Mortality Prediction by Kinetic Parameters of Lactate and S-Adenosylhomocysteine in a Cohort of Critically Ill Patients.

Tissue hypoxia is associated with the development of organ dysfunction and death in critically ill patients commonly captured using blood lactate. The kinetic parameters of serial lactate evaluations are superior at predicting mortality compared with single values. S-adenosylhomocysteine (SAH), which is also associated with hypoxia, was recently established as a useful predictor of septic organ dysfunction and death. We evaluated the performance of kinetic SAH parameters for mortality prediction compared with lactate parameters in a cohort of critically ill patients. For lactate and SAH, maxima and means as well as the normalized area scores were calculated for two periods: the first 24 h and the total study period of up to five days following ICU admission. Their performance in predicting in-hospital mortality were compared in 99 patients. All evaluated parameters of lactate and SAH were significantly higher in non-survivors compared with survivors. In univariate analysis, the predictive power for mortality of SAH was higher compared with lactate in all forms of application. Multivariable models containing SAH parameters demonstrated higher predictive values for mortality than models based on lactate parameters. The optimal models for mortality prediction incorporated both lactate and SAH parameters. Compared with lactate, SAH displayed stronger predictive power for mortality in static and dynamic application in critically ill patients.

S-Adenosylhomocysteine Is a Useful Metabolic Factor in the Early Prediction of Septic Disease Progression and Death in Critically Ill Patients: A Prospective Cohort Study.

A common final pathway of pathogenetic mechanisms in septic organ dysfunction and death is a lack or non-utilization of oxygen. Plasma concentrations of lactate serve as surrogates for the oxygen-deficiency-induced imbalance between energy supply and demand. As S-adenosylhomocysteine (SAH) was shown to reflect tissue hypoxia, we compared the ability of SAH versus lactate to predict the progression of inflammatory and septic disease to septic organ dysfunction and death. Using univariate and multiple logistic regression, we found that SAH but not lactate, taken upon patients' inclusion in the study close to ICU admission, significantly and independently contributed to the prediction of disease progression and death. Due to the stronger increase in SAH in relation to S-adenosylmethionine (SAM), the ratio of SAM to SAH, representing methylation potential, was significantly decreased in patients with septic organ dysfunction and non-survivors compared with SIRS/sepsis patients (2.8 (IQR 2.3-3.9) vs. 8.8 (4.9-13.8); p = 0.003) or survivors (4.9 (2.8-9.5) vs. 8.9 (5.1-14.3); p = 0.026), respectively. Thus, SAH appears to be a better contributor to the prediction of septic organ dysfunction and death than lactate in critically ill patients. As SAH is a potent inhibitor of SAM-dependent methyltransferases involved in numerous vital biochemical processes, the impairment of the SAM-to-SAH ratio in severely critically ill septic patients and non-survivors warrants further studies on the pathogenetic role of SAH in septic multiple organ failure.

Inhibition of lysyl oxidases synergizes with 5-azacytidine to restore erythropoiesis in myelodysplastic and myeloid malignancies.

Limited response rates and frequent relapses during standard of care with hypomethylating agents in myelodysplastic neoplasms (MN) require urgent improvement of this treatment indication. Here, by combining 5-azacytidine (5-AZA) with the pan-lysyl oxidase inhibitor PXS-5505, we demonstrate superior restoration of erythroid differentiation in hematopoietic stem and progenitor cells (HSPCs) of MN patients in 20/31 cases (65%) versus 9/31 cases (29%) treated with 5-AZA alone. This effect requires direct contact of HSPCs with bone marrow stroma components and is dependent on integrin signaling. We further confirm these results in vivo using a bone marrow niche-dependent MN xenograft model in female NSG mice, in which we additionally demonstrate an enforced reduction of dominant clones as well as significant attenuation of disease expansion and normalization of spleen sizes. Overall, these results lay out a strong pre-clinical rationale for efficacy of combination treatment of 5-AZA with PXS-5505 especially for anemic MN.

