures have suggested that the fall in E2 might contribute to developing oxidative stress (OS) which in turn is believed to play an important role in PO pathogenesis. The scarcity of human studies focusing on this issue prompted us to investigate the effects of the reproductive and post-reproductive phase of women’s life on OS and bone health. Methods: Serum parameters of oxidative challenge (lipid hydroperoxides and protein advanced oxidation products) and antioxidant defence (total serum antioxidants levels) along with bone mineral density (BMD) at femoral neck and lumbar spine were assessed in a sample of 191 women (98 pre- and 93 post-menopausal, of whom 30 osteoporotic). Results: Pearson’s correlation analysis unveiled that spinal BMD was negatively correlated with lipid hydroperoxides in overall postmenopausal subsample (r=–0.251, p=0.012), while no significant link between these two variables was detected in women in reproductive age (r=–0.022, p=0.833). Noteworthy, stepwise multiple regression analysis showed that the association found in post-menopausal women retained significance after adjusting for potential confounding factors (p=0.001). Conclusions: Our data showed that markers of oxidative challenge are associated with bone loss in women in post-menopausal status. We suggest that menopause-related estrogen withdrawal might contribute to make bone more vulnerable to oxidative injury thereby increasing the risk of PO development.

Background: Voriconazole and itraconazole are two broad-spectrum antifungal triazole derivates administered for the prevention and in the treatment of invasive fungal infections. Their broad inter- and intra-individual pharmacokinetic variability and the high probability of drug-drug interactions justify therapeutic drug monitoring. Methods: After liquid-liquid extraction with tert-butyl methyl ether, chromatographic separation was achieved on a Zorbax Eclipse XDB-C18 column using gradient elution with 10 mM ammonium formate and acetonitrile. Detection was performed by a tandem mass spectrometer coupled to LC via an atmospheric pressure chemical ionization (APCI) and quantification was performed using selected reaction monitoring (SRM) transitions Results: Total run time was 4.5 min. The method was validated for concentrations ranging from 0.05 to 10 μg/mL for voriconazole and from 0.025 to 5 μg/mL for itraconazole and hydroxyitraconazole, respectively. The intra- and inter-day correlation coefficients of variation were <7.7%–<9.2%, respectively. The accuracy ranged from 92.6% to 109%. Conclusions: A rapid and simple liquid chromatography-atmospheric pressure chemical ionization-tandem mass spectrometry (LC-APCI-MS/MS) method has been developed and validated to measure voriconazole itraconazole and hydroxyitraconazole in human plasma. This method is successfully applied to samples from patients receiving antifungal treatment.

Background: The study aim was to compare the performance of three different methods used for determining urinary glycosaminoglycans (GAG) levels in spot and 24-h urine samples. Methods: Performance characteristics were studied for cetylpyridinium chloride (CPC), and manual and automated dimethylmethylene blue (DMB) methods. Results: For automated DMB method, within-run precisions were 9.10% and 1.98%, and between-day precisions were 13.0% and 5.81% in low- and high-urine pools, respectively. The method was linear up to 100 mg/L of GAG concentration. The detection limit of the method was 0.71 mg/L. Mean recovery was 95.7%. Conclusions: The automated DMB method was found to give better performance characteristics than cetylpyridinium chloride (CPC) and manual DMB methods. It is a fast, cheap, simple and reliable method and can be applied in many diseases in which GAG is used as a screening test.

Background: Food allergy is a common problem in France involving 4%–6% of toddlers. As opposed to IgE-mediated cow’s milk allergy (CMA), delayed-onset CMA, mostly, non-IgE-mediated, remains difficult to diagnose in toddlers. Our study assessed the diagnostic performances of intestinal permeability and of fecal markers, in comparison with the standard allergic work-up in children referred for CMA diagnosis. Methods: Twenty-five consecutive children, mean age (standard deviation) 6.3 months (4.8) with digestive and/or extra-digestive manifestations suggesting CMA, were prospectively studied based on a standardized allergic work-up (specific cow’s protein IgE and IgG, skin prick test, atopy patch test and oral open cow’s milk challenge) and digestive work-up including fecal microbiota analysis, intestinal permeability determination (urinary lactitol/mannitol ratio) and fecal markers measurement, i.e., α 1 -antitrypsin, tumor necrosis factor-α, calprotectin, β-defensin2, secretory IgA and eosinophil-derived neurotoxin (EDN). Receiver operating characteristic (ROC) curves were calculated for all markers in order to define cut-off levels. Results: The cow’s milk challenge was positive in 11 children and negative in 14. The global test performances, i.e., the number of true positive+negative cases/the total number of cases, were 76% for intestinal permeability; 72% for fecal EDN; contrasting with atopy patch test, 68%; IgE, 60%; skin prick test, 55% and IgG, 52%. Conclusions: In this routine diagnosis allergy work-up for CMA in toddlers, the best efficacy was seen for intestinal permeability compared to IgE, IgG, skin prick test and atopy patch test. Moreover, fecal EDN in a single spot sample displayed a similar performance.

