Learning curve of achieving competency in emergency endoscopy in upper gastrointestinal bleeding: how much experience is necessary?

OBJECTIVES: The management of upper gastrointestinal bleeding (UGIB) has seen rapid advancements with revolutionising innovations. However, insufficient data exist on the necessary number of emergency endoscopies needed to achieve competency in haemostatic interventions. DESIGN: We retrospectively analysed all oesophagogastroduodenoscopies with signs of recent haemorrhage performed between 2015 and 2022 at our university hospital. A learning curve was created by plotting the number of previously performed oesophagogastroduodenoscopies with signs of recent haemorrhage against the treatment failure rate, defined as failed haemostasis, rebleeding and necessary surgical or radiological intervention. RESULTS: The study population included 787 cases with a median age of 66 years. Active bleeding was detected in 576 cases (73.2%). Treatment failure occurred in 225 (28.6%) cases. The learning curve showed a marked decline in treatment failure rates after nine oesophagogastroduodenoscopies had been performed by the respective endoscopists followed by a first plateau between 20 and 50 procedures. A second decline was observed after 51 emergency procedures followed by a second plateau. Endoscopists with experience of <10 emergency procedures had higher treatment failure rates compared with endoscopists with >51 emergency oesophagogastroduodenoscopies performed (p=0.039) or consultants (p=0.041). CONCLUSIONS: Our data suggest that a minimum number of 20 oesophagogastroduodenoscopies with signs of recent haemorrhage is necessary before endoscopists should be considered proficient to perform emergency procedures independently. Endoscopists might be considered as advanced-qualified experts in managing UGIB after a minimum of 50 haemostatic procedure performed. Implementing recommendations on minimum numbers of emergency endoscopies in education programmes of endoscopy trainees could improve their confidence and competency in managing acute UGIB.

High-dose chemotherapy and autologous haematopoietic stem-cell transplantation in older, fit patients with primary diffuse large B-cell CNS lymphoma (MARTA): a single-arm, phase 2 trial.

BACKGROUND: Available treatments for older patients with primary diffuse large B-cell CNS lymphoma (PCNSL) offer progression-free survival of up to 16 months. We aimed to investigate an intensified treatment of high-dose chemotherapy and autologous haematopoietic stem-cell transplantation (HSCT) in older patients with PCNSL. METHODS: MARTA was a prospective, single-arm, phase 2 study done at 15 research hospitals in Germany. Patients aged 65 years or older with newly diagnosed, untreated PCNSL were enrolled if they had an Eastern Cooperative Oncology Group performance status of 0-2 and were fit for high-dose chemotherapy and autologous HSCT. Induction treatment consisted of two 21-day cycles of high-dose intravenous methotrexate 3·5 g/m2 (day 1), intravenous cytarabine 2 g/m2 twice daily (days 2 and 3), and intravenous rituximab 375 mg/m2 (days 0 and 4) followed by high-dose chemotherapy with intravenous rituximab 375 mg/m2 (day -8), intravenous busulfan 3·2 mg/kg (days -7 and -6), and intravenous thiotepa 5 mg/kg (days -5 and -4) plus autologous HSCT. The primary endpoint was progression-free survival at 12 months in all patients who met eligibility criteria and started treatment. The study was registered with the German clinical trial registry, DRKS00011932, and recruitment is complete. FINDINGS: Between Nov 28, 2017, and Sept 16, 2020, 54 patients started induction treatment and 51 were included in the full analysis set. Median age was 71 years (IQR 68-75); 27 (53%) patients were female and 24 (47%) were male. At a median follow-up of 23·0 months (IQR 16·8-37·4), 23 (45%) of 51 patients progressed, relapsed, or died. 12-month progression-free survival was 58·8% (80% CI 48·9-68·2; 95% CI 44·1-70·9). During induction treatment, the most common grade 3-5 toxicities were thrombocytopenia and leukopenia (each in 52 [96%] of 54 patients). During high-dose chemotherapy and autologous HSCT, the most common grade 3-5 toxicity was leukopenia (37 [100%] of 37 patients). Treatment-related deaths were reported in three (6%) of 54 patients, all due to infectious complications. INTERPRETATION: Although the primary efficacy threshold was not met, short induction followed by high-dose chemotherapy and autologous HSCT is active in selected older patients with PCNSL and could serve as a benchmark for comparative trials. FUNDING: Else Kröner-Fresenius Foundation, Riemser Pharma, and Medical Center-University of Freiburg.