ISO 15189 is a sufficient instrument to guarantee high-quality manufacture of laboratory developed tests for in-house-use conform requirements of the European In-Vitro-Diagnostics Regulation.

The EU In-Vitro Diagnostic Device Regulation (IVDR) aims for transparent risk-and purpose-based validation of diagnostic devices, traceability of results to uniquely identified devices, and post-market surveillance. The IVDR regulates design, manufacture and putting into use of devices, but not medical services using these devices. In the absence of suitable commercial devices, the laboratory can resort to laboratory-developed tests (LDT) for in-house use. Documentary obligations (IVDR Art 5.5), the performance and safety specifications of ANNEX I, and development and manufacture under an ISO 15189-equivalent quality system apply. LDTs serve specific clinical needs, often for low volume niche applications, or correspond to the translational phase of new tests and treatments, often extremely relevant for patient care. As some commercial tests may disappear with the IVDR roll-out, many will require urgent LDT replacement. The workload will also depend on which modifications to commercial tests turns them into an LDT, and on how national legislators and competent authorities (CA) will handle new competences and responsibilities. We discuss appropriate interpretation of ISO 15189 to cover IVDR requirements. Selected cases illustrate LDT implementation covering medical needs with commensurate management of risk emanating from intended use and/or design of devices. Unintended collateral damage of the IVDR comprises loss of non-profitable niche applications, increases of costs and wasted resources, and migration of innovative research to more cost-efficient environments. Taking into account local specifics, the legislative framework should reduce the burden on and associated opportunity costs for the health care system, by making diligent use of existing frameworks.

Integrative Diagnostics: The Time Is Now-A Report From the International Society for Strategic Studies in Radiology.

Enormous recent progress in diagnostic testing can enable more accurate diagnosis and improved clinical outcomes. Yet these tests are increasingly challenging and frustrating; the volume and diversity of results may overwhelm the diagnostic acumen of even the most dedicated and experienced clinician. Because they are gathered and processed within the "silo" of each diagnostic discipline, diagnostic data are fragmented, and the electronic health record does little to synthesize new and existing data into usable information. Therefore, despite great promise, diagnoses may still be incorrect, delayed, or never made. Integrative diagnostics represents a vision for the future, wherein diagnostic data, together with clinical data from the electronic health record, are aggregated and contextualized by informatics tools to direct clinical action. Integrative diagnostics has the potential to identify correct therapies more quickly, modify treatment when appropriate, and terminate treatment when not effective, ultimately decreasing morbidity, improving outcomes, and avoiding unnecessary costs. Radiology, laboratory medicine, and pathology already play major roles in medical diagnostics. Our specialties can increase the value of our examinations by taking a holistic approach to their selection, interpretation, and application to the patient's care pathway. We have the means and rationale to incorporate integrative diagnostics into our specialties and guide its implementation in clinical practice.

Lyve-1 deficiency enhances the hepatic immune microenvironment entailing altered susceptibility to melanoma liver metastasis.

BACKGROUND: Hyaluronan receptor LYVE-1 is expressed by liver sinusoidal endothelial cells (LSEC), lymphatic endothelial cells and specialized macrophages. Besides binding to hyaluronan, LYVE-1 can mediate adhesion of leukocytes and cancer cells to endothelial cells. Here, we assessed the impact of LYVE-1 on physiological liver functions and metastasis. METHODS: Mice with deficiency of Lyve-1 (Lyve-1-KO) were analyzed using histology, immunofluorescence, microarray analysis, plasma proteomics and flow cytometry. Liver metastasis was studied by intrasplenic/intravenous injection of melanoma (B16F10 luc2, WT31) or colorectal carcinoma (MC38). RESULTS: Hepatic architecture, liver size, endothelial differentiation and angiocrine functions were unaltered in Lyve-1-KO. Hyaluronan plasma levels were significantly increased in Lyve-1-KO. Besides, plasma proteomics revealed increased carbonic anhydrase-2 and decreased FXIIIA. Furthermore, gene expression analysis of LSEC indicated regulation of immunological pathways. Therefore, liver metastasis of highly and weakly immunogenic tumors, i.e. melanoma and colorectal carcinoma (CRC), was analyzed. Hepatic metastasis of B16F10 luc2 and WT31 melanoma cells, but not MC38 CRC cells, was significantly reduced in Lyve-1-KO mice. In vivo retention assays with B16F10 luc2 cells were unaltered between Lyve-1-KO and control mice. However, in tumor-free Lyve-1-KO livers numbers of hepatic CD4+, CD8+ and regulatory T cells were increased. In addition, iron deposition was found in F4/80+ liver macrophages known to exert pro-inflammatory effects. CONCLUSION: Lyve-1 deficiency controlled hepatic metastasis in a tumor cell-specific manner leading to reduced growth of hepatic metastases of melanoma, but not CRC. Anti-tumorigenic effects are likely due to enhancement of the premetastatic hepatic immune microenvironment influencing early liver metastasis formation.

