Volume 50, Issue 6 - 2nd IFCC Ortho Clinical Diagnostics (OCD) Conference “Disease and the Clinical Laboratory Pregnancy Related Disorders: Present Perspectives and Emerging Challenges”

Pre-eclampsia (PE) and other hypertensive disorders of pregnancy (HDP) are a leading cause of adverse outcomes. Their pathophysiology remains elusive, hampering the development of efficient prevention. The onset of HDP and PE and the severity of their clinical manifestations are heterogeneous. The advent of preventive measures, such as low-dose aspirin that targets high-risk women, emphasizes the need of better prediction. Until recently, only environmental information and maternal risk factors were considered, with equivocal predictive value. No validated screening procedures were available to identify at-risk women despite the emergence of Doppler ultrasonography parameters for the uterine artery (e.g., pulsatility index and bilateral notching) and pathophysiological biochemical markers (e.g., angiogenesis, inflammation, and endothelial dysfunction). Owing to its heterogeneity and lack of specific, sensitive markers among those studied so far (>200), PE is unlikely to be detected early by a single predictive parameter. Systematic reviews have concluded that no single test fulfilling World Health Organization criteria for biomarker selection can diagnose/predict a disease. However, by combining antenatal risk factors, clinical parameters, as well as biophysical and biochemical markers into multivariate algorithms, the risk of PE can be estimated with performance levels that could reach clinical utility. Performance characteristics of selected algorithms will be presented and discussed with respect to transferability to different geographic and healthcare environments.

Preeclampsia (PE), which is defined as new onset hypertension after 20 weeks of pregnancy accompanied by proteinuria, is characterized by inadequate placentation, oxidative stress, inflammation and widespread endothelial dysfunction. A link between PE and long-term risk of cardiovascular disease (CVD) was suggested by retrospective studies, which found that PE was associated with a 2–3-fold risk of CVD later in life, with a 5–7-fold risk in the case of severe and/or early-onset PE. Recently, meta-analyses and prospective studies have confirmed the association between PE and the emergence of an unfavorable CVD risk profile, in particular a 3–5-fold increased prevalence of the metabolic syndrome only 8 years after the index pregnancy. PE and CVD share many risk factors, including obesity, hypertension, dyslipidemia, hypercoagulability, insulin resistance and both entities are characterized by endothelial dysfunction. PE and CVD are complex traits sharing common risk factors and pathophysiological processes, but the genetic link between both remains to be elucidated. However, recent evidence suggests that genetic determinants associated with the metabolic syndrome, inflammation and subsequent endothelial dysfunction are involved. As the evidence now supports that PE represents a risk factor for the emergence of the metabolic syndrome and CVD later in life, the importance of long-term follow-up assessment of CVD risk beginning early in women with a history of PE must be considered and translated into new preventive measures.

Fetal DNA is present at an approximate mean fractional concentration of 10% in the plasma of pregnant women. The detection of paternally-inherited DNA sequences that are absent in the maternal genome, e.g., Y chromosomal sequences for fetal sexing and the RHD gene for blood group genotyping, is well established. The recent emergence of single molecule counting technologies, such as digital polymerase chain reaction and massively parallel sequencing has allowed circulating fetal DNA to be used for the non-invasive prenatal diagnosis of fetal chromosomal aneuploidies and monogenic diseases. With large scale clinical validation and further reduction in costs, it is expected that the analysis of circulating fetal DNA will play an increasingly important role in the future practice of prenatal diagnosis.

Many countries are considering the expansion of their newborn bloodspot screening programmes. Whereas some countries screen for very few conditions, others are planning to screen for dozens. While advances in technology may facilitate this expansion, they must not lead it at the expense of considerations of the possible harms of this expansion. This article reviews some of the potential disbenefits of this expansion and outlines the ethical issues that should be considered.

The performance of prenatal screening tests for the identification of trisomy 21 (Down syndrome) has markedly improved since the 1970s and early 1980s when maternal age was the sole mode of screening the general pregnant population. With the discovery of second trimester serum markers in the 1980s and 1990s and implementation of double, triple, and quad marker testing; the discovery of first trimester serum and ultrasound markers in the 1990s and implementation of the combined test; and the development of the integrated test and sequential screening strategies over the past decade, the performance of screening has improved to a detection rate of 90%–95% at a false positive rate of 2%–5%. In this review, I will describe the advances in prenatal screening for trisomy 21, present current screening strategies, and discuss guidelines published by professional societies and regulatory bodies, with a focus on current prenatal screening practice in the USA.

