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.

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.

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.

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.

Assessing the Quality of Serological Testing in the COVID-19 Pandemic: Results of a European External Quality Assessment (EQA) Scheme for Anti-SARS-CoV-2 Antibody Detection.

External quality assessment (EQA) is a key instrument for achieving harmonization, and thus a high quality, of diagnostic procedures. As reliable test results are crucial for accurate assessment of SARS-CoV-2 infection prevalence, vaccine response, and immunity, and thus for successful management of the ongoing COVID-19 pandemic, the Reference Institute for Bioanalytics (RfB) was the first EQA provider to offer an open scheme for anti-SARS-CoV-2 antibody detection. The main objectives of this EQA were (i) to gain insights into the current diagnostic landscape and the performance of serological tests in Europe and (ii) to provide recommendations for diagnostic improvements. Within the EQA, a blinded panel of precharacterized human serum samples with variable anti-SARS-CoV-2 antibody titers was provided for detection of anti-SARS-CoV-2 IgG, IgA, and IgM antibodies. Across the three distribution rounds in 2020, 284 laboratories from 22 countries reported a total of 3,744 results for anti-SARS-CoV-2 antibody detection using more than 24 different assays for IgG. Overall, 97/3,004 results were false for anti-SARS-CoV-2 IgG, 88/248 for IgA, and 34/124 for IgM. Regarding diagnostic sensitivity and specificity, substantial differences were found between the different assays used, as well as between certified and noncertified tests. For cutoff samples, a drop in the diagnostic sensitivity to 46.3% and high interlaboratory variability were observed. In general, this EQA highlights the current variability of anti-SARS-CoV-2 antibody detection, technical limitations with respect to cutoff samples, and the lack of harmonization of testing procedures. Recommendations are provided to help laboratories and manufacturers further improve the quality of anti-SARS-CoV-2 serological diagnostics.

Implementation of the new EU IVD regulation - urgent initiatives are needed to avert impending crisis.

Laboratory medicine in the European Union is at the dawn of a regulatory revolution as it reaches the end of the transition from IVDD 98/79/EC (https://eur-lex.eur-opa.eu/legal-content/EN/TXT/?uri=CELEX%3A31998L0079&qid=1628781352814) to IVDR 2017/746 https://eur-lex.europa.eu/eli/reg/2017/746. Without amendments and contingency plans, implementation of the IVDR in May 2022 will lead the healthcare sector into uncharted waters due to unpreparedness of the EU regulatory infrastructure. Prospective risk analyses were not made by the European Commission, and if nothing happens it can be anticipated that the consequences will impact all stakeholders of the medical test pipeline, may seriously harm patients and may prevent caregivers from making appropriate clinical decisions due to non-availability of medical tests. Finally, it also may discourage manufacturers and academia from developing specialty tests, thereby hampering innovation in medical diagnostic care. We hereby inform laboratory professionals about the imminent diagnostic collapse using testimonies from representative stakeholders of the diagnostic supply chain and from academia developing innovative in-house tests in domains of unmet clinical needs. Steps taken by the EFLM Task Force on European Regulatory Affairs, under the umbrella of the Biomedical Alliance in Europe, will be highlighted, as well as the search for solutions through dialogue with the European Commission. Although we recognize that the IVDR promotes positive goals such as increased clinical evidence, surveillance, and transparency, we need to ensure that the capabilities of the diagnostic sector are not damaged by infrastructural unpreparedness, while at the same time being forced to submit to a growing bureaucratic and unsupportive structure that will not support its "droit d'exister".

SARS-CoV-2 Antibody Testing - How to Increase the Positive Predictive Value.

BACKGROUND: Despite increasing COVID-19 infection rates, low overall prevalence resulting in a poor positive predictive value (PPV) of serological tests requires strategies to increase specificity. We therefore investigated a dual diagnostic strategy and evaluated the correlation between the severity of a SARS-CoV-2 infection and the detectable immune-response. METHODS: Participants were systematically categorized into positive and control cohorts and a probability score of COVID-19 was calculated based on clinical symptoms. Six hundred eighty-two serum samples were analyzed using a highly specific high-throughput system. Combining the serological test result and probability score was performed as a dual diagnostic strategy. RESULTS: Specificity of 99.61% and sensitivity of 86.0% were the basis of our approach. A dual diagnostic strategy led to increased pre-test probability and thus to a test specificity of 100%. In a flu-like symptomatic population, we estimated a COVID-prevalence of 4.79%. Moreover, we detected significantly higher antibody values in patients with fever than without fever. CONCLUSIONS: Based on sensitivity and specificity results of our study being in line with previous findings, we demonstrated a dual assessment strategy including a symptom-based probability score and serological testing to increase the PPV. Moreover, the presence of fever seems to trigger a stronger immune-response.

