Volume 52, Issue 2

Lung cancer is one of the most common malignancies in the world and one of the leading causes of death from cancer. In the search for molecules that may be involved in lung tumor induction and progression, the receptor of advanced glycation end products (RAGE) comes across as a critical regulator of lung physiology. RAGE is a multiligand receptor that presents a differential expression pattern in lung epithelial cells compared to other cell types being gradually increased from fetal to birth and adult life. Under stress conditions, RAGE expression and activation are rapidly elevated resulting in chronic inflammation, which, in turn, in many instances, promotes epithelial cell malignant transformation. RAGE overexpression in normal lung alveolar type I epithelial cells is followed by rapid downregulation upon malignant transformation, being associated with increased aggressiveness. This is a striking paradox, since in every other cell type the pattern of RAGE expression follows the opposite direction, suggesting the involvement of RAGE in the well-functioning of lung cells. Additionally, RAGE has been attributed with the role of adhesion molecule, since it can stabilize mature alveolar epithelial cells to their substrate (basal lamina) by interacting electrostatically with other molecules. However, the reduction of RAGE observed in lung tumorigenesis interrupts cell-to-cell and cell-to-substrate communication, which is a critical step for cancer cell induction, progression and migration. This review addresses the differential properties of RAGE in lung physiology and carcinogenesis, providing evidence of therapeutic possibilities.

Cardiac troponins have changed our understanding of myocardial infarction profoundly during the past 20 years. In fact release of cardiac troponin into the blood has become part of the definition of myocardial infarction. Diagnostics and treatment have taken these new insights into account. The novel high-sensitive assays for cardiac troponins that are available recently clearly constitute a significant further improvement of analytical performance. In addition, the still incomplete clinical data available indicate that these assays will also further improve patient care in many ways. In summary, cardiac troponins are a great example of the potential impact of analytical performance of diagnostic tests on patient care and patient outcome.

The diagnosis of acute coronary syndrome (ACS) has challenged the minds of cardiologists, emergency physicians and laboratorists for decades. A major breakthrough has, however, occurred at the dawn of the third millennium, with development, commercialization and introduction into clinical practice of troponin immunoassays. A novel generation of these methods, conventionally defined as “high-sensitivity” (HS), has more recently emerged. These latest generation assays are characterized by improved analytical sensitivity, which would theoretically allow earlier and more efficient diagnosis of ACS. Despite the considerable amount of information gathered over the past few years about the clinical use of conventional and HS immunoassays, several doubts persist and – according to our personal perspective – the evidence that the latest generation methods would represent a real breakthrough in management of patients in short-stay units such as the emergency department is still an unresolved issue. Beside the mystifying nomenclature that characterizes several commercial tests, recent evidence suggests that the diagnostic performance of some contemporary sensitive methods would equal those of HS immunoassays for early diagnosis, serial assessment and even prognostication of patients. Conversely, the better diagnostic specificity of conventional methods may represent an advantage for triaging patients in overcrowded emergency departments. There is hence a tangible threat that the measurement of troponin with HS methods would become more or less an “expensive cholesterol of the third millennium”, and this risk must be carefully considered in a world of limited resources. So, our answer to the question if we do really need HS troponin immunoassays in the emergency department is “maybe not”.

Background: The determination of urinary cortisol/cortisone ratio is of clinical utility in cases of Cushing’s syndrome, apparent mineralocorticoid excess, and also provides information on 11β-hydroxysteroid dehydrogenase (11β-HSD) type 2 activity. It is therefore of utmost importance to ensure accurate cortisol and cortisone measurement and establish appropriate reference ranges. Methods: After the isotopic dilution of urine, sample cleanups were obtained with on-line solid-phase extraction and cortisol and cortisone, separated using a Zorbax Eclipse XDB-C18 HPLC analytical column, were analyzed by tandem mass spectrometry with an electrospray ionization source in positive ion mode operation. Results: The method was linear, with concentrations of up to 625 and 1125 nmol/L and lower limit of quantitation (LLOQ) of 5 and 6 nmol/L, for cortisol and cortisone, respectively. Within-run and between-run coefficients of variation were <5% and 6% for cortisol and 6% and 8% for cortisone, respectively. No ion suppression was observed. The non-parametric reference range for the cortisol/cortisone ratio was 0.14–1.09. Conclusions: A simple and sensitive liquid chromatography tandem mass spectrometry method was developed and validated for the measurement of cortisol and cortisone in urine. Our findings indicate that the proposed analytical method is suitable for routine purposes and useful in many pathological conditions.