Definition of factors associated with negative antibody response after COVID-19 vaccination in patients with hematological diseases.

COVID-19 in patients with hematological diseases is associated with a high mortality. Moreover, preventive vaccination demonstrated reduced efficacy and the knowledge on influencing factors is limited. In this single-center study, antibody levels of the SARS-CoV-2 spike protein were measured ≥ 2 weeks after 2nd COVID-19 vaccination with a concentration ≥ 0.8 U/mL considered positive. Between July and October 2021, in a total of 373 patients (median age 64 years, 44% women) with myeloid neoplasms (n = 214, 57%), lymphoid neoplasms (n = 124, n = 33%), and other diseases (n = 35, 10%), vaccination was performed with BNT162b2 (BioNTech), mRNA-1273 (Moderna), ChADOx1 (AstraZeneca), or a combination. A total of 229 patients (61%) were on active therapy within 3 months prior vaccination and 144 patients (39%) were previously treated or treatment naïve. Vaccination-related antibody response was negative in 56/373 patients (15%): in 39/124 patients with lymphoid neoplasms, 13/214 with myeloid neoplasms, and 4/35 with other diseases. Active treatment per se was not correlated with negative response. However, rituximab and BTK inhibitor treatment were correlated significantly with a negative vaccination response, whereas younger age and chronic myeloid leukemia (CML) disease were associated with positive response. In addition, 5 of 6 patients with myeloproliferative neoplasm (MPN) and negative vaccination response were on active treatment with ruxolitinib. In conclusion, a remarkable percentage of patients with hematological diseases had no response after 2nd COVID-19 vaccination. Multivariable analysis revealed important factors associated with response to vaccination. The results may serve as a guide for better protection and surveillance in this vulnerable patient cohort.

Liquid Profiling for Cancer Patient Stratification in Precision Medicine-Current Status and Challenges for Successful Implementation in Standard Care.

Circulating tumor DNA (ctDNA), accurately described by the term liquid profiling (LP), enables real-time assessment of the tumor mutational profile as a minimally invasive test and has therefore rapidly gained traction, particular for the management of cancer patients. By LP, tumor-specific genetic alterations can be determined as part of companion diagnostics to guide selection of appropriate targeted therapeutics. Because LP facilitates longitudinal monitoring of cancer patients, it can be used to detect acquired resistant mechanisms or as a personalized biomarker for earlier detection of disease recurrence, among other applications. However, LP is not yet integrated into routine care to the extent that might be expected. This is due to the lack of harmonization and standardization of preanalytical and analytical workflows, the lack of proper quality controls, limited evidence of its clinical utility, heterogeneous study results, the uncertainty of clinicians regarding the value and appropriate indications for LP and its interpretation, and finally, the lack of reimbursement for most LP tests. In this review, the value proposition of LP for cancer patient management and treatment optimization, the current status of implementation in standard care, and the main challenges that need to be overcome are discussed in detail.

Liquid profiling of circulating tumor DNA in colorectal cancer: steps needed to achieve its full clinical value as standard care.