Pre-eclampsia (PE) is a consequence of an abnormal placental invasion. Uterine artery Doppler (Ut-AD) is directly related to trophoblastic invasion of the spiral arteries, which occurs before 18 weeks’ gestation. A correct interpretation of Ut-AD indices and waveform patterns requires a rigorous and standardized methodology, in particular for the definition of notches. To date, aspirin is the only treatment associated with a decreased incidence of PE, and early identification of women at risk is crucial to optimize its use. The diagnostic performance of Ut-AD as a screening test should take into account the characteristics of the population studied. In women at high-risk of PE (i.e., women with a previous history of PE), results vary from a detection rate of 63%, with 25% false-positive results for all forms of PE, to 91% detection with 5% false-positive for early PE if repeated measurements, combined with maternal characteristics, are performed. Multicenter randomized clinical trials failed to demonstrate a benefit from administering aspirin in low-risk women with abnormal Ut-AD. In unselected populations, the use of Ut-AD, alone or integrated into algorithms including maternal characteristics, cannot be recommended for clinical practice at any gestational age. Combination with biological markers is a new field of research that could improve the performance of Ut-AD.

The preterm premature rupture of membranes (PPROM) is a common condition in pregnant women and is associated with significant maternal and perinatal morbidity. Most of the time, the diagnosis is done during physical examination. However, in 10%–20% of equivocal cases, biological markers are needed to confirm the diagnosis, especially when leakage of fluid is low or intermittent. In these cases, a quick and reliable diagnosis is necessary for applying the appropriate measures to reduce perinatal complications. The prognosis in PPROM is linked to maternal inflammatory markers that might predict perinatal infection, and therefore be helpful to decide the timing of the delivery. Nevertheless, further research is needed to identify robust biological markers for the diagnosis of PPROM in equivocal cases and for the prognosis.

Background: Age-related macular degeneration (AMD) is a multifactorial disease for which an involvement of alterations in the retinal ABC transporter gene (ABCA4) is still debated. Oxidative stress in retinal pigment epithelial cells has been postulated to contribute to the pathogenesis of the disease. Mitochondrial ferritin (FtMt), an iron-sequestering protein, is expressed in cell types characterized by high metabolic activity and oxygen consumption, including human retina, suggesting a role in protecting mitochondria from iron-dependent oxidative damage. Based on these findings we wanted to investigate whether mutations in this gene could be found in AMD patients. Methods: Mutational scanning of the FTMT gene was performed in a cohort of 50 patients affected by age-related macular degeneration. The ABCA4 gene was also scanned in one patient carrying an FtMt mutation. In silico analyses were carried out on the identified variants. The recombinant form of FtMt variant was expressed in Escherichia coli and biochemically characterized. Results: One patient was found to be heterozygous for two previously unreported genetic changes: a complex FtMt mutation (c.437_450delinsCT: delAGGACATCAAGAAGinsCT) and a missense p.Leu973Phe (c.2919G>T) mutation in exon 20 of ABCA4 . Computational analyses predicted a severe structural impairment for FtMt variant and a mild destabilizing effect for ABCA4. E. coli expression of recombinant FtMt variant yielded a highly insoluble protein that could not be renatured under in vitro conditions suitable for wild-type ferritins. Conclusions: Our findings suggest that the FtMt mutation may determine a condition similar to haploinsufficiency resulting in a reduced protection from iron-dependent oxidative stress in mitochondria.