Background: Neutrophil gelatinase-associated lipocalin (NGAL) serum values have been shown to increase in preeclampsia. The goal of the present study was to evaluate changes in urinary NGAL concentrations during uncomplicated pregnancy and in cases of preeclampsia and hypertension. Methods: Fifty-one pregnant women who developed preeclampsia and 28 diagnosed with essential or gestational hypertension were investigated for urinary NGAL concentrations during pregnancy. As controls, 100 healthy pregnant women with uncomplicated singleton pregnancies were randomly selected. Urinary NGAL as well as urinary creatinine and albumin were measured by a standardized clinical chemistry platform (ARCHITECT ® ; Abbott Diagnostics, Abbott Park, IL, USA). Results: Urinary NGAL concentrations increased during pregnancy in healthy pregnant women, whereas this increase was not detected in preeclampsia. In order to correct for diuresis, spot urine concentrations were also determined as NGAL/creatinine ratio. NGAL/creatinine ratio in pregnancy week 36–38 was significantly lower in preeclampsia than in healthy pregnant women or pregnant women with hypertension. NGAL urinary concentrations did not correlate with albumin concentration in urine. Conclusions: Urinary NGAL is not a valuable early biomarker for preeclampsia.

Background: In this study we have looked at the reliability of a multi-sugar test in a pediatric patient population and its accuracy at small urine volumes to evaluate intestinal permeability. Methods: Out of 117 subjects enrolled, 31 were healthy and 86 were sick. A solution containing lactulose, rhamnose, sucrose, and sucralose was administered to subjects who were on fasting; the urine excreted during 5 h was collected and measured. Samples were analyzed by gas chromatography-tandem mass spectrometry and results were expressed as percentage of sugar recoveries and lactulose/rhamnose (L/R) ratio. Results: The analyses showed a clear effect of low urinary volumes (≤240 mL) particularly affecting rhamnose excretion in healthy subjects and sucrose and sucralose recovery in diseased children. Despite the low rhamnose recovery, as lactulose is not similarly affected, the diagnostic reliability of L/R ratio is well preserved at low diuresis conditions. However, this ratio can be useful to discriminate acute conditions vs. clinical remissions only at high urine volumes. Data also suggest potential diagnostic applicability of sucrose and sucralose in children at high urine volumes. Conclusions: In conclusion, the multi-sugar test has a good predictivity in pediatric subjects but results must be carefully interpreted in the face of reduced diuresis.

Background: Recently, measurement of amino terminal propeptide of type III procollagen (PIIINP) was introduced as a part of the hepatic cirrhotic marker enhanced liver fibrosis™ test on the automated ADVIA Centaur ® immunoassay platform (Siemens Healthcare Diagnostics Inc., Tarrytown, NY, USA). In this article, we show that the Centaur PIIINP may be used in place of the much more labor-intensive RIA method, and we present an age stratified reference interval. Methods: We analyzed four control samples 20 times over a period of 5 days. Centaur PIIINP assay measurements were compared with the widely used UniQ PIIINP RIA assay (Orion Diagnostica, Espoo, Finland) using 55 patient samples (range=3.7–43.3 µg/L). Furthermore, we established a reference interval based on samples from 287 blood donors. Results: In the concentration range 2.5–11.9 µg/L, the total imprecision was below 8%. Comparison with the UniQ PIIINP RIA assay yielded: Centaur PIIINP µg/L=1.9×(UniQ PIIINP RIA)+0.6 µg/L, r 2 =0.94. The reference interval for the Centaur PIIINP assay showed no gender difference but was stratified by age [4.0–11.0 µg/L (18–40 years) and 3.5–9.5 µg/L (41–70 years)]. Conclusions: We conclude that the Centaur PIIINP assay is suitable for routine use with our newly defined reference interval. The results obtained by Centaur correlates well with those obtained by the previously employed RIA, though the absolute values are higher.

