Peripheral Vein Blood Samples Research Articles

Human Gonads Do Not Contribute to the Circulating Pool of 11-Oxygenated Androgens.

Androstenedione (A4) and testosterone (T) are produced by both the adrenal glands and the gonads. The adrenal enzyme 11β-hydroxylase (CYP11B1) executes the final step in cortisol synthesis; CYP11B1 also uses A4 and T as substrates, generating 11-hydroxyandrostenedione and 11-hydroxytestosterone, respectively. It has been suggested that CYP11B1 is expressed in the gonads, yet the circulating levels of all 11-oxygenated androgens (11-oxyandrogens) are similar in males and females of reproductive ages, despite enormous differences in T. To assess the gonadal contribution to the circulating pool of 11-oxyandrogens. We used liquid chromatography-tandem mass spectrometry to measure 13 steroids, including traditional and 11-oxyandrogens in: (I) paired gonadal and peripheral vein blood samples obtained during gonadal venograms from 11 patients (7 women), median age 37 (range 31-51 years); and (II) 17 women, median age 57 (range 41-81 years) before and after bilateral salpingo-oophorectomy (BSO). We also compared CYP11B1, 17α-hydroxylase/17,20-lyase (CYP17A1), and 3β-hydroxysteroid dehydrogenase type 2 (HSD3B2) mRNA expression in adrenal, ovarian, and testicular tissue. A4, T, estradiol, estrone, progesterone, 17α- and 16α-hydroxyprogesterone were all higher in gonadal veins vs. periphery (p < 0.05 for all), while four 11-oxyandrogens were similar between matched gonadal and peripheral vein samples. Equally, in women who underwent BSO, A4 (median [interquartile range]: 59.7 [47.7-67.6] ng/dL vs. 32.7 [27.4-47.8] ng/dL, p < 0.001) and T (24.1 [16.4-32.3] vs.15.5 [13.7-19.0] ng/dL, p < 0.001) declined, while 11-oxyandrogens remained stable. Gonadal tissue displayed negligible CYP11B1 mRNA. Despite producing substantial amounts of A4 and T, human gonads are not relevant sources of 11-oxyandrogens.

Spatial heterogeneity in epithelial to mesenchymal transition properties of circulating tumor cells associated with distant recurrence in pancreatic cancer patients.

BackgroundThe spatial heterogeneity of epithelial to mesenchymal transition (EMT)-related circulating tumor cells (CTCs) within the circulatory system and its potential clinical relevance remain unclear in pancreatic cancer (PC) patients. We aimed to map the distribution of EMT-related CTCs along the spreading pathway and investigate the prognostic significance due to the potential spatial heterogeneity in the count and phenotypic properties of CTCs.MethodsBoth portal vein (PoV) and peripheral vein (PV) blood samples were collected from 39 PC patients. CTCs were isolated by using a CD45 negative enrichment method, and EMT-related phenotypes in CTCs were analyzed by 4-channel immunofluorescence. The correlations of CTCs with patient characteristics and recurrence-free survival (RFS) were analyzed.ResultsBoth the number {median CTC total count, 10 [6–16] in PoV vs. 5 [1–7] in PV per mL, P<0.0001} and EMT status of CTCs [median mesenchymal CTC (M-CTC) percentage, 0.33 (0.13–0.52) in PoV vs. 0.2 (0–0.4) in PV, P=0.0211] showed significant spatial heterogeneity during dissemination from the PoV to the PV. Univariate analysis adjusting for patient age and sex revealed that CTC total count and M-CTC percentage in PoV samples could be risk factors for RFS in PC patients (P=0.003 and P=0.001, respectively), and ROC curve analysis found that both of these factors had good performance in distinguishing patients with early distant recurrence (within 6 months), with the optimal cut-off values of 14 cells/mL (AUROC =0.893, sensitivity =0.857, specificity =0.813, P=0.001) and 0.545 (AUROC =0.795, sensitivity =0.714, specificity =0.906, P=0.016), respectively.ConclusionsMultivascular assessment of EMT-related CTCs suggested profound dynamic alterations in total count and phenotypes during dissemination, and the spatial heterogeneity of CTCs in circulation could help establish novel prognosis markers in PC patients.

Transient Existence of Circulating Mesenchymal Stem Cells in the Deep Veins in Humans Following Long Bone Intramedullary Reaming.