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.

SARS-CoV-2 antibody testing-questions to be asked.

Severe acute respiratory syndrome coronavirus 2 infection and development of coronavirus disease 2019 presents a major health care challenge of global dimensions. Laboratory diagnostics of infected patients, and the assessment of immunity against severe acute respiratory syndrome coronavirus 2, presents a major cornerstone in handling the pandemic. Currently, there is an increase in demand for antibody testing and a large number of tests are already marketed or are in the late stage of development. However, the interpretation of test results depends on many variables and factors, including sensitivity, specificity, potential cross-reactivity and cross-protectivity, the diagnostic value of antibodies of different isotypes, and the use of antibody testing in identification of acutely ill patients or in epidemiological settings. In this article, the recently established COVID-19 Task Force of the German Society for Clinical Chemistry and Laboratory Medicine (DGKL) addresses these issues on the basis of currently available data sets in this rapidly moving field.

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.

Results of the first pilot external quality assessment (EQA) scheme for anti-SARS-CoV2-antibody testing.

Objectives Assessment of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection prevalence and immunity is cornerstones in the fight against COVID-19 pandemic. For pandemic control, reliable assays for the detection of anti-SARS-CoV-2 antibodies are required. This pilot external quality assessment (EQA) scheme aimed to independently assess the participants' clinical performance of anti-SARS-CoV-2 testing, to identify shortcomings in clinical practice and to evaluate the suitability of the scheme format. Methods The EQA scheme consisted of eight serum samples with variable reactivity against SARS-CoV-2 intended for the analysis of anti-SARS-CoV-2 immunoglobulin (Ig)G, IgA, and IgM antibodies. Laboratories reported: (1) results for each sample and the respective method, (2) raw data from replicate testing of each sample. Results The 16 selected pilot EQA participants reported 294 interpreted results and 796 raw data results from replicate testing. The overall error rate for the anti-SARS-CoV-2 IgG, IgA, and IgM tests was 2.7, 6.9, and 16.7%, respectively. While the overall diagnostic specificity was rated as very high, sensitivity rates between 67 and 98% indicate considerable quality differences between the manufacturers, especially for IgA and IgM. Conclusions Even the results reported by the small number of participants indicate a very heterogeneous landscape of anti-SARS-CoV-2 serological testing. Differences of available tests and the individual performance of laboratories result in a success rate of 57.1% with one laboratory succeeding for all three antibody-classes. These results are an incentive for laboratories to participate in upcoming open EQA schemes that are needed to achieve a harmonization of test results and to improve serological testing.

The novel seizure quality index for the antidepressant outcome prediction in electroconvulsive therapy: association with biomarkers in the cerebrospinal fluid.

For patients with depression treated with electroconvulsive therapy (ECT), the novel seizure quality index (SQI) can predict the risk of non-response (and non-remission)-as early as after the second ECT session-based the extent of several ictal parameters of the seizure. We aim to test several CSF markers on their ability to predict the degree of seizure quality, measured by the SQI to identify possible factors, that could explain some variability of the seizure quality. Baseline CSF levels of metabolites from the kynurenine pathway, markers of neurodegeneration (tau proteins, ß-amyloids and neurogranin), elements of the innate immune system, endocannabinoids, sphingolipids, neurotrophic factors (VEGF) and Klotho were measured before ECT in patients with depression (n = 12) to identify possible correlations with the SQI by Pearson's partial correlation. Negative, linear relationships with the SQI for response were observed for CSF levels of T-tau (rpartial = - 0.69, p = 0.019), phosphatidylcholines (rpartial = - 0.52, p = 0.038) and IL-8 (rpartial = - 0.67, p = 0.047). Regarding the SQI for remission, a negative, linear relationship was noted with CSF levels of the endocannabinoid AEA (rpartial = - 0.70, p = 0.024) and CD163 (rpartial = - 0.68, p = 0.029). In sum, CSF Markers for the innate immune system, for neurodegeneration and from lipids were found to be associated with the SQI for response and remission after adjusting for age. Consistently, higher CSF levels of the markers were always associated with lower seizure quality. Based on these results, further research regarding the mechanism of seizure quality in ECT is suggested.