The analysis of circulating tumor DNA (ctDNA) is at the threshold of implementation into standard care for colorectal cancer (CRC) patients. However, data about the clinical utility of liquid profiling (LP), its acceptance by clinicians, and its integration into clinical workflows in real-world settings remain limited. Here, LP tests requested as part of routine care since 2016 were retrospectively evaluated. Results show restrained request behavior that improved moderately over time, as well as reliable diagnostic performance comparable to translational studies, with an overall agreement of 91.7%. Extremely low ctDNA levels at < 0.1% in over 20% of cases, a high frequency of concomitant driver mutations (in up to 14% of cases), and ctDNA levels reflecting the clinical course of disease were revealed. However, certain limitations hampering successful translation of ctDNA into clinical practice were uncovered, including the lack of clinically relevant ctDNA thresholds, appropriate time points of LP requests, and integrative evaluation of ctDNA, imaging, and clinical findings. In conclusion, these results highlight the potential clinical value of LP for CRC patient management and demonstrate issues that need to be addressed for successful long-term implementation in clinical workflows.

Bone marrow sinusoidal endothelium controls terminal erythroid differentiation and reticulocyte maturation.

Within the bone marrow microenvironment, endothelial cells (EC) exert important functions. Arterial EC support hematopoiesis while H-type capillaries induce bone formation. Here, we show that BM sinusoidal EC (BM-SEC) actively control erythropoiesis. Mice with stabilized ß-catenin in BM-SEC (Ctnnb1OE-SEC) generated by using a BM-SEC-restricted Cre mouse line (Stab2-iCreF3) develop fatal anemia. While activation of Wnt-signaling in BM-SEC causes an increase in erythroblast subsets (PII-PIV), mature erythroid cells (PV) are reduced indicating impairment of terminal erythroid differentiation/reticulocyte maturation. Transplantation of Ctnnb1OE-SEC hematopoietic stem cells into wildtype recipients confirms lethal anemia to be caused by cell-extrinsic, endothelial-mediated effects. Ctnnb1OE-SEC BM-SEC reveal aberrant sinusoidal differentiation with altered EC gene expression and perisinusoidal ECM deposition and angiocrine dysregulation with de novo endothelial expression of FGF23 and DKK2, elevated in anemia and involved in vascular stabilization, respectively. Our study demonstrates that BM-SEC play an important role in the bone marrow microenvironment in health and disease.

Coupling size exclusion chromatography to ultracentrifugation improves detection of exosomal proteins from human plasma by LC-MS.

OBJECTIVES: Exosomes are small lipid bilayer vesicles that are defined by their endocytic origin and size range of 30-140 nm. They are constantly produced by different cell types, by both healthy and abnormal cells, and can be isolated from almost all body fluids.Little information exists in isolating exosomes from plasma due to the complexity of its content and the presence of contaminating plasma proteins. DESIGN AND METHODS: We carried-out liquid chromatography-mass spectrometry (LC-MS/MS) analyses of plasma-derived vesicles from 4 healthy donors obtained by 2 coupled methodologies: Ultracentrifugation (UC) coupled with size-exclusion chromatography (SEC) to isolate and subsequently enrich exosomes.We compared the proteins detected by UC alone and UC coupled with SEC. RESULTS: In the coupled UC + SEC methodology we found 52.25% more proteins enriched in exosomes as CD9, Annexins, YWHAZ (14-3-3 family) and others, than by using UC alone. There is also a reduction of 98.8% of contaminating plasma proteins by coupling UC and SEC in comparison to using UC alone. CONCLUSIONS: We conclude that exosomes can be successfully isolated from plasma using a very simple combination of standard methods, which could largely improve the proteomics profiling of plasma exosomes.

Intraocular epidermal growth factor concentration, axial length, and high axial myopia.