Background: Age-related macular degeneration (AMD) is a complex disorder causing irreversible central vision loss. Complement Factor H ( CFH ) and age-related maculopathy susceptibility 2 ( ARMS2 ) are now widely accepted as important AMD susceptibility genes. In particular, two specific variants, CFH p.Y402H and ARMS2 p.A69S, have been reported as strongly AMD associated. In order to perform the genetic screening of these single nucleotide polymorphisms (SNPs), we describe a high resolution melting analysis (HRM) as a rapid closed tube mutation scanning assay. Methods: To validate HRM genotyping, 94 DNA samples from AMD patients (previously genotyped by sequence analysis) were analyzed. PCR amplification and melting curve analysis were performed in the LightCycler 480 Real-Time PCR System. In order to evaluate the accuracy of the HRM assay, we performed a blinded study of 20 unknown independent samples. Results: We correctly genotyped all samples. In fact, all samples corresponded to the previous genotype assignments. Conclusions: Early identification of individuals with genetic risk variants CFH p.Y402H and ARMS2 p.A69S is clinically important for the definition of AMD status. High-resolution DNA melting is homogenous, accurate and rapid method for CFH and ARMS2 genotyping.

Background: The aim of our study was to establish an unlabeled-probe high-resolution melting (HRM) approach to the detection of Kirsten RAS ( KRAS ) codon 12 and 13 mutations in pancreatic adenocarcinoma (PA) tissues as a novel and effective diagnostic technique. Methods: We tested the sensitivity and specificity of this genotyping approach in cell lines with known KRAS mutations using 166 bp amplicons and 37 bp wild-type probe to detect KRAS codon 12 and 13 mutations. We screened 49 PA tissues to be subsequently sequenced to confirm the mutations. Simultaneously, we tested the specimens using Sanger sequencing and then used target-DNA cloning and sequencing for verification. Results: It was found that unlabeled-probe HRM was reliable in detecting 3% of mutant cell lines DNA diluted with that of the wild-type, whereas Sanger sequencing could only discriminate 20% mutant cell ratios. In detecting 49 specimens, the former was capable of detecting 23 mutations (46.9%); and the latter could observe 15 (30.6%). For further verification, T-A DNA cloning and sequencing was applied to the differences, with the results matching those of the unlabeled-probe HRM. Conclusions: It was concluded that the unlabeled-probe HRM approach can be a sensitive and accurate screening technique to detect KRAS codon 12 and 13 mutations in diagnosing and treating PA.

Background: Add-on test requests, where a clinician requests further test assays on an existing specimen, contribute disproportionately to pathology service workload. However, little research has quantified the volume, rates, source or types of add-on tests. This study provides a descriptive analysis of add-on testing within a pathology service serving five hospitals. Methods: We analyzed 6 months of test data extracted from a pathology service in metropolitan Sydney, Australia. Add-on requests were analyzed in terms of total volume and as a proportion of all test requests and test assays; ten most frequently requested add-on test types for clinical chemistry and hematology; by patient registration category; and proportions of add-on requests received within 1-, 4-, 8-, and 24-h of specimen collection. Results: Add-on test requests constituted 3.7% (n=19,541) of the total 529,361 test requests. Clinical chemistry and hematology add-on requests accounted for 76.9% of all add-on requests. The add-on request rate was higher in the clinical chemistry (5.4%) than in hematology (1.3%). Patients who entered hospital via the emergency department had the highest rates of add-on requests. A total of 79.5% of add-on requests across the pathology service were made within 24-h of specimen collection. Conclusions: The volume of add-on requests is substantial and varies considerably by test type and patient registration category thus impacting differentially upon pathology service departments. While some add-on requests are unavoidable in clinical practice, others are precipitated by inadequate information at the point of care. Improving appropriate utilization of add-on testing will reduce their burden on pathology services.

Background: A growing body of evidence suggests that the apoptotic process is dysregulated in schizophrenia. However, only a few studies have evaluated apoptotic markers in vivo in patients or their cell cultures. Methods: Serum concentrations of Fas receptor (Fas/APO-1) and Fas ligand (FasL) were measured by ELISA techniques. The differences were tested according to the patients’ demographic, clinical and drug treatment characteristics. The clinical accuracy of the examined markers was assessed using receiver operating characteristic (ROC) curve analysis. Results: In this case-controlled study both sFas/APO-1 and FasL were significantly higher in the patients with schizophrenia than in the controls. An increase in apoptotic markers was independent of the symptomatology, drug treatment, heredity, the first onset of the disease, the duration of the psychotic disease as well as the tobacco abuse. A significant negative correlation between the duration of the disease and sFasL concentration was found. At the same time, a significant positive correlation was found between sFasL and lymphocyte caspase-3 activity. ROC curve analysis showed that sFasL was the most strongly associated with the presence of schizophrenia. Conclusions: We can conclude that the extrinsic apoptotic pathway is dysregulated in schizophrenia and sFasL may be a clinically useful disease predictor.