The biology of mesenchymal stem cells (MSCs) in humans is incompletely understood and a possible role of systemically circulating cells in health and autoimmune disease remains controversial. Physiological movement of bone marrow MSCs to sites of injury would support the rationale for intravenous administration for relocation to damaged organs. We hypothesized that biophysical skeletal trauma rather than molecular cues may explain reported MSC circulation phenomena. Deep-femoral vein (FV) and matched peripheral vein blood samples (PVBs) were collected from patients undergoing lower-limb orthopaedic procedures during surgery (tibia using conventional sequential reaming, n = 9, femur using reamer/irrigator/aspirator (RIA), n = 15). PVBs were also taken from early (n = 15) and established (n = 12) rheumatoid arthritis (RA) patients and healthy donors (n = 12). Colony-forming unit-fibroblasts (CFU-Fs) were found in 17/36 FVBs but only 7/74 PVBs (mostly from femoral RIA); highly proliferative clonogenic cells were not generated. Only one colony was found in control/RA samples (n = 28). The rare CFU-Fs’ MSC nature was confirmed by phenotypic: CD105+/CD73+/CD90+ and CD19−/CD31−/CD33−/CD34−/CD45−/CD61−, and molecular profiles with 39/80 genes (including osteo-, chondro-, adipo-genic and immaturity markers) similar across multiple MSC tissue controls, but not dermal fibroblasts. Analysis of FVB-MSCs suggested that their likely origin was bone marrow as only two differences were observed between FVB-MSCs and IC-BM-MSCs (ACVR2A, p = 0.032 and MSX1, p = 0.003). Stromal cells with the phenotype and molecular profile of MSCs were scarcely found in the circulation, supporting the hypothesis that their very rare presence is likely linked to biophysical micro-damage caused by skeletal trauma (here orthopaedic manipulation) rather than specific molecular cues to a circulatory pool of MSCs capable of repair of remote organs or tissues. These findings support the use of organ resident cells or MSCs placed in situ to repair tissues rather than systemic administration.

Circulating tumor cells detection in tumor draining vein of breast cancer patients

Circulating tumor cells (CTCs) in tumor draining vein blood (DB) are potential sources for liquid biopsy. However, the identification of CTCs in DB of breast cancer has not been attempted. In this study, we investigated the feasibility of CTC detection in DB of breast cancer patients using a newly developed filtration-based microfluidic CTC detection device. Samples of peripheral vein blood (PB) and DB drawn from the lateral thoracic vein of the resected breast tissue were collected during the perioperative period. We investigated 41 breast cancer patients who underwent breast surgery with axillary lymph node dissection. DB was successfully collected in 36 patients (87.8%), with a mean amount of 0.85 ml. CTCs were detected in 58.3% of PB samples and 80.6% of DB samples. DB had significant higher number of CTCs compared with PB (p < 0.001). CTCs were detected in 75.0% of DB samples and 50.0% of PB samples from patients achieving pathological complete response after neoadjuvant chemotherapy. These results suggest that abundant CTCs are released into the DB of breast cancer patients, indicating that CTCs in DB would be alternative sources for liquid biopsy and potential indicators for monitoring of treatment response and prognosis in breast cancer patients.

Enumerating Circulating Tumor Cells with a Self-Assembled Cell Array (SACA) Chip: A Feasibility Study in Patients with Colorectal Cancer.

Colorectal cancer (CRC) is the second most common cause of cancer-related death worldwide. Detecting and enumerating circulating tumor cells (CTCs) in patients with colorectal cancer emerged as an important prognostic tool which provides a direct estimate of metastatic potential. Improving the turnaround time and decreasing sample volume is critical for incorporating this liquid biopsy tool into routine practice. The objective of the current study was to validate the clinical feasibility of a self-assembled cell array (SACA) chip, a CTC counting platform with less than 4 h turnaround time, in patients with newly diagnosed colorectal cancers. In total, 179 patients with newly diagnosed colorectal cancers from a single institute were enrolled. Epithelial cell adhesion molecule positive (EpCAM(+)), cluster of differentiation 45 negative (CD45(−)) cells were isolated and enumerated from 2 mL of peripheral vein blood (PB) and inferior mesenteric vein blood (IMV) samples obtained during surgery. We found that the CTC count in PB but not IMV correlates with disease stages. Neoadjuvant chemotherapy did not lead to decreased CTC count in both types of blood samples. With cutoffs of four CTCs per 2 mL of blood, and serum carcinoembryonic antigen (CEA) level of 5 ng/mL, patients with non-metastatic disease were more likely to experience recurrence if they had high PB CTC count and high serum CEA concentration (odds ratio, 8.9). Our study demonstrates the feasibility of enumerating CTCs with a SACA chip in patients with colorectal cancer.

Analysis of blood lead level of children living around lead-zinc mines

Objective: To investigate the blood lead level of children living around lead-zinc mining areas, in order to analyze its distribution features of the population. Methods: In August 2016, three natural villiages around lead-zinc mines were selected randomly as survey sites, which were grouped A, B and C according to each distances from the center field, totally 1 379 children from the three villages were selected by cluster random sampling method as the investigation subjects, and their elbow peripheral vein blood samples were collected to determine the blood lead level by graphite furnace atomic absorption spectrometry. The distribution features of blood lead in different district, gender and age groups were compared and analyzed. Results: The median and quartile range of blood lead were 0.066 and 0.058 mg/L respectively. The blood lead levels of 341 children were esceeding 100 μg/L and that account for 24.73%. The median blood lead showed statistical significant differrnce among three groups (P<0.01) , and the medians from high to low were: 0.075 mg/L (A) , 0.065 mg/L (B) and 0.059 mg/L (C) respectively. The average blood lead of male was higher than that of female (P<0.01) . There was significant difference in blood lead levels among different aged groups (P<0.01) , and the high blood lead rate dicreased with the increase of age (P<0.01) . Results of multivariate logistic regression analysis showed that there was a high risk of excessive lead in the blood lead of boys aged 1-6 years from the center of the area. Conclusion: The blood lead level of children living around lead-zinc mining areas is relatively high. There are difference in district distribution, gender and age.