Background: Hematopoietic chimerism analysis is important in the follow-up of patients undergoing allogeneic stem cell transplantation. PCR of short tandem repeats is mainly used for monitoring chimerism after transplantation. Validation studies and precision of assay’s performance with respect to different mixed chimerism stages is not fully addressed. The aim of the present study was to assess the impact of several microsatellite analytical parameters in the quantification of hematopoietic chimerism after allogeneic hematopoietic stem cell transplantation and to analyze the overall analytical process through the application of internal quality control procedures. Methods: Artificial DNA mixtures prepared in known proportions and patients samples were analyzed using three microsatellites, together with amplification of amelogenin gene and fluorescence in situ hybridization (FISH) for X and Y chromosomes. Limit of detection, analytical and clinical sensitivity, stochastic threshold and precision profiling was established. Levey-Jennings charts and Westgard rules were applied for quality control evaluation. Results: Analytical and clinical sensitivity of the microsatellite markers was between 0.5% and 1.6%. Amelogenin detection and FISH for X and Y chromosomes showed a similar sensitivity. Severe allelic imbalance resulted in up to 50% difference between the calculated and corrected mixed chimerism. Systematic errors were identified using Levey-Jennings charts and Westgard rules. Conclusions: Analysis of hematopoietic chimerism performance is a critical step to better understand potential intrinsic errors that may impact the final hematopoietic chimerism results. Implementing quality control tools, such as Levey-Jennings charts together with Westgard rules can identify systematic and random errors so corrective actions can be performed.

Background: Antibodies against low-density lipoproteins (LDLs) that have been oxidized are associated with development of atherosclerotic lesions. In individuals infected with human immunodeficiency virus type 1 (HIV-1) with or without therapy, dyslipidemia and increased cardiovascular risk are observed. Methods: Serum levels of IgG antibodies against oxidized LDLs (IgG anti-oxLDL Abs) were determined by assay in 151 HIV-1-infected patients. Of these, 42 patients did not receive anti-retroviral therapy (ART-naïve), whereas 109 received highly active anti-retroviral therapy (HAART) consisting of lopinavir/ritonavir (LOP/r; n=50), efavirenz (EFV; n=30) and nevirapine (NVP; n=29) associated with nucleoside reverse transcriptase inhibitors. HIV-1 seronegative individuals (n=43) participated in the study. The following parameters were quantified: total cholesterol and its fractions, atherogenic indices (AIs), apolipoproteins A1 and B100, high sensitivity C-reactive protein, CD4 + and CD8 + T cells, and HIV-1-RNA. Results: Levels of IgG anti-oxLDL Abs were significantly higher (p<0.05) in the LOP/r group compared with the EFV and/or NVP and the seronegative group: median 0.32 (0.15, 0.58; 95% confidence interval) vs. 0.25 (0.13, 0.53) vs. 0.18 (0.04, 0.38), respectively. HIV-1-infected ART-naïve patients (n=42) presented antibodies levels similar to those observed for the LOP/r group, 0.33 (0.13, 0.63; p>0.05). The levels of IgG anti-oxLDL Abs correlated with an increase in AIs (r=0.216; p=0.036) and triglycerides (r=0.220; p=0.044) in the LOP/r group, and AIs in the ART-naïve group (r=0.300; p=0.046). Conclusions: Patients treated with LOP/r showed higher levels of IgG anti-oxLDL Abs compared with patients treated with EFV or NVP regimens, and these levels were associated with an increase in AIs.