Background: Enumeration and characterization of circulating tumor cells (CTCs) can provide information on patient prognosis and treatment efficacy. However, CTCs are rare, making their isolation a major technological challenge. We developed a technique for enrichment, and subsequent characterization of CTCs based on efficient depletion of human leukocytes. Methods : The technique (CanPatrol TM CTC enrichment) we developed is based on red blood cell lysis to remove erythrocytes, followed by depletion of CD45+ leukocytes using a magnetic bead separation method, and subsequent isolation of CTCs by virtue of their larger size, compared with leukocytes. We also demonstrated that fluorescence in situ hybridization (FISH) and genetic abnormalities analysis could be performed on the isolated CTCs. Results: The spiking experiments showed that the average efficacy of leukocytes depletion was 99.98% and the average tumor cells recovery was not lower than 80%. FISH could be used to perform ALK gene rearrangement analysis on the collected NCI-H2228 cells, and EGFR Exon 19 deletion was detected by PCR-based analysis in isolated HCC827 cells. The in vivo feasibility of this technique had been demonstrated in patients with non-small cell lung cancer, breast, colon, and esophageal cancers. CTCs were detected in 13 of 59 blood samples. Tumor microemboli was also detected in three breast cancer samples. Conclusions: The technique we developed allowed isolation and characterization of circulating epithelial tumor cells that do not express classical epithelial antigens. This potentially leads to a more accurate enumeration of the number of CTCs and is suitable for application to a broad range of cancers.

Background: Conventional cytological examination has limited sensitivity for detecting tumor cells in serous body cavity effusions and therefore, adjuvant techniques are necessary for a reliable diagnosis. Flow cytometry has proven benefit in these circumstances. The aim of our study was to explore the feasibility of CELL-DYN Sapphire, an advanced hematology analyzer with flow cytometric capabilities, for detecting tumor cells in serous body fluids, using CD326 monoclonal antibodies, which are directed against the epithelial marker EpCAM. Methods: One hundred and five serous fluids (39 peritoneal and 66 pleural effusions) were analyzed by the CELL-DYN Sapphire using monoclonal antibody combinations CD3/CD19 and CD45/CD326. Of all samples a cytospin preparation was made and microscopically examined; the pathology findings served as a reference. Results: Using a threshold of 1% CD326+ cells, CELL-DYN Sapphire identified nine out of 12 cases with tumor cells in the serous effusions (sensitivity 75%), whereas routine cytology found eight cases (sensitivity 67%). The combination of immunophenotyping and cytology identified all 12 cases with tumor cells in the effusion fluid (sensitivity 100%). The specificities were 92% and 100%, respectively. Conclusions: We demonstrated that it is feasible to run an immunophenotypic assay on CELL-DYN Sapphire for detecting tumor cells in serous body fluids. In addition, this study confirmed that a combination of conventional cytology and flow cytometry had a very high diagnostic yield in cases of carcinomatous effusions.