The immune mechanism of intestinal tract Toll-like receptor in mediating EV71 virus type severe hand-foot-and-mouth disease and the MAPK pathway.

Immunological response is thought to play a crucial role in the development of a severe hand-foot-and-mouth disease (HFMD) infection in children, but the mechanisms remain largely unknown. This study was designed to help in elucidating the immunopathological pathways involved in the disease by quantifying Toll-like receptor (TLR) mRNAs, MAPK factors and cytokine levels in children experiencing the disease. A total of 86 enterovirus 71 (EV71)-infected HFMD children (49 with mild and 27 with severe disease), along with 30 healthy children were involved in the study. Peripheral vein blood samples were collected from each individual, and used to isolate peripheral blood mononuclear cells (PBMCs) for mRNA extraction and sera for measuring levels of tumor necrosis factor (TNF)-α, interferon (IFN)-γ, interleukin (IL)-6 and IL-10. The average expression levels of TLR3, TLR4, TLR7 and TLR8 mRNA in PBMCs of children with severe HFMD were significantly higher than those in the other children, the lowest values were found in the healthy control group (P<0.05). The expression levels of TLR2 and TLR9 mRNA were not significantly different (P>0.05) among the groups. Additionally, the expression levels of TNF-α, IFN-γ, IL-6 and IL-10 in the serum of the children in the severe group were significantly higher than those in the other two groups, and the lowest values were again found in the control group (P<0.05). Pearson correlation analysis showed that the TLR3, TLR4, TLR7 and TLR8 mRNA levels in PBMCs were positively correlated with the TNF-α, IFN-γ, IL-6 and IL-10 levels in the serum (P<0.05). Furthermore, the expression levels of the ERK, JNK and p38 mRNA in PBMCs of children in the severe group were significantly higher than those in the other two groups, with the lowest values being in the control group (P<0.05). Pearson correlation analysis showed that the TLR3, TLR4, TLR7 and TLR8 mRNA levels in PBMCs were positively correlated with ERK, JNK and p38 mRNA levels (P<0.05). The results of our study seem to indicate that the high expression levels of TLR3, TLR4, TLR7 and TLR8 induced in severe EV71 HFMD regulate the expression of cytokines by MAPK signaling pathway and negatively affect the ability of the organism to resolve the infection. Further studies are needed to test the hypothesis that immuno-modulation would be an effective treatment approach in pediatric cases of severe HFMD.

Establishment of normal reference values for thromboelastography in Chinese elderly in Beijing

Objective To determine the normal reference values for thromboelastography (TEG) in Chinese healthy elderly people in Beijing and compare them with those provided by the manufacturer and with people under the age of 55 years. Methods The healthy elderly people aged over 65 years were enrolled from March 2014 to April 2014. 4 ml of peripheral vein blood samples were taken from each person. All subjects underwent the laboratory tests of prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT) and fibrinogen (FIB). TEG parameters including reaction time (R), coagulation time (K), angle (alpha) and maximum amplitude (MA) were determined by using a Heamoscope 5000 device. Results A total of 119 healthy elderly subjects were enrolled in this study. The mean values (95% CI) of TEG parameters for them were R: 6.29 min (4.4-8.2 min), K: 1.63 min (0.8-2.5 min), α-Angle: 66.09° (55.0-77.1°), MA: 63.43mm (54.5-72.3 mm). There were significant differences in the values of R, K and α-Angle between the elderly and non-elderly groups (P<0.001 or 0.05). There were significant differences in the values of K, α-Angle and MA between males and females (P<0.05 or 0.001). According to the normal reference values provided by the manufacturer, 16.1% (19/119) of the healthy elderly volunteers had at least one abnormal parameter beyond its normal range. 3% (3/119) of the healthy elderly people were diagnosed as coagulation abnormalities, yielding a specificity of 84%. Compared with normal reference values provided by the manufacturer, the value of R was lower and the value of MA was higher in the healthy elderly people in Beijing. Conclusions The normal reference values for TEG should be established based on the local population characteristics. Key words: Thrombelastography; Reference values

Enhanced Enrichment and Purification of Blood-Derived T-Cells Using a Novel Hydrogel Technology