Background: Phospholipid-dependent coagulation tests for lupus anticoagulant (LA) are considered an important step for the diagnosis of anti-phospholipid syndrome; however, LA laboratory detection is difficult because of many variables. Five hospital laboratories, located in a North-Italy area and using the same method for LA testing, cooperated to standardise sample treatment and analytical procedure in order to define the upper values for LA negativity. Methods: In total, 200 normal subjects (40 for each centre) were studied for six LA functional assays, using the same procedure, reagent lot and analyser type. The first tests done were LA screen and LA confirm assays, based on diluted Russell’s Viper Venom Time, with low and high phospholipid content, respectively. The second tests performed were silica clotting time screen and confirm assays, based on activated partial thromboplastin time, with low and high phospholipid content, respectively. Finally, two mixing assays were executed for both screening assays, diluting patient sample with a pool prepared with plasma collected from the study population. Results: Data analysis demonstrated a difference between centres for all assays when results were expressed in seconds; the difference disappeared when results are normalised with the local mean normal value of each centre and are expressed as a normalised ratio. The study population was normally distributed; so the value corresponding to 99th percentile was used as limit value for LA negativity. Values expressed as normalised ratio, for LA and silica clotting time screenings were 1.22 and 1.23, respectively. Conclusions: The study allowed us to define a uniform approach to LA testing and evaluation for laboratories employing the same methods.

Background: Multiple organ dysfunction syndrome (MODS) observed in patients with sepsis and in non-septic patients organ failure (OF) is associated with a high mortality rate. We investigated whether new coagulation assays [quantification of procoagulant phospholipids (PPL) activity, functional assays measuring the activity of thrombomodulin (TMa) or tissue factor (TFa) and thrombin generation using calibrated automated thrombography (CAT)] could constitute new tools to better understand the physiopathology of MODS and have any prognostic value. Methods: We measured TMa, TFa, PPL and CAT in 32 healthy controls, 24 patients with sepsis and 26 patients with non-septic OF. We compared these parameters with usual coagulation assays [prothrombin time, activated partial thromboplastin time, protein C (PC), protein S, D-Dimers (D-Di), soluble thrombomodulin (sTM)] and markers of inflammation (IL-6, CRP). Samples were collected within 24 h of the diagnosis. Results: TMa, TFa, PPL, the lag time and time to thrombin peak levels were increased in both groups of patients. For both groups D-Di, IL-6, CRP and endogenous thrombin potential (ETP) were higher in non-survivors than in survivors, while PC and PPL were lower in non-survivors than in survivors. TMa increase was more marked in non-survivors patients with OF, while the ratio TMa/sTM was low in non-survivors with sepsis. Received operating characteristic (ROC) curve analysis indicated that thrombin peak and ETP were the more powerful discriminating factors in patients with sepsis or non-septic OF, respectively. Conclusions: PPL, TMa and CAT assays could represent promising tools to identify patients with increased risk of mortality in MODS and could procure insights into pathogenesis of MODS.

Background: Point of care (POC) testing for fetal scalp blood lactate is a more user friendly and more successful approach compared to scalp pH for intrapartum fetal monitoring. The aim of this study was to assess the analytical specificity and clinical reliability of three POC lactate methods. Methods: The analytical performance of three POC lactate methods was compared to Cobas 6000 (Roche Diagnostics) laboratory reference method: Lactate Pro from Arkray, GEM 4000 from Instrumentation Laboratory and StatStrip Lactate from Nova Biomedical. The clinical performance and influences on accuracy and decision making criteria for the three POC methods was assessed with umbilical cord samples and compared to the laboratory reference method. The influence of varying ranges of hemoglobin, pH and partial oxygen pressure (pO 2 ) on the accuracy of results was assessed. Results: Although all three POC methods showed good correlation with the reference method for the umbilical cord sample population (r=0.989, 0.973 and 0.980, respectively), Lactate Pro and Gem 4000 showed a significant negative bias compared to the reference method. The degree of bias meant a significant readjustment of decision making criteria was required for fetal lactate use. The accuracy of the Lactate Pro results was affected by hemoglobin and to a lesser extent pH. Conclusions: The three electrochemical POC devices can measure fetal lactate reliably. StatStrip Lactate showed a closer correlation and concordance to our laboratory reference method. The results of this study indicate the requirement for predetermining the reliability of POC lactate methods before use present in fetal and perinatal settings.