PURPOSE: Various molecules such as dopamine have been found to be associated with axial elongation in experimental studies. Here, we examined whether intraocular EGF is associated with axial length in myopic patients. METHODS: The hospital-based investigation included patients of European descent without optic nerve, retinal, or macular diseases except for myopic maculopathy. Using aqueous humor samples collected during surgery, the EGF concentration was examined applying a cytometric bead array. High myopia was defined by an axial length of ≥ 27.0 mm. RESULTS: The study included a non-highly myopic group of 11 patients (mean age, 72.9 ± 10.8 years; mean axial length, 24.3 ± 1.1 mm) and a highly myopic group of three patients (age, 81.11 ± 12.3 years; axial length, 29.5 ± 1.3 mm), with one of them having pathologic myopic maculopathy. In multivariable linear regression analysis, higher EGF concentration was correlated with the highly myopic versus non-highly myopic group (beta, 1.24; non-standardized correlation coefficient B, 6.24; 95% confidence interval (CI), 0.10,12.4;P = 0.047) after adjusting for axial length. The amount of intraocular EGF was significantly higher in the highly myopic group than in the non-highly myopic group (89.1 ± 40.8 pg versus 34.1 ± 13.2 pg; P = 0.005), and it was highest in the eye with myopic maculopathy (135 pg). CONCLUSIONS: The intraocular amount of EGF is higher in highly myopic versus non-highly myopic eyes.

Longitudinal assessment of anti-SARS-CoV-2 antibody dynamics and clinical features following convalescence from a COVID-19 infection.

INTRODUCTION: The longevity of antibody levels against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the duration of immunity are current topics of major scientific interest. Antibody kinetics during the acute phase are well studied, whereas the long-term kinetics are yet to be determined, with contradictory results from the studies to date. Here, we present a longitudinal analysis of the serological responses to a SARS-CoV-2 infection following convalescence and the association with post-COVID syndrome (PCS). MATERIALS AND METHODS: A total of 237 serum samples were prospectively collected from 61 participants who had had a SARS-CoV-2 infection, which was confirmed using quantitative reverse-transcription polymerase chain reaction (qRT-PCR). For each participant, anti-nucleocapsid (N) and anti-spike subunit 1 receptor binding domain (RBD/S1) immunoglobulin (Ig) levels were regularly determined over a period of 8 months. COVID-19-associated symptoms were assessed using a standardized questionnaire at study entry and again after 6 months. RESULTS: Antibodies were detectable in 56 of the 61 participants. No substantial decline in anti-SARS-CoV-2 pan-Ig levels was observed for the duration of the follow-up period. Antibody levels correlated positively with the disease severity, body mass index, fever, and smoking status. It was found that 46.8% of the participants suffered from PCS, with olfactory and gustatory dysfunctions being the most commonly reported symptoms. CONCLUSION: The results demonstrate stable anti-SARS-CoV-2 antibody titers and thus may indicate a long-lasting immunity. The results are in line with recently published data and provide further insight concerning asymptomatic to mildly-affected patients, the association with clinical features, and the frequency of PCS.

Direct comparison study between droplet digital PCR and a combination of allele-specific PCR, asymmetric rapid PCR and melting curve analysis for the detection of BRAF V600E mutation in plasma from melanoma patients.

Background In metastatic melanoma, 40%-50% of patients harbor a BRAF V600E mutation and are thereby eligible to receive a combined BRAF/MEK inhibitor therapy. Compared to standard-of-care tissue-based genetic testing, analysis of circulating tumor DNA (ctDNA) from blood enables a comprehensive assessment of tumor mutational status in real-time and can be used for monitoring response to therapy. The aim of our study was to directly compare the performance of two highly sensitive methodologies, droplet digital PCR (ddPCR) and a combination of ARMS/asymmetric-rapid PCR/melting curve analysis, for the detection of BRAF V600E in plasma from melanoma patients. Methods Cell-free DNA (cfDNA) was isolated from 120 plasma samples of stage I to IV melanoma patients. Identical plasma-cfDNA samples were subjected to BRAF V600E mutational analysis using in parallel, ddPCR and the combination of ARMS/asymmetric-rapid PCR/melting curve analysis. Results BRAF V600E mutation was detected in 9/117 (7.7%) ctDNA samples by ddPCR and in 22/117 (18.8%) ctDNA samples by the combination of ARMS/asymmetric- rapid PCR/melting curve analysis. The concordance between these two methodologies was 85.5% (100/117). The comparison of plasma-ctDNA analysis using ddPCR and tissue testing revealed an overall agreement of 79.4% (27/34), while the corresponding agreement using the combination of ARMS/asymmetric-rapid PCR/melting curve analysis was 73.5% (25/34). Moreover, comparing the detection of BRAF-mutant ctDNA with the clinics, overall agreement of 87.2% (48/55) for ddPCR and 79.2% (42/53) was demonstrated. Remarkably, the duration of sample storage was negatively correlated with correctness of genotyping results highlighting the importance of pre-analytical factors. Conclusions Our direct comparison study has shown a high level of concordance between ddPCR and the combination of ARMS/asymmetric-rapid PCR/melting curve analysis for the detection of BRAF V600E mutations in plasma.