Background: Protein S100B is soon in clinical use as a sensitive marker after mild traumatic head injury in adults. Initial studies of S100B in pediatric head injury have shown promising results. Venous sampling can be challenging in children and capillary samples are often a preferred option. The aim of the study was to investigate the relation between capillary, venous and arterial measurements of protein S100B, primarily by determining whether capillary S100B differ from venous and if capillary S100B can predict venous S100B levels, and secondarily, if arterial S100B samples can substitute venous samples in severely brain-injured patients. Methods: Venous, arterial and capillary blood samples for S100B were collected simultaneously once a day for a maximum of 6 days. Patients were ≥18 years old and admitted to neurointensive care due to severe brain pathology. Results: Capillary S100B samples were on average 0.08 μg/L higher than venous S100B samples. Prediction of venous concentration from capillary samples yielded a prediction error of 0.07 μg/L. The mean difference between venous and arterial samples was 0.01 μg/L. The mean prediction error was 0.03 μg/L. Conclusions: Capillary and venous serum S100B are not interchangeable, and should be considered as two separate, although related, variables. Arterial measurements of S100B can successfully predict the corresponding venous concentration.

Background: This study aims to investigate the effect of increased serum adiponectin concentration during smoking cessation on soluble intercellular adhesion molecule-1 (sICAM-1) concentration. Methods: One hundred and sixty-six eligible smokers were assessed at baseline and were followed up at the 1st, 5th, and 9th weeks after smoking cessation. Demographic data, body weight and blood pressure of these participants were obtained; serum glucose biochemical data, sICAM-1 and adiponectin concentrations were measured. Repeated measures analysis paired t-tests and generalized estimating equations for balanced repeated measures were used for statistical analyses. Results: Forty-one individuals completed the 2-month smoking cessation program. The mean cigarette consumption dramatically decreased (p<0.0001) and the cotinine concentration also decreased significantly (p<0.0001) among the quitters. Serum adiponectin concentration significantly increased (p=0.0186) and sICAM-1 significantly decreased (p<0.0001) in quitters after smoking cessation. The elevation of serum adiponectin concentration significantly correlated with lowering of sICAM-1 (p=0.0001) concentration. Body weight changes at the end of 2-month smoking cessation was inversely correlated with adiponectin increment from baseline (p=0.0003). Conclusions: An increase in serum adiponectin concentration is an independent factor correlated with lowering of sICAM-1 concentration during smoking cessation.

Background: CD36 is a widely expressed cell surface receptor, that among other ligands binds lipoproteins, and its function has been implicated in many of the complications belonging to the metabolic syndrome. We have previously identified a circulating form of CD36 and established an in-house ELISA assay for measurement of CD36 in plasma. Plasma CD36 was elevated in insulin resistant obese and diabetic patients, and in patients with unstable atherosclerotic plaques. The objective of this study was to compare two new commercial CD36 ELISA assays and our in-house ELISA assay. Methods: CD36 was measured in 30 plasma samples from 10 individuals by the in-house and the two commercial assays (Cusabio Biotech and Adipobioscience). Results: Our results demonstrate that there is an absolute incongruity between the three assays. The incongruity could not be explained by different pre-analytical procedures in assay protocols. Conclusions: The lack of correlation indicates that measurement of CD36 levels in plasma is not trivial. The two commercial assays are not appropriate for CD36 detection in plasma and it seems unlikely that the established pathophysiological association with elevated plasma CD36 can be reproduced.

Background: The determination of urinary neopterin using high performance liquid chromatography (HPLC) has routinely been performed in Innsbruck since the middle of the 1980s. The aim was to compare the HPLC method with a commercially available radioimmunoassay. Methods: Neopterin to creatinine ratios were calculated in the urine of 33 healthy volunteers (33±7.3 years) collected at eight given time points. Neopterin and creatinine were simultaneously determined by HPLC and by radioimmunoassay (RIA; BRAHMS; neopterin) and an enzymatic method (Roche; creatinine), respectively. Assays were compared with Bland-Altman plots and Passing-Bablok regression analysis. Results: Neopterin to creatinine ratios ranged from 62 to 490 μmol/mol (HPLC) and from 17 to 425 μmol/mol (RIA-enzymatic combined). The ratios were on average 17.3% lower using the combined RIA-enzymatic method compared to the HPLC method. Neopterin concentrations measured by RIA were only slightly lower (on average 4.9%) than by the HPLC and the regression analysis revealed a good accordance with linearity. In contrast, creatinine concentrations by the enzymatic method were on average 12.8% higher than by HPLC. Conclusions: There is a marked bias between HPLC and the combined RIA-enzymatic method for measurement of urinary neopterin to creatinine ratios which has to be considered when methods are switched during the follow-up of patients.