Background: The aim of the study was to retrospectively investigate whether parameters of routine point-of-care testing (POCT) predict hospital mortality in critically ill surgical patients on admission to the intensive care unit (ICU). Methods: Arterial blood analyses of 1551 patients on admission to the adult surgical ICU of the Technical University Munich were reviewed. POCT was performed on a blood gas analyser. The association between acid-base status and mortality was evaluated. Metabolic acidosis was defined by base excess (BE) <–2 mmol/L and, wherever applicable, subdivided into lactic acidosis by lactate >50% of BE, anion gap (AG)-acidosis by AG >16 mmol/L, hyperchloraemic acidosis by chloride >115 mmol/L. Metabolic alkalosis was defined by BE ≥3 mmol/L. Logistic regression analysis identified variables independently associated with mortality. Results: Overall mortality was 8.8%. Mortality was greater in male patients (p=0.012). Mean age was greater in non-survivors (p<0.0005). Nine hundred and eighty-six patients showed no metabolic acid-base disorder (mortality 7.3%), thereof 26 patients with pCO 2 >55 mm Hg (mortality 23.1%). Three hundred and seventy-seven patients presented with acidosis (mortality 11.4%), thereof 163 patients with lactic acidosis (mortality 19%). Mortality for alkalosis (174 patients) was 12.1%. Mean blood glucose level for non-survivors was higher compared to survivors (p<0.0005). Logistic regression analysis identified lactate, glucose, age, male gender as independent predictors of mortality. Conclusions: Lactate and glucose on ICU admission independently predict mortality. BE and AG failed as prognostic markers. Lactic acidosis showed a high mortality rate implying that lactate levels should be obtained on ICU admission. Prevalence of hyperchloraemic acidosis was low. Metabolic alkalosis was associated with an increased mortality. Further studies on this disturbance and its attendant high mortality are warranted.

Background: In cancer patients, including women with a diagnosis of ovarian cancer, cancer antigen 125 (CA125) is used to evaluate the presence of peritoneal involvement. The aims of the present study were to assess CA125 reference intervals and reference change values (RCV) in postmenopausal reference women, postmenopausal women breast cancer free, reference men and cancer free men. Methods: The series consisted of 433 subjects: 105 postmenopausal breast cancer free women and 56 cancer free men in addition to a total of 272 reference subjects (145 postmenopausal women and 127 men). Repeated CA125 measurements were made in a subset of 149 women and 54 men to calculate RCV and index of individuality. Serum CA125 levels were evaluated by a chemiluminescent assay. Results: In postmenopausal reference women, the mean CA125 value and 2.5th–97.5th percentiles were 6.70, 2.60–11.00 kU/L, respectively, with a unidirectional RCV of 38.4%. In postmenopausal breast cancer free women, the mean CA125 value and 2.5th–97.5th percentile were 7.45, 4.09–10.92 kU/L, respectively, with a RCV of 34.5%. The difference between the means was statistically significant (t=–3.02, p=0.003). In the two male subgroups, the difference between the means for CA125 was not statistically significant (t=0.43, p=0.665). On considering the entire male population, the mean CA125 value and 2.5th–97.5th percentiles were 7.50 and 2.40–13.2 kU/L, respectively, while the unidirectional RCV was 34.3%. In all the studied groups, the indices of individuality were equal to or below 0.6. Conclusions: The extremely low index of individuality found underlines the importance of using the RCV instead of absolute values as a parameter when interpreting the CA125 data in the monitoring and follow-up of patients with ovarian cancer.

Background: Interleukins, interferons and oxidative DNA products are important biomarkers assessing the inflammations and tissue damages caused by toxic materials in the body. We tried to evaluate distributions, reference values and age related changes of blood levels of inflammatory cytokines, C-reactive protein (CRP), IgE and urine levels of 8-hydroxy-2′-deoxyguanosine (8-OHdG) among workers in a cohort study evaluating the health influences of toner particles. Methods: A total of 1366 male workers under age 50 years (age 19–49 years; 718 exposed and 648 not exposed to toner particles) in a cross sectional study of 1614 (categorized as 809 exposed and 805 not exposed, age 19–59 years) workers in a photocopier company has been followed prospectively as the cohort. Blood levels of interleukin (IL)-4, IL-6, IL-8, interferon-γ (IFN-γ), CRP, IgE and urine 8-OHdG were measured annually for 5 years. Results: Reference values of the biomarkers are; CRP: 0.01–0.63×10 -2 g/L, IgE: 6–1480 IU/mL, IL-4: 2.6–76.1 pg/mL, IL-6: 0.4–4.9 pg/mL and 8-OHdG: 1.5–8.2 ng/mgCr. We could not evaluate reference values for IL-8 and IFN-γ because most of the values were below the sensitivity limits (2.0 pg/mL and 0.1 IU/mL, respectively). There were no differences of the biomarker levels between the toner exposed and the control workers. We observed a statistically significant age related decrease of serum IL-4 levels. Conclusions: This is the first report assessing the distributions and reference values of inflammatory biomarker levels in a large scaled cohort. We observed age related changes of some of the biomarkers. We could not detect any differences of the studied biomarker values between the toner exposed and the control workers.