Abstract Introduction: The rapid growth of cell-based immunotherapy research in academic and industry laboratories has highlighted a clear need for an optimal method to isolate specific immune cell populations from heterogeneous samples with high viability, purity and recovery efficiency. This is a particular concern in the area of CAR T-cell immunotherapy research, since isolated cells must be both viable and free of any magnetic bead labels for clinical use (N Engl J Med 2014; 371:1507-17). Herein, we report a novel approach for isolating desired T-cell populations from heterogeneous samples, including peripheral and umbilical cord blood, using a biologically-friendly hydrogel technology (QuickGel™) with an interior magnetic bead carrier (MagCloudz™). MagCloudz™ have been engineered with an exterior streptavidin binding surface, which readily allows for target cell isolation using a biotinylated antibody directed against the cellular marker of interest. Methods: Target cells were isolated from human samples and analyzed for marker expression and viability as follows. CD3+ T-cells were first labeled with biotinylated antibody and then bound to the MagCloudz™ streptavidin during a short incubation step. These target CD3+ T-cells were then separated from the undesired cell populations using a magnetic stand. Any cells non-specifically adhered to the MagCloudz™ were removed during a wash step and then the CD3+ T-cells were released from the MagCloudz™ using a specially designed buffer, which dissolved the QuickGel™ and effectively de-coupled the T-cells from the magnetic bead carriers. The magnetic beads were then removed using the magnetic stand. CD3/CD45 expression on isolated T-cells were determined by flow cytometry. Viability of recovered cells was determined by 7-AAD and Annexin-V staining. Results: We demonstrate successful CD3+/CD45+ T-cell enrichment from both peripheral and umbilical cord vein blood samples. In peripheral blood, flow cytometry analysis indicated that CD3+/CD45+ T-cell populations were enriched 1.8-fold from 56% in the starting population to 98.3% CD3+/CD45+ in the isolated T-cell population, with recovery &gt;80% and viability &gt;90%. In umbilical cord blood, CD3+/CD45+ T-cells were enriched 4.8-fold from 17.5% in the starting population to 83.7% CD3+/CD45+ in the isolated T-cell population. Viability of the recovered T-cells was 96%, making them ideal for use in further functional studies (ongoing). Conclusions: These data demonstrate that the novel MagCloudz™ with QuickGel™ approach described here has the potential to drastically improve the enrichment, purity and viability of isolated of immune cell populations from whole blood. We expect that this approach will reduce the cost and time of bio-processing for immunotherapy research and provide a means to deliver magnetic label-free cells for clinical applications such as CAR T-Cell therapy (N Engl J Med 2014; 371:1507-17). Disclosures Brehm: The Jackson Laboratory: Consultancy.

Effects of low-flow sevoflurane anesthesia on renal function in low birth weight infants.

BackgroundLow-flow sevoflurane anesthesia has been shown to influence renal function in rats, but not in adult humans. Presently, no study has assessed the effects of sevoflurane on renal function in low birth weight infants. Our aim was to study the renal function in low birth weight infants undergoing surgery with low-flow sevoflurane anesthesia.MethodsForty infants graded as American Society of Anesthesiologists (ASA) grade I or II undergoing abdominal surgery were selected. After the induction of anesthesia, they received sevoflurane semi-closed inhalation anesthesia with an oxygen flow rate of 1 L/minute. According to patient vital signs, in-tidal sevoflurane concentration was maintained at 2.5%–4.0%. Peripheral vein blood samples and urine specimens were obtained before surgery (T0), at the end of surgery (T1), and 24 (T2), 48 (T3), and 72 hours (T4) after surgery. Serum creatinine (Cr), blood urea nitrogen (BUN), urinary retinol binding protein (RBP), and β-N-acetyl-glucosaminidase (NAG) levels were determined at these time points. Also, a temperature probe was inserted into the center of a soda lime canister and temperature readings were obtained.ResultsThere were no significant differences in Cr and BUN before and after surgery (P > 0.05). However, RBP and NAG levels increased after surgery (P < 0.05), but returned to preoperative levels 72 hours (T4) after surgery. The highest soda lime temperature was 37.3 ± 3.1°C.ConclusionsLow-flow sevoflurane semi-closed inhalation anesthesia has no significant effect on the renal function of low birth weight infants.

Influence of surgical resection of hepatocellular carcinoma(HCC) for hematogenous dissemination of HCC cells and its effect on recurrence and metastasis: 3 years prospective study.