Background: Tests for fecal calprotectin are usually either enzyme-linked immunosorbent assays (ELISA) or a time-resolved fluorimetric immunoassay (TRFIA). These time-consuming tests are performed only once every 1 or 2 weeks. Before the results of the tests are known most patients have already undergone colonoscopy. A rapid test, performed on outpatients, could minimize the number of necessary colonoscopies. To establish optimal cut-off values minimizing the necessity for colonoscopies, we compared two commercially available rapid tests with a quantitative TRFIA. Methods: Fecal samples were collected from 85 patients with lower gastrointestinal complaints. Calprotectin was measured using quantitative TRFIA as well as using two rapid tests: Prevent ID CalDetect and Quantum Blue calprotectin. We used the TRFIA method as the golden standard with a cut-off value of 50 μg/g. The percentage correct classification, sensitivity, specificity and positive and negative predictive value were calculated for both rapid tests at various cut-off levels. Results: Correlation between both of the rapid tests with TRFIA was significant. Quantum Blue calprotectin (κ 0.77) correlated better than Prevent ID CalDetect (κ 0.46). Optimal cut-off levels for Prevent ID CalDetect and Quantum Blue calprotectin rapid tests were 15 μg/g and 40 μg/g with a reduction in the number of necessary colonoscopies of 39% and 62%, respectively. Conclusions: The Quantum Blue calprotectin rapid test demonstrated better analytical performance than the Prevent ID CalDetect in reducing the number of colonoscopies. Furthermore, the former test has the advantage of using a point of care reader for quantitative measurement and for establishing an optimal cut-off level.

Background: The immediate impact of blood gas test results on patient care favors the use of blood gas analyzers as point-of-care-testing (POCT) devices. We performed an analytical performance evaluation of four cartridge-type blood gas analyzers for the determination of pH, partial carbon dioxide pressure (pCO 2 ), partial oxygen pressure (pO 2 ), ionized calcium (iCa 2+ ), potassium (K + ), glucose, lactate and total hemoglobin (tHb), in comparison with a traditional blood gas analyzer. Methods: The analyzers included in the study are: RP405, GEM Premier 4000, ABL90 FLEX and Cobas b 123. The ABL700 served as comparator. For each instrument the imprecision was estimated according to the CLSI EP5-A2. Based on the CLSI EP9-A2 evaluation protocol, a method comparison was performed using patient samples. Obtained data were compared against preset quality specifications, based on ABL700 performance and biological variation. Results: The precision of the RP405 and ABL90 FLEX was remarkably better than the preset criteria based on ABL700 performance. The GEM appears to have the worst precision. The RP405 yielded the best overall performance, with exception for tHb and iCa 2+ . Noteworthy is the very good performance of the glucose determination on RP405. The ABL90 FLEX showed the best performance for pH, K + and iCa 2+ measurements. For tHb determination the Cobas b123 revealed the best results. Regarding practicability, all instruments were found to be user friendly. Conclusions: Globally, all four cartridge-type blood gas analyzers produced clinically acceptable results. The analytical performance, together with the ease of use and low maintenance time of the instruments, demonstrate that these analyzers are perfectly suitable for both POCT and laboratory use.