Timing of endoscopy in patients with elevated lactate levels and acute upper gastrointestinal bleeding; a retrospective comparative study.

BACKGROUND/AIMS: While current guidelines recommend performing endoscopy within 24 h in case of acute upper gastrointestinal bleeding (AUGIB), the precise timing remains an issue of debate. Lactate is an established parameter for risk stratification in a variety of medical emergencies. This study evaluated the predictive ability of elevated lactate levels in identifying patients with UGIB, who may benefit from emergent endoscopy. METHODS: We retrospectively analyzed all patients with elevated lactate levels, who presented to our emergency department between 01 January 2015 and 31 December 2019 due to suspected AUGIB. RESULTS: Of 134 included cases, 81.3% had an Charlson comorbidity index of ≥3 and 50.4% presented with shock. Fifteen (11.2%) patients died and mortality rates rose with increasing lactate levels. Emergent endoscopy within 6 h (EE) and non-EE were performed in 64 (47.8%) and 70 (52.2%) patients, respectively. Patients who underwent EE had lower systolic blood pressure (107.6 mmHg vs. 123.2 mmHg; p = 0.001) and received blood transfusions more frequently (79.7% vs 64.3%; p = 0.048), but interestingly need for endoscopic intervention (26.6% vs 20.0%; p = 0.37), rebleeding (17.2% vs. 15.7%; p = 0.82) and mortality (9.4% vs. 11.4%; p = 0.7) did not differ significantly. CONCLUSION: In conclusion, our findings support the recommendations of current guidelines to perform non-EE after sufficient resuscitation and management of comorbid illnesses.

Efficacy of endoscopic therapy and long-term outcomes of upper gastrointestinal tumor bleeding in patients with esophageal cancer.

BACKGROUND: Upper gastrointestinal bleeding (UGIB) from malignancies is associated with a poor outcome. Only a small number of studies on gastrointestinal tumor bleeding have been published so far, focusing mainly on bleeding from gastric cancer. Since the information on patients with UGIB from esophageal cancer appears insufficient, this study aimed to present clinical and endoscopic findings, treatment options as well as clinical outcomes such as rebleeding and survival of those patients. METHODS: This retrospective analysis included all patients admitted with UGIB from esophageal cancer at our university hospital during a 10-year period. RESULTS: 45 patients were analyzed of whom 26 (57.8%) already had cancer stage IV at index bleeding. 22 (48.9%) patients presented with hemodynamic instability and 30 (66.7%) patients received blood transfusions. Active bleeding was present in 24 (53.3%) patients, of whom 20 (83.3%) received endoscopic therapy. Successful hemostasis was achieved in 18 (90%) of 20 patients with Argon plasma coagulation used most frequently (52.4%). Early and delayed rebleeding occurred in 5 (12.5%) and 11 (27.5%) of all inoperable patients, respectively. Intake of anticoagulation or anti-platelet drugs were risk factors for delayed rebleeding and the median overall survival after index bleeding was 1.2 months. CONCLUSION: UGIB from esophageal cancer occurred most frequently in advanced tumor stages and was associated with significant blood loss. Even though initial endoscopic therapy was effective, rebleeding occurred in a significant number of patients. Those taking anticoagulants or anti-platelet drugs should be closely monitored for rebleeding. The overall survival after index bleeding was poor.

Evaluation of Ultrasound-based Surveillance for Hepatocellular Carcinoma in Patients at Risk: Results From a German Multicenter Retrospective Cohort Study.