Aims was to study whether hepatal surgery leads to hematogenous dissemination of hepatocellular carcinoma (HCC) cells and determine period of its persistence by nested reverse transcription-polymerase chain reaction (RT-PCR). Peripheral vein blood (5ml) samples were obtained from 54 HCC patients of T1N0M0 and 6 HCC patients of T2N0M0 stage and 20 patients with liver cavernous hemangioma(LCH) before operation and 48h,72h and 1 week after surgical resection of hepatoma between Janurary 1st, 2007 and December 31th, 2010. We detected alpha-fetoprotein (AFP) messenger RNA(mRNA) by nested RT-PCR perioperatively.FPmRNA of all 60 cases of HCC patients and 20 cases of LCH were negative before hepatal surgery, 28 of 60 HCC cases became positve at 48h after surgery (46.7%), 16 of 60 HCC cases remained positive at 72h postoperatively(26.7%), none of 60 HCC patients AFPmRNA was detected at 1 week after hepatal surgery(0.0%). None of LCH patients were detected AFPmRNA after hepatal surgery(0.0%). For 28 HCC patients with AFPmRNA positive after hepatal surgery, 4 HCC patients developed intrahepatic tumor recurrences in 1st year (4/28, 14.3%),6 HCC patients relapsed in the second year(6/28,21.4%),10 HCC patients relapsed in the third year(10/28,35.7%). For 32 HCC patients with AFPmRNA negative group postoperation, 5 cases relapsed in 1st year (5/32, 15.6%),7 cases developed intrahepatic tumor recurrences in the second year (7/32, 21.9%),11 cases relasped in the third year (11/32, 34.4%,P>0.05),none of HCC patients occured distal metastasis after surgical resection of hepatoma. None of LCH patients relapsed postoperation within three years.Hepatal surgery may cause HCC cells spreading into peripheral blood shortly, but it may be not concerning with recurrence or metastasis of HCC.

Diagnostic accuracy of arterial line blood gas measurements as an estimate of arteriovenous fistula recirculation

We studied the diagnostic accuracy of blood gas determination as a novel method for the estimation of arteriovenous fistula (AVF) recirculation (RC). In 25 patients on chronic haemodialysis, with failure of a previously well functioning native AVF (mean two-needle urea-based RC: 41 ± 10%), arterial line (AL) as well as a peripheral vein (PV) blood samples drawn by the end of a 4 h haemodialysis session, before and after the surgical repair of their AVF. Compared to PV samples, patients with RC had significantly higher AL blood pCO2 and pO2 values (P < 0.001) and lower AL blood pH and K(+) values (P < 0.001), findings that were reversed after the surgical restoration of adequate AVF function. On regression analysis, urea RC values were correlated positively with AL pCO2 values (r = 0.683, P < 0.001) and negatively with AL pH values (r = 0.896, P < 0.001). AL pCO2 > 40 mmHg was shown to have the best sensitivity and AL pH < 7.25 the best specificity. RC index, that is, the AL pCO2 /pH ratio, was found to have superior test characteristics compared to pH and pCO2 (sensitivity 95% and specificity 88% for values >5.5) making it a powerful diagnostic as well as screening tool. We propose the regular AL blood gas measurement as a novel method of AVF function surveillance and RC diagnosis. AL blood pH < 7.25, pCO2 > 40 mmHg and RC index > 5.5, escorted by rather high pO2 and low K(+) by the end of dialysis session, but probably earlier as well, signify an important RC (>20%) and warrant further investigation of AVF patency.