Cytokine-mediated cellular immune activation in electroconvulsive therapy: A CSF study in patients with treatment-resistant depression.

Objectives: Evidence points towards an important relationship between the antidepressant effects of electroconvulsive therapy (ECT) and the modulation of the immune system. To further elucidate this interplay, we performed a study on the effects of the antidepressant treatment by ECT on 25 cytokines in patients with depression.Methods: We measured 25 different cytokines (interleukin (IL)-1ß, IL-1RA, Il-2, IL-2R, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12 (p40/p70), IL-13, IL-15, IL-17, tumor necrosis factor-α, interferon (IFN)-α, IFN-γ, granulocyte-macrophage colony-stimulating factor, macrophage inflammatory protein (MIP)-1α, MIP-1ß, IFN-γ-induced protein 10 (IP-10), monokine induced by IFN-γ, Eotaxin, Rantes and monocyte chemoattractant protein 1) in the cerebrospinal fluid (CSF) and blood of 12 patients with a severe and treatment-resistant depressive episode before and after a course of ECT.Results: CSF levels of IP-10, IL-5 and IL-8 were elevated after ECT and more ECT sessions were associated with the differences of CSF levels before and after ECT of IFN-γ, IL-2RA, Rantes, IL-6 and IL-1ß. Responders and/or remitters had a decrease of CSF levels of IL-17, MIP-1α, Rantes and IL-2R during ECT. CSF IP-10 levels increased less during ECT in patients who had a remission.Conclusions: Although the sample size was small, we found different effects of the ECT treatment per se and of the antidepressant action induced by ECT in CSF and blood.

Circulating monocytes and tumor-associated macrophages express recombined immunoglobulins in glioblastoma patients.

BACKGROUND: Glioblastoma is the most common and malignant brain tumor in adults. Glioblastoma is usually fatal 12-15 months after diagnosis and the current possibilities in therapy are mostly only palliative. Therefore, new forms of diagnosis and therapy are urgently needed. Since tumor-associated macrophages are key players in tumor progression and survival there is large potential in investigating their immunological characteristics in glioblastoma patients. Recent evidence shows the expression of variable immunoglobulins and TCRαß in subpopulations of monocytes, in vitro polarized macrophages and macrophages in the tumor microenvironment. We set out to investigate the immunoglobulin sequences of circulating monocytes and tumor-associated macrophages from glioblastoma patients to evaluate their potential as novel diagnostic or therapeutic targets. RESULTS: We routinely find consistent expression of immunoglobulins in tumor-associated macrophages (TAM) and circulating monocytes from all glioblastoma patients analyzed in this study. However, the immunoglobulin repertoires of circulating monocytes and TAM are generally more restricted compared to B cells. Furthermore, the immunoglobulin expression in the macrophage populations negatively correlates with the tumor volume. Interestingly, the comparison of somatic mutations, V-chain usage, CDR3-length and the distribution of used heavy chain genes on the locus of chromosome 14 of the immunoglobulins from myeloid to B cells revealed virtually no differences. CONCLUSIONS: The investigation of the immunoglobulin repertoires from TAM and circulating monocytes in glioblastoma-patients revealed a negative correlation to the tumor volume, which could not be detected in the immunoglobulin repertoires of the patients' B lymphocytes. Furthermore, the immunoglobulin repertoires of monocytes were more diverse than the repertoires of the macrophages in the tumor microenvironment from the same patients suggesting a tumor-specific immune response which could be advantageous for the use as diagnostic or therapeutic target.