Background and Aims: Hepatocellular carcinoma (HCC) surveillance in patients at risk is strongly recommended and usually performed by ultrasound (US) semiannually with or without alfa-fetoprotein (AFP) measurements. Quality parameters except for surveillance intervals have not been strictly defined. We aimed to evaluate surveillance success and risk factors for surveillance failure. Methods: Patients with ≥1 US prior to HCC diagnosis performed at four tertiary referral hospitals in Germany between 2008 and 2019 were retrospectively analyzed. Surveillance success was defined as HCC detection within Milan criteria. Results: Only 47% of 156 patients, median age 63 (interquartile range: 57-70) years, 56% male, and 96% with cirrhosis, received recommended surveillance modality and interval. Surveillance failure occurred in 29% and was significantly associated with lower median model for end-stage liver disease (MELD) score odds ratio (OR) 1.154, 95% confidence interval (CI): 1.027-1.297, p=0.025) and HCC localization within right liver lobe (OR: 6.083, 95% CI: 1.303-28.407, p=0.022), but not with AFP ≥200 µg/L. Patients with surveillance failure had significantly more intermediate/advanced tumor stages (93% vs. 6%, p<0.001), fewer curative treatment options (15% vs. 75%, p<0.001) and lower survival at 1 year (54% vs. 75%, p=0.041), 2 years (32% vs. 57%, p=0.019) and 5 years (0% vs. 16%, p=0.009). Alcoholic and non-alcoholic fatty liver disease (OR: 6.1, 95% CI: 1.7-21.3, p=0.005) and ascites (OR: 3.9, 95% CI: 1.2-12.6, p=0.021) were independently associated with severe visual limitations on US. Conclusions: US-based HCC surveillance in patients at risk frequently fails and its failure is associated with unfavorable patient-related outcomes. Lower MELD score and HCC localization within right liver lobe were significantly associated with surveillance failure.

Increased accuracy of FNA-based cytological diagnosis of pancreatic lesions by use of an ethanol-based fixative system: A STROBE compliant study.

EUS-guided fine needle aspiration cytology (FNA) is the gold standard of evaluation of solid pancreatic lesions. However, accuracy is generally low. The aim of this study was to compare the diagnostic yield of conventional cytology (CC) with liquid-based cytological analysis using an ethanol based fixative system (LBC) without onsite cytopathological assessment. We performed a retrospective evaluation in patients referred to the Department of Interdisciplinary Endoscopy at Jena University Hospital for FNA of pancreatic masses between 2008 and 2015. LBC preservation of specimen was introduced in April 2011. Gold standard was defined as a surgically obtained histology or a patient follow-up of at least 1 year for diagnosis or exclusion of malignancy. 172 patients were included into the final analysis. Mean age was 64.8 years (SD 12.4 years), 105 patients were male. 107 lesions were malignant, while 65 lesions were benign. 89 specimens were evaluated by CC, whereas 83 specimens were processed by LBC. Liquid-based cytology performed significantly better than conventional cytology in terms of sensitivity (87.8% vs 67.2% (P = .021)), specificity (100% vs 87.1% (P = .047)) negative predictive value (NPV) (85% vs 58.7% (P = .009)) and accuracy (92.8% vs 74.2% (P = .001)). We observed no learning curve after implementation of LBC Liquid based cytology is a simple and inexpensive technique that helps improving sensitivity, specificity, NPV and accuracy over conventional cytology in fine needle aspirates from patients with pancreatic lesions. Therefore, this real-world evidence shows, that EUS-FNA specimen processing should be performed using LBC to achieve best possible results.

Adenoma detection rate using narrow-band imaging is inferior to high-definition white light colonoscopy in screening and surveillance colonoscopies in daily clinical care: A randomized controlled trial.