703 CIRCULATING TUMOR CELLS CAN BE DETECTED IN PATIENTS WITH TESTICULAR GERM CELL TUMORS

You have accessJournal of UrologyPenis/Testis/Urethra: Benign & Malignant Disaese (I)1 Apr 2013703 CIRCULATING TUMOR CELLS CAN BE DETECTED IN PATIENTS WITH TESTICULAR GERM CELL TUMORS Christian Ruf, Paulina Nastaly, Pascal Becker, Hendrik Isbarn, Friedemann Honecker, Klaus Pantel, Sabine Riethdorf, Dirk Hoeppner, Margit Fisch, Walter Wagner, and Sascha Ahyai Christian RufChristian Ruf Hamburg, Germany More articles by this author , Paulina NastalyPaulina Nastaly Hamburg, Germany More articles by this author , Pascal BeckerPascal Becker Hamburg, Germany More articles by this author , Hendrik IsbarnHendrik Isbarn Hamburg, Germany More articles by this author , Friedemann HoneckerFriedemann Honecker Hamburg, Germany More articles by this author , Klaus PantelKlaus Pantel Hamburg, Germany More articles by this author , Sabine RiethdorfSabine Riethdorf Hamburg, Germany More articles by this author , Dirk HoeppnerDirk Hoeppner Berlin, Germany More articles by this author , Margit FischMargit Fisch Hamburg, Germany More articles by this author , Walter WagnerWalter Wagner Hamburg, Germany More articles by this author , and Sascha AhyaiSascha Ahyai Hamburg, Germany More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2013.02.261AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES The available serum tumor markers (AFP, β-HCG, LDH) are increased in only approximately 60% of testicular germ cell tumors (TGCT) patients. Therefore, additional markers would facilitate clinical diagnosis, staging and treatment of these patients. Detection of circulating tumor cells (CTC) has been associated with poor prognosis in other malignancies (e.g. Prostate-, Colon- and Breastcancer), however, little is known about the presence of CTC in blood of TGCT patients. METHODS In order to select the most appropriate markers for CTCs detection, the expression of different epithelial and germ cell-specific markers was examined in 4 different TGCT cell lines (TCam-2/2102Ep/NCCIT/NT2) as well as 12 histologically different primary testicular tumors. Peripheral and testicular vein blood samples from 73 and 12 patients, respectively, were collected and examined for CTCs. All samples were enriched for mononuclear cells using Ficoll density gradient centrifugation and CTCs were detected by alkaline phosphatase (AP) enzymatic activity and immunocytochemistry using anti-keratin, anti-EpCAM and anti-SALL4-antibodies. Additionally, peripheral blood samples were tested for the presence of CTCs by the automated CellSearch® system. Patients with ≥1 CTC/sample were classified as CTC-positive. To compare results for testicular and peripheral blood, CTC yields were calculated as number of CTC per 1 ml of blood. RESULTS Based on the previous analyses, double immunofluorescent staining for SALL4 and keratin, as well as analysis of AP activity were performed to detect CTCs. According to both detection systems û the one based on selected markers and automated CellSearch®, 7 (58.3%) testicular vein and 13 (17.8%) peripheral blood samples showed presence of at least one CTC. The CTC number ranged from 5 to 108/ml and 0.13 to 2.18/ml of testicular vein and peripheral blood, respectively. CTC were detected in seminoma and non-seminoma, in clinically metastasized and non-metastasized stages. CONCLUSIONS This is the first study to demonstrate CTC detection in TGCT patients. The proposed detection systems seem to be specific and sensitive for the identification of CTC. An association between CTC and histological subtype as well as metastatic stage is under investigation and will be presented at the meeting. © 2013 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 189Issue 4SApril 2013Page: e289 Peer Review Report Advertisement Copyright & Permissions© 2013 by American Urological Association Education and Research, Inc.MetricsAuthor Information Christian Ruf Hamburg, Germany More articles by this author Paulina Nastaly Hamburg, Germany More articles by this author Pascal Becker Hamburg, Germany More articles by this author Hendrik Isbarn Hamburg, Germany More articles by this author Friedemann Honecker Hamburg, Germany More articles by this author Klaus Pantel Hamburg, Germany More articles by this author Sabine Riethdorf Hamburg, Germany More articles by this author Dirk Hoeppner Berlin, Germany More articles by this author Margit Fisch Hamburg, Germany More articles by this author Walter Wagner Hamburg, Germany More articles by this author Sascha Ahyai Hamburg, Germany More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Mutations in the D-loop region and increased copy number of mitochondrial DNA in human laryngeal squamous cell carcinoma

The effects of D-loop mutations and the mtDNA copy number alterations in LSCC are poorly understood. Herein, we investigated the features and roles of somatic mutations of the D-loop region and copy number alterations in mtDNA of LSCC. Using direct sequencing and real-time quantitative PCR, we examined D-loop mutations and mtDNA copy number in LSCC tissues, paracancerous normal tissues and peripheral vein blood samples from 40 LSCC patients. A student's t test, ANOVA test and χ(2) test were used to analyze association among mutations, mtDNA copy number alterations with clinicopathologic parameters. The results revealed that 21 tumors (52.5 %) had somatic mtDNA D-loop mutations with a total of 34 mutations. Among them, 28 (82.4 %) and 6 (17.6 %) were located in HVII and HVI, respectively. D-loop mutations correlated with tumor differentiation and p53 mutation (P < 0.05), and increased mtDNA copy number. In addition, mtDNA copy number in tumor tissues and paracancerous normal tissues were all significantly higher than in peripheral blood (P < 0.05). The copy number of mtDNA in the cases which carried D-loop mutation was significantly higher than that of the negative cases (P < 0.05). These results suggest that the mtDNA D-loop in LSCC is an unstable region with a high frequency of somatic mutation and polymorphisms. Together with the increase in mtDNA copy number, these factors may play a role in carcinogenesis of the larynx.

Increased intracardiac vascular endothelial growth factor levels in patients with paroxysmal, but not persistent atrial fibrillation

Although inflammation appears to play a pivotal role in the pathophysiology of atrial fibrillation (AF), the source of inflammation is unknown. We hypothesized that multilevel measurement of several inflammatory proteins in AF patients would help assess the extent and the source of inflammation. Thirty-nine patients with paroxysmal AF, 33 with persistent AF, and 9 control patients with Wolff-Parkinson-White syndrome were enrolled. Peripheral, left atrial, coronary sinus, and pulmonary vein blood samples were obtained during catheterization. Serum levels of vascular endothelial growth factor (VEGF), interleukin-8 (IL-8), soluble intercellular adhesion molecule 1 (sICAM-1), and transforming growth factor-β1 (TGF-β1) were measured at the four sampled sites. Interleukin-8, sICAM-1, and TGF-β1 levels did not differ among groups at any of the sampled sites. Peripheral VEGF levels were higher in both paroxysmal and persistent AF patients than in controls (P ≤ 0.03). Left atrial VEGF levels were higher in paroxysmal AF (P = 0.05), but not in persistent AF (P = 0.32), compared with controls. Coronary sinus and pulmonary vein VEGF levels did not differ significantly among groups. Low levels of several inflammatory markers in both paroxysmal and persistent AF patients suggest that the inflammatory process is of low grade, if present. In the context of normal pulmonary vein VEGF levels, the heart itself is the most likely source of high left atrial VEGF levels in paroxysmal AF patients; however, this disorder appears to be a transient event in the natural history of AF.