Background: The aim of this study was to determine the influence of inflammation on acute phase protein and epithelial-mesenchymal transition (EMT) in buccal cancer. Methods: Western blotting was carried out to investigate the expression of haptoglobin and epithelial-mesenchymal transition in oral cancer cell lines with or without IL-6 stimulation. We studied patients with buccal cancer patients without distant metastasis at diagnosis. Correlation between cellular haptoglobin, EMT, and clinical characteristics of buccal cancer was analyzed to assess the prognostic value of cellular haptoglobin level and EMT. The relationship of haptoglobin, and EMT expression with survival was assessed using Cox proportional hazard models. Results: Western blotting analysis showed that increased haptoglobin protein was associated with overexpression of vimentin. Under IL-6 stimulation, overexpression of haptoglobin, EMT-associated motile phenotype was noted in OC2 cell lines. Overexpression of haptoglobin was also associated with an increased risk for locoregional recurrence [hazard ratio (HR) 1.04; p=0.011] after adjusting for age, gender, disease site, stage, and treatment modality. Conclusions: Increased cellular expression of haptoglobin is associated with EMT in oral cancer cell lines and this phenomenon could be exaggerated with IL-6. Cellular expression of haptoglobin is related to locoregional recurrence rate in buccal cancer patients.

Background: We compared two recently developed immunoassays for serum thymidine kinase 1 (TK1) activity: one manual assay (DiviTum, Biovica ® ) and one fully automated assay (Liaison, Diasorin ® ). Methods: The study included 368 women: 149 healthy blood donors (control), 59 patients with benign breast disease (BBD) and 160 patients with primary breast cancer (BC). Results: A regression analysis of the Liaison (y) and DiviTum (x) assays for all three groups yielded the equation y=3.93+0.03x (r=0.85, n=368). The r-value in BC was higher than in control and BBD (0.90 vs. 0.81 and 0.64). The correlation between the two assays for TK1 values above the cut-off was higher compared to that below (0.88 and 0.59). Breakdown of the BBD group into subgroups with proliferative and non-proliferative lesions was effective only with the measurement of TK1 with DiviTum assay (p=0.03). The TK1 activity determined preoperatively in BC patients with DiviTum and Liaison assays was significantly associated with T-stage (for both p=0.01), presence of vascular invasion (p=0.002 and p=0.02), lack of estrogen receptor (ER) (p=0.001 and p=0.01) and progesterone receptor (PR) (p=0.01 and p=0.03) expression. Only TK1 analyzed with the DiviTum assay was associated with tumor grade and molecular subtype of BC (p=0.02 and p=0.003). Multivariate Cox proportional hazards analyses demonstrated that T-stage, PR status and TK1 activity measured by both methods (DiviTum, RR=3.0, p=0.02 and Liaison, RR=3.1, p=0.01) were independent predictors of disease recurrence. Conclusions: In spite of differences observed between TK1 activity measured by the DiviTum and Liaison assays, both of them may be used for recurrence prediction in preoperative evaluation of BC patients.

Background: The present study was undertaken to evaluate a new sensitive thyroglobulin (Tg) mini-recovery test (Tg-mrec) for the detection of potential interferences in sera from patients with differentiated thyroid carcinoma (DTC) and low Tg levels. Methods: 167 DTC patients with serum Tg <2 μg/L were enrolled. Both TgAb and Tg-mrec measurements were performed on the automated Kryptor ® platform. Serum pretreatment in proprietory blocking tubes was perfomed to screen for heterophile antibody interferences. The concordance rates between tests were evaluated. Results: One case of over-recovery occurred in a patient with discordant Tg results in different immunoassays. The prevalences of a positive TgAb test and a reduced recovery rate were 15%–12%, respectively. Serum TgAb and Tg-mrec tests were both positive in 16 patients, both negative in 138 patients and discordant in 13 patients, respectively. The concordance rate between the Tg-mrec test and the TgAb assay was 92% (Cohens’ kappa 0.894; 95% confidence interval: 0.82–0.93, p<0.001). In all, among seven clinically relevant interferences, three were found by TgAb, five by Tg-mrec and six by using both tests. Conclusions: The automated Kryptor ® Tg-mrec test has a complimentary value to TgAb immunoassay testing in the detection of potential interferences in Tg measurements in patients with DTC and low Tg values (i.e., <2 μg/L). Tg-mrec may detect interferences from TgAb not measured directly by an individual TgAb assay or from other interfering substances, such as heterophile antibodies.