BACKGROUND: Despite recent advances in endoscopic technology adenoma miss rate still is up to 20% contributing to interval cancers. Improved imaging modalities have been introduced to increase adenoma detection rate (ADR). Recently, narrow-band imaging (NBI) (Exera II series, Olympus Corporation) was not significantly better than high-definition white light colonoscopy (HD-WLC). An improved second generation of NBI (190-NBI) is characterized by better illumination of the bowel lumen and may be associated with a higher ADR. METHODS: We performed a prospective randomized study on patients referred to the Jena University Hospital for screening or surveillance colonoscopy between January 2015 and April 2017. Participating endoscopists were divided into 2 subgroups depending on their individual experience. Colonoscopy was performed by use of HD-WLC or 190-NBI upon withdrawal. RESULTS: Five hundred fifty-three patients participated in the study. Eighty patients were excluded (insufficient bowel cleansing [n = 34], anticoagulation precluding polypectomy [n=15], partial colonic resection [n=9], other reasons [n = 22]). Mean age was 66.9 years (standard deviation 10.3 years), and 253 patients were male (53.5%). Bowel preparation and withdrawal time were not different. ADR among all subgroups was 39.4% using HD-WLC, but only 29.1% were using 190-NBI (P = .02). Number of polyps per patient was lower using 190-NBI than with HD-WLC (0.58 vs 0.86; P = .02). Subgroup analysis revealed that 190-NBI was inferior to HD-WLC only in unexperienced endoscopists. CONCLUSION: In our stud,y ADR was lower by use of 190-NBI. These differences persisted only in unexperienced investigators. 190-NBI seems to be more challenging regarding ADR, requiring more intensive training prior to implementing this technology in daily clinical care. REGISTRATION: ClinicalTrials.gov (identifier: NCT03081975).

Perinatal Obesity Induces Hepatic Growth Restriction with Increased DNA Damage Response, Senescence, and Dysregulated Igf-1-Akt-Foxo1 Signaling in Male Offspring of Obese Mice.

Maternal obesity predisposes for hepato-metabolic disorders early in life. However, the underlying mechanisms causing early onset dysfunction of the liver and metabolism remain elusive. Since obesity is associated with subacute chronic inflammation and accelerated aging, we test the hypothesis whether maternal obesity induces aging processes in the developing liver and determines thereby hepatic growth. To this end, maternal obesity was induced with high-fat diet (HFD) in C57BL/6N mice and male offspring were studied at the end of the lactation [postnatal day 21 (P21)]. Maternal obesity induced an obese body composition with metabolic inflammation and a marked hepatic growth restriction in the male offspring at P21. Proteomic and molecular analyses revealed three interrelated mechanisms that might account for the impaired hepatic growth pattern, indicating prematurely induced aging processes: (1) Increased DNA damage response (γH2AX), (2) significant upregulation of hepatocellular senescence markers (Cdnk1a, Cdkn2a); and (3) inhibition of hepatic insulin/insulin-like growth factor (IGF)-1-AKT-p38-FoxO1 signaling with an insufficient proliferative growth response. In conclusion, our murine data demonstrate that perinatal obesity induces an obese body composition in male offspring with hepatic growth restriction through a possible premature hepatic aging that is indicated by a pathologic sequence of inflammation, DNA damage, senescence, and signs of a possibly insufficient regenerative capacity.

Optical flow analysis reveals that Kinesin-mediated advection impacts the orientation of microtubules in the Drosophila oocyte.

The orientation of microtubule (MT) networks is exploited by motors to deliver cargoes to specific intracellular destinations and is thus essential for cell polarity and function. Reconstituted in vitro systems have largely contributed to understanding the molecular framework regulating the behavior of MT filaments. In cells, however, MTs are exposed to various biomechanical forces that might impact on their orientation, but little is known about it. Oocytes, which display forceful cytoplasmic streaming, are excellent model systems to study the impact of motion forces on cytoskeletons in vivo. Here we implement variational optical flow analysis as a new approach to analyze the polarity of MTs in the Drosophila oocyte, a cell that displays distinct Kinesin-dependent streaming. After validating the method as robust for describing MT orientation from confocal movies, we find that increasing the speed of flows results in aberrant plus end growth direction. Furthermore, we find that in oocytes where Kinesin is unable to induce cytoplasmic streaming, the growth direction of MT plus ends is also altered. These findings lead us to propose that cytoplasmic streaming - and thus motion by advection - contributes to the correct orientation of MTs in vivo. Finally, we propose a possible mechanism for a specialized cytoplasmic actin network (the actin mesh) to act as a regulator of flow speeds to counteract the recruitment of Kinesin to MTs.

Ethanol-based fixation is superior to conventional brush cytology in the evaluation of indeterminate biliary strictures by endoscopic retrograde cholangiography.