Peripheral levels of circulating progenitor cells correlate with area at risk and microvascular obstruction at cardiac magnetic resonance imaging in patients with ST-elevation myocardial infarction

Background Circulating progenitor cells (CPC) are bone marrowderived elements with reparative properties known to be mobilised in the context of acute coronary syndromes. Controversy persists over the determinants of their release in the acute phase of ST-elevation myocardial infarction (STEMI). We used cardiovascular magnetic resonance (CMR) to investigate this issue. Methods Fifteen STEMI patients undergoing successful primary percutaneous coronary intervention (pPCI) were included. Peripheral vein blood samples were drawn at 48 hours after pPCI to measure CPC (CD34+/ KDR +/CD45-). At 2-7 days after pPCI all patients underwent CMR assessment with SSFP, T2-STIR, first-pass perfusion and late gadolinium-enhancement imaging. Quantitative T2 area-at-risk (AAR, % of LV volume), infarct size (IS, % of LV volume), myocardial salvage (MS, calculated as AAR-IS) were calculated. Microvascular obstruction (MVO) was assessed both as perfusion defect score on first-pass imaging (PD, expressed as a sum of a 0 to 3 scoring for each of 16 LV segments) or as MVO score in the late enhancement images. Results CPC levels (expressed as %, number of cells per total number of cytometric events) were more strongly related to extent of AAR (rho=0.72, p=0.002) than to IS (rho=0.5, p=0.06), but did not correlate to MS (rho=0.002, p=0.9). Moreover, CPC were related to PD score (rho=0.54, p=0.03) but not to MVO score (rho=0.3, p=0.2). Conclusions Our data suggest that CPC are mobilized in STEMI patients in response to the total myocardial ischemic insult, as represented by the area-at-risk on T2-STIR. Furthermore, CPC positive correlation with PD suggests that they could be mobilized in attempt to overcome ongoing microvascular dysfunction.

Iron deficiency anemia and iron stores in adult and adolescent women in pregnancy

To determine whether primiparous adolescent pregnancy is associated with increased risk of iron deficiency anemia and iron deficiency when compared with older primiparas. Prospective cohort. Caratinga city, Minas Gerais State, Brazil. A total of 183 women, being 61 adolescents and 122 adults. Five peripheral vein blood samples were collected from each participant, at 9-16 and 29-36 weeks, upon admission for delivery, within the first hour after delivery and 30-60 days post-partum. We measured hemoglobin (Hb), transferrin saturation index and ferritin. Adolescent and adult women using chi-squared test, Student's t-test and hotelling test. There were significant differences in Hb concentration during the various sampling periods, for the whole cohort. The prevalence of anemia (Hb < 11 g/dl) did not differ significantly between adults and adolescents. The decrease in the mean transferrin levels was more intense in adolescents than in adults, especially during the second half of pregnancy. Low ferritin levels (<12 microg/l) were more frequent in adolescents than in adults during pregnancy and throughout the study period. Although there were no significant differences in the prevalence of iron deficiency anemia (Hb < 11 g/dl) in adolescents compared to adult women, low body iron stores and ferritin (< 12 microg/l) were significantly more frequent in adolescents.

Muscarinic cholinergic receptor antagonists enhanced the expression of CD(8)(+)CD(25)(+)Foxp(3)(+) regulatory T cells in stable COPD patients