Background: To establish stringent complete remission (SCR) in patients with multiple myeloma (MM), it is currently recommended to obtain a normal serum free light chains (sFLC) ratio. The appearance of serum oligoclonal bands (OB) after autologous stem cell transplantation (ASCT) is considered a favorable prognostic factor. The objective of this study was to examine sFLC for assessing SCR in patients with MM, and ASCT with OB. We also examined how capillary electrophoresis (CE) compares with agarose gel electrophoresis (Aga) in identifying oligoclonal bands. Methods: Out of 238 patients studied in our institution between April 1992 and December 2008 a serum protein electrophoresis (SPE) was performed by means of CE and sFLC determination on 37 patients with MM in complete remission (CR), ASCT and OB presence were assigned by conventional Aga electrophoresis and IF. Results: Statistically significant differences (SSD) were found when comparing CE vs. Aga, regarding BO visualization in SPE, favoring the latter. In connection with sFLC, the group of patients with an abnormal ratio presented elevated values in the γ-globulin zone of the SPE, whereas the group of patients with a normal ratio of sFLC presented with normal values resulting in SSD between the groups. Conclusions: It is essential to perform immunofixation to certify the presence of OB, especially if CE is used as it is difficult to distinguish them using this method. A normal sFLC was observed in most of the patients with OB and normal values of the SPE γ-globulin zone. The above-mentioned information might demonstrate a limitation of sFLC test in SCR evaluation for patients with MM, ASCT and CR if OB has been detected.

Background: A link between homocysteine (Hcy) and left ventricular ejection fraction (LVEF) emerged from recent studies but was yet not explored specifically in diabetic patients. This study aimed to assess the relationship between LVEF and Hcy in a cohort of adults with diabetes. Methods: LVEF was determined through the measure of left ventricular end-diastolic/end-systolic volumes in 409 consecutive asymptomatic patients with diabetes who underwent stress myocardial perfusion imaging. Clinical and biological parameters which were determinants of LVEF in univariate analyses with p<0.15 were included in a multivariate analysis. Results: In univariate analyses, factors significantly associated with a LVEF<55% were gender [women vs. men, odds ratio (OR)=0.22 (0.13; 0.38)], peripheral arterial disease [OR=2.49 (1.34; 4.62)], active smoking [OR=1.97 (1.16; 3.33)], silent myocardial ischemia (SMI) [OR=2.19 (1.25; 3.86)], the highest vs. the lowest tertile of creatinine [OR=2.08 (1.17; 3.68)], of albuminuria [OR=2.22 (1.27; 3.90)] and of Hcy [OR=1.83 (1.07; 3.13)]. No relationship was observed between blood pressure and decrease in LVEF. In the multivariate analysis, female gender was confirmed as being protective for having a LVEF<55%. Presence of SMI [OR=2.20 (1.14; 4.23)] and Hcy ≥15 μmol/L [OR=1.81 (1.06; 3.07)] were the two remaining significant factors associated with an increased relative risk of having LVEF<55%. A trend was only observed for the criteria active smokers. Conclusions: This study highlights an inverse relationship between Hcy and LVEF in patients with diabetes, independent of age, gender, SMI, smoking, blood pressure, renal function, folates, vitamin B12, lipid parameters and hepatic enzymes.

Background: Accumulating evidence suggests that elevated plasma homocysteine (Hcy), prevalent in hypertensive patients, affects oxidant/antioxidant balance of the body, and is linked to the development of atherosclerosis, inflammation, and endothelium injury. Our objective was to examine a hypothesis that Hcy is a predictor of total antioxidant status (TAS) and endothelial progenitor cells (EPCs), important in the repair of injured endothelium, in hypertensive patients. Methods: This study was conducted with newly diagnosed essential hypertension patients (n=42) and healthy controls (n=20). Anthropometric and biochemical characteristics, including plasma Hcy, lipids, TAS, and C-reactive protein (CRP) were quantified. Intima-media thickness (IMT) was assessed in carotid arteries. Blood derived EPCs were quantified using an in vitro culture assay. Results: Hcy, IMT, and CRP were significantly elevated while TAS and EPCs were significantly lower in hypertensive patients compared with controls. In multivariate regression analysis Hcy was a predictor of IMT of carotid artery and EPCs number. Conclusions: Our results suggest that Hcy might increase carotid artery IMT by reducing EPCs numbers. Possible involvement of Hcy in the reduction of EPCs number in hypertensive patients might be in part mediated by Hcy influence on the TAS.