Background: The focus during the diagnostic process for patients with acute chest pain is to discriminate patients who can be safely discharged from those who are at risk for an acute coronary syndrome (ACS). In this study the diagnostic value of the clinical examination is compared with laboratory testing of troponin. Methods: This study included 710 chest pain patients who presented at the ED of two hospitals in the Netherlands. Clinical examination and laboratory testing were combined in the recently developed HEART-score. The diagnostic values of clinical presentation, troponin and the HEART-score for a major adverse coronary event (MACE) and an ACS within 6 weeks were assessed. Furthermore, the improvement of HEART with the second troponin measurement after 6 h was assessed using the net reclassification improvement (NRI). Results: The use of HEART (AUC MACE : 0.77; AUC ACS : 0.82) obtains a higher diagnostic value than troponin (AUC MACE : 0.72; AUC ACS : 0.74) or clinical evaluation (AUC MACE : 0.69; AUC ACS : 0.74). Statistical significant different AUCs were obtained when HEART is compared to troponin or clinical evaluation (p<0.01). The use of the second troponin test (after 6 h of admission) within HEART resulted in an improvement of 8.0%. Conclusions: The HEART-score combines clinical evaluation and results from laboratory testing, which should be used together, to discriminate patients at risk of a cardiac event from patients who can be safely discharged. In addition, it is shown that a second troponin measurement slightly improves the discriminative ability of the HEART-score.

Background: The leading mechanisms responsible for the most prevalent and serious cardiac injuries include myocardiocyte stretch, myocardiocyte necrosis and cardiac fibrosis, which can now be reliably mirrored by measurement of natriuretic peptides, cardiospecific troponins and galectin-3, respectively. Although a large amount of knowledge has been gathered about the behavior and clinical significance of these biomarkers in patients with cardiac disorders, less information is available on their biology in paraphysiological conditions, including high-intensity endurance exercise. Methods: The study population consisted of 18 trained athletes, who performed a 60-km ultramarathon run. Blood was collected before the run (i.e., “baseline”) and immediately after the end of the ultramarathon (“post-marathon”) for measurement of serum high-sensitivity troponin I (TnI), NT-proBNP and galectin-3. Results: The concentration of all biomarkers measured in the post-marathon samples was remarkably increased as compared with the values obtained on baseline specimens. In particular, the median increase was 3.3 for TnI, 3.5 for NT-proBNP and 2.4 for galectin-3, respectively. The frequency of values exceeding the diagnostic threshold did not differ at baseline and after the ultramarathon for TnI (6% vs. 25%; p=0.15), instead was significantly increased for NT-proBNP (0% vs. 28%; p=0.016) and galectin-3 (0% vs. 67%; p<0.001). No significant correlation was found among the increase of any of the three biomarkers. Conclusions: The results of this study demonstrate that high-intensity endurance exercise is associated with biochemical abnormalities that may reflect adverse consequences on cardiac structure and biology.

Background: Cardiac troponin-specific autoantibodies (cTnAAb) can interfere with the measurement of cardiac troponin I (cTnI) by immunoassays used for the diagnosis of myocardial infarction (MI). Here, an improved version of a previous autoantibody assay was validated and used to evaluate the cTnAAb prevalence in a cohort of consecutive chest pain patients presenting to an emergency department. Methods: Admission samples from 510 patients with suspected MI were analyzed in parallel with two sandwich-type cTnAAb assays based on different cTnI epitopes used to capture cardiac troponin-bound cTnAAbs. Results: Sample-specific backgrounds were lower for the new assay than for the old assay (median 1225 vs. 2693 counts, p<0.001). Net signals of cTnAAb-positive samples were higher for the new assay than for the old assay (median 5076 vs. 3921 counts, p<0.001). Of all patients, 9.2% were cTnAAb-positive for the new assay and 7.3% for the old assay (p=0.013). Previous cardiac problems were not associated with cTnAAb status and cTnAAb status did not correlate with the 12-month outcome. Conclusions: With our new and more sensitive autoantibody assay, approximately one out of ten patients who presented to the initial cardiac triage had detectable amounts of cTnAAbs in the circulation. Because these cTnAAbs can interfere with state-of-the-art cTnI assays, their high prevalence should be acknowledged by clinical chemists, physicians, and kit manufacturers.