Therapeutic drug monitoring (TDM) of 5-fluorouracil (5-FU): new preanalytic aspects.

Background 5-Fluorouracil (5-FU) is frequently used for the treatment of gastrointestinal tumors. The pharmacological effect of 5-FU is influenced by genetic polymorphisms as well as differently dosed regimens. Currently, 5-FU is generally administered as a continuous infusion via an implanted port system using a body surface area (BSA)-based dose calculation. In order to optimize treatment, the area under the curve (AUC) can be estimated to allow for individual dose adjustment. A 5-FU AUC range between 20 and 30 [mg×h×L] is recommended. The aim of the current study was to assess if blood for AUC analysis could also be drawn at the side where the port system had been placed. Methods We collected EDTA blood samples of patients receiving infusional 5-FU simultaneously from different sampling points (right/left cubital vein). 5-FU concentrations were measured in a steady-state equilibrium based on nanoparticle immunoassay (My5-FU; Saladax). Results A total of 39 patients took part in this study. About half of the patients did not reach the target 5-FU concentration window (37% were under- and 16% of the patients were overdosed). Calculated median AUC was 23.3 for the right arm (range 5.8-59.4) and a median of 23.4 for the left arm (range 5.3-61.0). AUC values showed no difference between right compared to left arms (p=0.99). Conclusions In all, these results confirm that a high percentage of patients are not treated with 5-FU doses reaching suggested AUC levels of 20-30. The location of venepuncture, however, had no impact on the results of plasma 5-FU concentration.

Mass spectrometry based analytical quality assessment of serum and plasma specimens with patterns of endo- and exogenous peptides.

Background Inappropriate preanalytical sample handling is a major threat for any biomarker discovery approach. Blood specimens have a genuine proteolytic activity that leads to a time dependent decay of peptidic quality control markers (QCMs). The aim of this study was to identify QCMs for direct assessment of sample quality (DASQ) of serum and plasma specimens. Methods Serum and plasma specimens of healthy volunteers and tumor patients were spiked with two synthetic reporter peptides (exogenous QCMs) and aged under controlled conditions for up to 24 h. The proteolytic fragments of endogenous and exogenous QCMs were monitored for each time point by mass spectrometry (MS). The decay pattern of peptides was used for supervised classification of samples according to their respective preanalytical quality. Results The classification accuracy for fresh specimens (1 h) was 96% and 99% for serum and plasma specimens, respectively, when endo- and exogenous QCMs were used for the calculations. However, classification of older specimens was more difficult and overall classification accuracy decreased to 79%. Conclusions MALDI-TOF MS is a simple and robust method that can be used for DASQ of serum and plasma specimens in a high throughput manner. We propose DASQ as a fast and simple step that can be included in multicentric large-scale projects to ensure the homogeneity of sample quality.

Expression of combinatorial immunoglobulins in macrophages in the tumor microenvironment.

Recent evidence indicates the presence of macrophage subpopulations that express the TCRαß in chronic inflammatory diseases such as tuberculosis and atherosclerosis and in the tumor microenvironment. Here, we demonstrate that a second subpopulation of macrophages expresses rearranged heavy and light chain immunoglobulins. We identify immunoglobulin expression in human and murine monocytes, in ex vivo differentiated macrophages and macrophages from the tumor microenvironment of five randomly selected distinct human tumor entities. The immunoglobulin heavy and light chains are expressed in a small macrophage subfraction (~3-5%) as combinatorial and individual-specific immune receptors. Using Sanger sequencing and deep sequencing, we routinely find markedly restricted Ig repertoires in monocytes/macrophages compared to normal B cells. Furthermore, we report the complete Ig heavy and light chain sequences of a fully functional immunoglobulin from a single tumor-associated macrophage. These results demonstrate that Ig expression is a defining feature of monocytes and also macrophages in the tumor microenvironment and thus reveal an as yet unrecognized modus operandi of host defense in professional phagocytes.