Background: Matrix metalloproteinase 9 (MMP-9) and the endogenous inhibitor to MMP-9, tissue inhibitor of metalloproteinase 1 (TIMP-1), have important roles in tissue remodelling and are implicated in a number of diseases related to inflammation. The time course in activation and formation of MMPs and TIMPs during an inflammatory reaction is not fully known. This study investigates MMP-9 and TIMP-1 concentrations and MMP-9 activity at different time points after major surgery when a state of noticeable inflammation is expected. Methods: Serum MMP-9 and TIMP-1 concentrations and MMP-9 activity were analysed preoperatively and 4 and 30 days postoperatively in patients undergoing elective surgery (coronary artery bypass n=21; orthopaedic surgery, n=29). Results: Serum TIMP-1 and MMP-9 activity increased significantly 4 days after surgery (p<0.05 and p<0.01, respectively) and decreased again 30 days after surgery (p<0.01, respectively, compared to 4 days after surgery). Serum MMP-9 increased significantly 4 days after surgery (p<0.05) and was still high 30 days after surgery (p<0.01 compared to before surgery). The calculated MMP-9/TIMP-1 ratio was increased 30 days after surgery compared to before surgery (p<0.01). Conclusions: The inflammatory state induced by elective surgery is associated with increased TIMP-1 response and MMP-9 activity in serum within a few days which may be of importance for the postoperative heeling process. The further increase in MMP-9 concentrations at day 30 postoperative did not result in increased MMP-9 activity. Serum MMP-9 concentrations or the calculated MMP-9/TIMP-1 ratio do not entirely represent MMP-9 activity during surgery-induced inflammation.

Background: Circulating endothelial progenitor cells (EPCs) contribute to vascular endothelial repair. Endothelin (ET)-1 is associated with endothelial damage and atherogenesis. The experimental aim of this study was to determine, in vitro, the effects of ET-1 on the ability of EPCs to form colonies, migrate, release angiogenic growth factors and resist apoptosis. Methods: Peripheral blood samples were collected from 10 healthy adult humans. Cells with phenotypic EPC characteristics were isolated and EPC colony-forming capacity (CFU assay), migratory activity (Boyden chamber), release of angiogenic growth factors (enzyme immunoassay) and apoptosis (TUNEL assay) were determined in the absence and presence of ET-1 (100 pmol). Results: EPC colony-forming units (42±12 vs. 39±11), migratory capacity (910±146 vs. 936±148 AU) and release of vascular endothelial growth factor (202.8±68.1 vs. 204.8±69.8 pg/mL) and granulocyte-colony stimulating factor (1294.4±378.3 vs. 1136.1±310.3 pg/mL) were not significantly affected by ET-1. EPCs treated with ET-1 demonstrated a 20% increase (p<0.05) in cellular apoptosis. The proapoptotic effect of ET-1 was abolished with ET receptor blockade as well as with apocynin, a nicotinamide adenine dinucleotide phosphate (NADPH) inhibitor. Conclusions: These results indicate that ET-1 does not affect EPC colony formation, migratory capacity or angiogenic growth factor release, but does increase EPC susceptibility to apoptosis through an NADPH-dependent mechanism. Increased EPC apoptosis may contribute to the proatherogenic effects of ET-1.

Background: Impaired cardiac function leads to impaired renal function. We assessed renal function in pregnant patients with heart failure. Methods: This was a retrospective study. From 1999 to 2010, 42 pregnant patients with heart failure were classified into the single-pregnancy group and the twin or multifetation group. Clinical manifestations were assessed. Serum concentrations of creatinine and cystatin C were assessed. Estimated glomerular filtration rate (eGFR) based on serum concentrations of creatinine or cystatin C was completed. Results: There were 29 single pregnancies, 12 twin pregnancies, and one multifetation. Ten patients in the twin pregnancy or multifetation group had in-vitro fertilization. The concentration of creatinine was 84.6±33.8 μmol/L and the creatinine-based eGFR was 87.2±34.9 mL/min per 1.73 m 2 . The percentage of patients with a creatinine based eGFR<60 mL/min per 1.73 m 2 was 23.8%. The concentration of cystatin C was 1.5±0.7 mg/L and the cystatin C-based eGFR was 65.2±45.8 mL/min per 1.73 m 2 . The percentage of patients with a cystatin C-based eGFR<60 mL/min per 1.73 m 2 was 52.4%. Conclusions: Serum concentrations of cystatin C and cystatin C-based eGFR are important indicators of renal impairment in pregnant patients with heart failure.