The aim of this study was to compare the diagnostic yield of conventional cytology (CC) with ethanol-based fixation, a cytological analysis using an ethanol based fixative system including a cell block procedure (EBF) to evaluate indeterminate biliary strictures (IBStr). We also compared additionally taken fluorescence-guided forceps biopsies (FB) with EBF concerning a potential additive diagnostic benefit.Early detection and accurate diagnosis are crucial for patients with suspected carcinoma within the biliary tree to preserve curative treatment options but diagnostics and patient care in the evaluation of IBStr are still challenging. ERC-guided brush cytology is the gold standard of nonsurgical evaluation of IBStr. However, accuracy is generally low. New specimen processing's are needed to higher the diagnostic yield in the evaluation of IBStr.We performed a retrospective evaluation in 404 patients referred for further diagnosis of IBStr. Gold standard was defined as surgically obtained histology or patient follow-up of at least 1 year to diagnose or exclude malignancy.Three hundred thirty-four patients were included into the final analysis. One hundred seventy-two strictures were malignant, 162 strictures benign. One hundred seventeen specimens were evaluated by CC, 217 processed by EBF. EBF performed significantly better in terms of sensitivity (24.6% vs 60%, P < .001) and accuracy (59.0% vs 75.1%, P = .006). Fifty-eight FB were additionally taken and showed a numerically improved sensitivity compared to EBF alone (80% vs 62.9%, P = .19).EBF is a simple and inexpensive technique that substantially improved sensitivity and accuracy in the evaluation of IBStr. FB specimen did not significantly improve diagnostic yield.

In Vitro Evaluation of Mechanical Properties of Segmented Esophageal Self-Expandable Metal Stents: Innovative Test Methods Are Needed.

Background: Self-expanding metal stents (SEMSs) in different geometric shapes are well established treatment options in diseases of the esophagus. Mechanical properties and stent design may have an impact on patient comfort, migration rate, and removability. In this in vitro study, we evaluated mechanical properties of three segmented SEMSs (segSEMSs) for the esophagus with regard to distinct stent sections. Materials and Methods: Radial forces were measured using a testing method distinguishing between circumferential radial and local radial force. The center parts of the segSEMSs were measured for circumferential radial forces without being affected by the flared ends. Axial forces were measured at 20° bending. Results: Circumferential radial force measurements over the full stent length showed substantial differences against measurements of the center parts of the stents as the flared ends falsify test results by up to 53%. Although circumferential radial forces of the center parts were about the same (<10% variances) for all segSEMSs, local radial forces showed considerable differences of up to 26%. One segSEMS showed high axial forces, whereas the other two only needed half of the force (up to 53%) to be bent to 20°. Conclusion: Flared ends of segSEMSs have a substantial impact on radial force measurements and therefore alter test results, confirmed by our separated center part test of segSEMSs. Our innovative setup whereby we compressed the stent in an asymmetric manner (local radial force) and evaluated sections of stents separately, indeed revealed differences to circumferential measurements, leading to a more in-depth knowledge of stent characteristics.

Mathematical imaging methods for mitosis analysis in live-cell phase contrast microscopy.

In this paper we propose a workflow to detect and track mitotic cells in time-lapse microscopy image sequences. In order to avoid the requirement for cell lines expressing fluorescent markers and the associated phototoxicity, phase contrast microscopy is often preferred over fluorescence microscopy in live-cell imaging. However, common specific image characteristics complicate image processing and impede use of standard methods. Nevertheless, automated analysis is desirable due to manual analysis being subjective, biased and extremely time-consuming for large data sets. Here, we present the following workflow based on mathematical imaging methods. In the first step, mitosis detection is performed by means of the circular Hough transform. The obtained circular contour subsequently serves as an initialisation for the tracking algorithm based on variational methods. It is sub-divided into two parts: in order to determine the beginning of the whole mitosis cycle, a backwards tracking procedure is performed. After that, the cell is tracked forwards in time until the end of mitosis. As a result, the average of mitosis duration and ratios of different cell fates (cell death, no division, division into two or more daughter cells) can be measured and statistics on cell morphologies can be obtained. All of the tools are featured in the user-friendly MATLAB®Graphical User Interface MitosisAnalyser.