Background: We compared B-type natriuretic peptide (BNP) levels, clinical and laboratory findings in early-onset preeclampsia (EOP), late-onset preeclampsia (LOP) and healthy pregnant groups. Methods: We studied 40 healthy pregnant and 40 preeclamptic patients. Preeclamptics were divided in two groups, the EOP group (n=20) and LOP group (n=20), according to gestational age at the onset of disease. The distinction criterion for early- vs. late-onset was set as week 34 of gestation. The concentration of the BNP levels was measured by a sandwich fluorescence immunoassay. For statistical analysis of the clinical and laboratory findings non-parametric methods were applied. Results: BNP levels were higher in EOP [61.35 (36.95–93.25) pg/mL] and LOP patients [32.4 (19.15–39.2) pg/mL] than in healthy pregnant women [10.05 (6.08–16.03) pg/mL] (both p<0.001). Furthermore, EOPs had significantly higher BNP levels as compared to LOP patients (p<0.001). A BNP cut-off <24.5 pg/mL had a negative-predictive value of 85.1% excluding preeclampsia. There was a significant inverse correlation between plasma BNP levels of EOP patients and sodium (p<0.05) and total protein concentrations (p<0.05). In the EOP group, a significant positive correlation was observed between plasma levels of BNP and hematocrit (p<0.05), serum potassium (p<0.05), urea (p<0.05) and 24-h proteinuria (p<0.05). Conclusions: BNP levels were significantly higher in EOP than in LOP patients. The cut-off value <24.5 pg/mL seems to be a powerful discriminative indicator excluding preeclampsia. The amount of proteinuria and total protein levels correlate with the elevation of the BNP levels. In EOP the extent of proteinuria is higher than in the LOP.

Background: The glucokinase regulator gene ( GCKR ) rs780094 has been shown to be strongly associated with some metabolic traits and atherosclerotic parameters, while the association between GCKR rs780094 and carotid intima-media thickness (CIMT) has not been fully investigated in the general population. The associations between the GCKR rs780094 genotype and metabolic traits including CIMT were examined in a Japanese community-dwelling population. Methods: A total of 2491 Japanese adults (907 men and 1584 women) who participated in a medical screening program for the general population from 29 to 94 years of age during 2008 to 2010 were enrolled. GCKR rs780094 was genotyped by the TaqMan polymerase chain reaction method, and associations with metabolic markers including CIMT were evaluated. Results: GCKR rs780094 AA genotype was significantly associated with higher TG (p<0.001 vs. GG), lower HDL-C (p=0.021 vs. GG), and lower HbA 1c (p=0.023 vs. GG). The AA genotype showed significantly thinner CIMT (p=0.001 vs. GX). These associations were seen only in men. Conclusions: GCKR rs780094 was associated with TG, HDL-C, and HbA 1c levels, as well as with CIMT in Japanese community-dwelling men, but not women.

Background: Many epidemic outbreaks of Chikungunya fever (CHIKF) have been reported throughout the world including India after its reemergence in 2005. The immuno protective role of envelope proteins during Chikungunya virus (CHIKV) infection has been reported. With the aim of identifying the immunodominant epitopes within the envelope protein we investigated the detailed analysis of fine specificity of antibody response in different individuals during CHIKV infection. Methods: The peptides corresponding to the full length of E1, E2 and E3 proteins of S27 strain of CHIKV were synthesized and their seroreactivity with CHIKV positive patients’ sera collected from different epidemic regions of India was determined using indirect ELISA. Results: The data analysis reveals many potent epitopes throughout the length of envelope E2 protein thus displaying it as the most promising antigen for diagnostic purpose. We found that the main IgG isotype response to envelope protein was predominantly of subclass IgG3. Interestingly, most of the epitopes were found to be conserved for detecting IgM, IgG and IgG3 antibody response. Conclusions: Peptides E2P3, E2P7, E2P16 and E2P17 were revealed as the most immunodominant peptides that together can form the basis for designing an accurate, economical and easy to synthesize a peptide-based immunodiagnostic for CHIKV. This study provides new and important insight into the humoral response generated by CHIKV S27 strain during the early phase of infection.