to investigate the levels of peripheral CD(8)(+)CD(25)(+)Foxp(3)(+) regulatory T cells (CD(8)(+)CD(25)(+)Treg) in stable chronic obstructive pulmonary disease (COPD) patients, and the effect of muscarinic cholinergic receptor antagonist, tiotropium bromide, on the expression of CD(8)(+)CD(25)(+)Treg. from Oct. 2007 to Mar. 2008, 23 patients with stable moderate to severe COPD received tiotropium bromide inhalation (18 microg daily) for 3 months. Before and after the use of tiotropium bromide, lung function was performed and peripheral vein blood samples were collected from the patient. Lymphocytes were isolated by three-color labeled monoclonal antibodies flow cytometry to detect the quantity and percentage of CD(8)(+)T cell, CD(8)(+)CD(25)(+) T cell, CD(8)(+)CD(25)(+)Treg, CD(4)(+)T cell, CD(4)(+)CD(25)(+) T cell and CD(4)(+)CD(25)(+)Treg, respectively. Paired t test was used for comparison between data before and after treatment. in patients with stable COPD, after tiotropium bromide treatment, the percentage of CD(4)(+) T cells was increased from (28 +/- 10)% to (36 +/- 6)%, and the difference was significant (t = 3.20, P < 0.01). CD(4)(+)CD(25)(+) was decreased from (10 +/- 7)% to (4 +/- 3)% (t = 3.78, P < 0.01), and CD(8)(+)CD(25)(+)Treg was increased from (8 +/- 8)% to (21 +/- 21)% (t = 2.72, P < 0.05). At baseline, CD(8)(+) cells, CD(8)(+)CD(25)(+) cells and CD(4)(+)CD(25)(+)Treg were detectable in the peripheral blood, but no significant changes were observed after treatment. After treatment with tiotropium bromide, the lung function was markedly improved; FEV(1), FEV(1)/Pre%, and FEV(1)/FVC were increased from (1.0 +/- 0.3) L, (35 +/- 10)% and (41 +/- 8)% to (1.1 +/- 0.3) L, (40 +/- 11)% and (45 +/- 11)%, respectively (t = 2.65, 2.56 and 2.37, respectively, all P < 0.05). By linear correlation analysis, the quantity of changes of CD(4)(+) T cells and CD(4)(+)CD(25)(+) T cells were negatively correlated with the rate of change in CD(8)(+)CD(25)(+)Treg (r = -0.61, P < 0.05 and r = -0.72, P < 0.01, respectively). in the peripheral blood of patients with stable COPD, there was expression of CD(8)(+)CD(25)(+)Treg and CD(4)(+)CD(25)(+)Treg. The muscarinic receptor antagonist tiotropium bromide, promoted the amplification of CD(4)(+), inhibited the expression of CD(25)(+), and enhanced the expression of CD(8)(+)Treg, suggesting that muscarinic receptor antagonist tiotropium bromide may possess immunomodulation function.

Circulating Endothelial Cells

Congenital heart disease can be complicated by pulmonary arterial hypertension (PAH), the reversibility of which is often difficult to predict. We recently reported a lung biopsy study showing impaired apoptotic regulation of endothelial cells in irreversible PAH. The objective of the present study was to identify noninvasive biomarkers of endothelial turnover that could be used to identify congenital heart disease patients at risk of irreversible PAH. Circulating endothelial cells (CECs) isolated with CD146-coated beads and circulating CD34(+)CD133(+) progenitor cells (CPCs) were quantified in peripheral vein, pulmonary artery, and pulmonary vein blood samples from 26 patients with congenital heart disease (16 with reversible PAH [median age 2 years] and 10 with irreversible PAH [median age 9 years]) and 5 control patients. Surgical lung biopsy was performed in 19 cases. As expected, endothelial remodeling was observed in irreversible PAH but not in reversible PAH. CEC and CPC numbers were each similar in the 3 types of blood samples. CEC numbers were significantly higher in patients with irreversible PAH (median 57 CEC/mL) than in patients with reversible PAH and control subjects (median 3 CEC/mL in the 2 groups). In contrast, CPC numbers did not differ among patients with irreversible or reversible PAH and control subjects (median 84, 64, and 44 CPC/10(5) lymphocytes, respectively, in the 3 groups). Irreversible PAH in congenital heart disease is associated with endothelial damage and with increased circulating endothelial cell counts. The present study suggests that CECs could be a valuable tool to define therapeutic strategies in congenital heart disease patients with PAH.

Differential Time to Positivity and Quantitative Cultures for Noninvasive Diagnosis of Catheter-Related Blood Stream Infection in Children

Accurate diagnosis of catheter-related blood stream infection (CRBSI) is necessary to make a decision about removal of the catheter. Differential time to positivity (DTP) and the ratio of quantitative cultures (RQC) between central and peripheral blood cultures have not been evaluated against a strict standard in children, namely catheter tip culture. Our aim is to compare DTP and RQC in the diagnosis of catheter tip-confirmed catheter-related infection in children. Prospective study performed in 2 large hospitals in Santiago, Chile. Children with clinically suspected CRBSI had 2 peripheral and central vein blood samples obtained for automated culture in Bact/Alert and for quantitative cultures in 5% sheep blood agar plate. The catheter tip was cultured. Sensitivity, specificity, positive predictive value, negative predictive value, likelihood ratios (LR), and accuracy of DTP and RQC were compared against catheter tip-confirmed CRBSI. During a 3-year period, 344 clinically suspected CRBSIs were diagnosed in children of which 124 episodes met study criteria. Catheter tip culture-confirmed CRBSI in 25 (20%) of 124 episodes. A total of 34 microorganisms were cultured from 25 CRBSI; 8 of 25 (32%) episodes were polymicrobial. Staphylococcus aureus followed by coagulase-negative Staphylococcus were the most common microorganisms. For CRBSI, DTP and RQC reached a sensitivity of 75% versus 24% (P < 0.001), specificity of 86 versus 94%, positive predictive value of 58% versus 50%, negative predictive value of 93% versus 82%, LR of 5.48 versus 4.50, and accuracy of 0.84 versus 0.79. In children, DTP was better than RQC for diagnosis of catheter tip-confirmed CRBSI.