A stochastic model for the normal tissue complication probability (NTCP) and applicationss.

The normal tissue complication probability (NTCP) is a measure for the estimated side effects of a given radiation treatment schedule. Here we use a stochastic logistic birth-death process to define an organ-specific and patient-specific NTCP. We emphasize an asymptotic simplification which relates the NTCP to the solution of a logistic differential equation. This framework is based on simple modelling assumptions and it prepares a framework for the use of the NTCP model in clinical practice. As example, we consider side effects of prostate cancer brachytherapy such as increase in urinal frequency, urinal retention and acute rectal dysfunction.

Greenhouse gas emissions from green waste composting windrow.

The process of composting is a source of greenhouse gases (GHG) that contribute to climate change. We monitored three field-scale green waste compost windrows over a one-year period to measure the seasonal variance of the GHG fluxes. The compost pile that experienced the wettest and coolest weather had the highest average CH4 emission of 254±76gCday-1 dry weight (DW) Mg-1 and lowest average N2O emission of 152±21mgNday-1 DW Mg-1compared to the other seasonal piles. The highest N2O emissions (342±41mgNday-1 DW Mg-1) came from the pile that underwent the driest and hottest weather. The compost windrow oxygen (O2) concentration and moisture content were the most consistent factors predicting N2O and CH4 emissions from all seasonal compost piles. Compared to N2O, CH4 was a higher contributor to the overall global warming potential (GWP) expressed as CO2 equivalents (CO2 eq.). Therefore, CH4 mitigation practices, such as increasing O2 concentration in the compost windrows through moisture control, feedstock changes to increase porosity, and windrow turning, may reduce the overall GWP of composting. Based on the results of the present study, statewide total GHG emissions of green waste composting were estimated at 789,000Mg of CO2 eq., representing 2.1% of total annual GHG emissions of the California agricultural sector and 0.18% of the total state emissions.

Direct green waste land application: How to reduce its impacts on greenhouse gas and volatile organic compound emissions?

Direct land application as an alternative to green waste (GW) disposal in landfills or composting requires an understanding of its impacts on greenhouse gas (GHG) and volatile organic compound (VOC) emissions. We investigated the effects of two approaches of GW direct land application, surface application and soil incorporation, on carbon dioxide (CO2), nitrous oxide (N2O) and methane (CH4), and VOC emissions for a 12month period. Five treatments were applied in fall 2013 on fallow land under a Mediterranean climate in California: 30cm height GW on surface; 15cm height GW on surface; 15cm height GW tilled into soil; control+till; control+no till. In addition, a laboratory experiment was conducted to develop a mechanistic understanding of the influence of GW application on soil O2 consumption and GHG emission. The annual cumulative N2O, CO2 and VOC emissions ranged from 1.6 to 5.5kgN2O-Nha(-1), 5.3 to 40.6MgCO2-Cha(-1) and 0.6 to 9.9kgVOCha(-1), respectively, and were greatly reduced by GW soil incorporation compared to surface application. Application of GW quickly consumed soil O2 within one day in the lab incubation. These results indicate that to reduce GHG and VOC emissions of GW direct land application, GW incorporation into soil is recommended.

Medical Therapy of Active Ulcerative Colitis.

BACKGROUND: Medical therapy of mild and moderate ulcerative colitis (UC) of any extent is evidence-based and standardized by national and international guidelines. However, patients with steroid-refractory UC still represent a challenge. METHODS: A literature search using PubMed (search terms: ulcerative colitis, therapy, new, 1-2008-2015) resulted in 821 publications. For the current article, 88 citations were extracted including 36 randomized controlled studies, 18 reviews, and 8 meta-analyses. RESULTS: In steroid-refractory UC, early intensive therapy using anti-tumor necrosis factor (TNF) antibodies or the calcineurin inhibitors cyclosporine and tacrolimus is indicated in any case to prevent progression to a toxic megacolon and/or to avoid proctocolectomy. In patients with chronic disease activity, treatment with anti-TNF antibodies has a higher level of evidence than azathioprine therapy and should therefore be preferred. However, there is a subgroup of UC patients who may achieve prolonged steroid-free remission on azathioprine monotherapy. The importance of vedolizumab, a newly registered inhibiting antibody against integrin, has not yet been fully clarified since direct comparison studies are lacking, in particular in relation to anti-TNF antibodies. CONCLUSION: There is a great need for additional innovative therapies, especially in cases of primary non-response or secondary loss of response to anti-TNF antibodies. New small molecules (Janus kinase inhibitors) are promising with an acceptable safety profile and efficacy in UC. Further, strategies that target the intestinal microbiome are currently considered for patients with active or relapsing UC, and may in the future open up new therapeutic options.

Raman microspectroscopy: shining a new light on reproductive medicine.

BACKGROUND The last 20 years have seen an enormous upsurge in the number of publications reporting findings obtained by Raman spectroscopy, a non-invasive, non-destructive method which uses the inelastic scattering of light to provide a 'fingerprint' of the sample's chemical composition and constituents. Long neglected because of practical difficulties, the technique has been transformed by recent technological advances into a powerful analytical tool capable of opening avenues of investigation that were previously out of the reach of biomedical scientists. Beyond introducing the approach and describing its relative merits and weaknesses, the aim of this review is to provide a spur for discussion of what may become an invaluable tool for biomedical investigations. METHODS A comprehensive review of the literature was conducted searching PubMed and Ovid databases using numerous MeSH terms associated with reproductive medicine. Furthermore, the reference lists of all reported literature were explored. The searches were restricted to English language articles published in the last 50 years. RESULTS Beginning with simple characterizations of biologically and medically important substances, aided by increasing technological sophistication, the use of Raman spectroscopy in biomedicine has quickly expanded to the investigation of complex biochemical interactions, the assessment of organelles and now the evaluation of living cells and tissue. The first Raman investigations of reproductive organs were primarily oncological in nature; however, the past few years have seen an increase in the application of the technique for the assessment and evaluation of both male and female gametes. In particular, progress has been made in the characterization, identification and localization of sperm nuclear DNA damage. CONCLUSIONS The use of Raman spectroscopy has already provided many tantalizing glimpses into the potential that the technique has to answer many of the unresolved issues in investigative and therapeutic reproductive medicine. However, without stringent assessment and the clear representation of the methods' findings, their true meaning cannot be revealed nor should any conclusions be hastily derived. For the potential of Raman microspectroscopy to be truly realized, the dependability and reliability of the technique and its results can only be ascertained by multidisciplinary collaborations that undertake carefully conducted, controlled and analysed studies.

Raman microspectroscopic discrimination of TCam-2 cultures reveals the presence of two sub-populations of cells.

TCam-2 cells are the main in vitro model for investigations into seminomatous tumors. However, despite their widespread use, questions remain regarding the cells' homogeneity and consequently how representative they are of seminomas. We assess the TCam-2 cell line using routine and novel authentication methods to determine its homogeneity, identify any cellular sub-populations and resolve whether any changes could be due to generational differentiation. TCam-2, embryonal carcinoma cells (2102EP) and breast cancer cell (MCF7) lines were assessed using qRT-PCR, immunocytochemistry, flow cytometry and short tandem repeat analyses. Raman maps of individual cells (minimum of 10) and single scan spectra from 200 cells per culture were obtained. TCam-2s displayed the characteristic marker gene expression pattern for seminoma, were uniform in size and granularity and short tandem repeat analysis showed no contamination. However, based only on physical parameters, flowcytometry was unable to differentiate between TCam-2 and 2102EPs. Raman maps of TCam-2s comprised three equally distributed, distinct spectral patterns displaying large intercellular single spectral variation. All other cells showed little variation. Principal component, cluster and local spectral angle analyses indicated that the TCam-2s contained two different types of cells, one of which comprised two subgroups and was similar to some 2102EP cells. Protein expression corroborated the presence of different cells and generational differences. The detailed characterization provided by the Raman spectra, augmented by the routine methods, provide substantiation to the long-held suspicion that TCam-2 are not homogeneous but comprise differing cell populations, one of which may be embryonal carcinoma in origin.