Brain-Derived Neurotrophic Factor in the Cerebrospinal Fluid Increases During Electroconvulsive Therapy in Patients With Depression: A Preliminary Report.

OBJECTIVE: Preclinical evidence suggests a role for brain-derived neurotrophic factor (BDNF) in the mode of action of electroconvulsive therapy (ECT). Clinical data regarding BDNF levels in serum or plasma are more inconsistent. We measured BDNF levels from the cerebrospinal fluid (CSF) in patients with major depression before and shortly after a course of ECT. METHODS: Cerebrospinal fluid and serum BDNF levels were determined using commercially available enzyme-linked immunosorbent assay (ELISA) kits. RESULTS: We included 9 patients with a severe depressive episode within a major depressive disorder into the study. The CSF BDNF concentrations at baseline were lower compared with those CSF BDNF levels after the complete ECT treatment (P = 0.042), whereas no such a constellation was found for serum BDNF. No associations between the BDNF levels and the amount of individual ECT sessions or the reduction of the depressive symptoms were found. CONCLUSIONS: For the first time, it has been shown that CSF BDNF concentrations increase during a course of ECT in patients with a severe unipolar depressive episode, which is in line with the neurotrophin hypothesis as a mode of action of ECT, although it was not possible to demonstrate either a dose-effect relation or a relationship with the actual antidepressant effects in our small sample. Major limitation is the small sample size.

Diagnostics 4.0: the medical laboratory in digital health.

During recent years, the digital revolution has changed the face of societies including industrial production, economies and peoples' social lives. From these changes we may extrapolate the developments that digitization of health care will bring to medicine in general and laboratory medicine in particular. Disruptive technologies will fundamentally change the way laboratory tests are going to be ordered, carried out and interpreted in the future, and test results from various sources need to be curated to be of added value for the patient's condition. Wearables and implantables will quantify the concentrations for an unknown number of laboratory parameters, and the data will be stored in cloud services at the fingertips of the patient as the sovereign of his/her health care data. A 24/7 online availability of health services will strengthen predictive medicine and may enable a vastly improved preventive health care that is supported by deep-learning algorithms for clinical decision-making not only on behalf of the physician, but also the empowered patient (e.g. health bots). This will likely shift the current role of Laboratory Medicine as a central provider of diagnostic information from a "hidden champion" towards a higher visibility redefining the patient-physician-laboratory relationship. For example, accessing digital health data will allow Laboratory Medicine to more efficiently contribute to the medical dialog than is often the case today. From this perspective, this will require major readjustments in the way we execute our profession, and it will also need new concepts of education and continuous professional development.

Digital networks for laboratory data: potentials, barriers and current initiatives.

Medical care is increasingly delivered by multiple providers across healthcare sectors and specialties, leading to a fragmentation of the electronic patient record across organizations and vendor IT systems. The rapid uptake of wearables and connected diagnostic devices adds another source of densely collected data by the patients themselves. Integration of these data sources opens up several potentials: a longitudinal view of laboratory findings would close the gaps between individual provider visits and allow to more closely follow disease progression. Adding non-laboratory data (e.g. diagnoses, procedures) would add context and support clinical interpretation of findings. Case-based reasoning and disease-modelling approaches would allow to identify similar patient groups and classify endotypes. Realization of these potentials is, however, subject to several barriers, including legal and ethical prerequisites of data access, syntactic and semantic integration, comparability of items and user-centered presentation. The German Medical Informatics Initiative is presented as a current undertaking that strives to address these issues by establishing a national infrastructure for the secondary use of routine clinical data.

Rapid susceptibility testing of multi-drug resistant Escherichia coli and Klebsiella by glucose metabolization monitoring.

Background The increasing number of multi-drug resistant (MDR) bacteria provides enormous challenges for choosing an appropriate antibiotic therapy in the early phase of sepsis. While bacterial identification has been greatly accelerated by the introduction of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), the antibiotic susceptibility testing (AST) remains time-consuming. Here, we present a rapid susceptibility testing method for testing Gram-negative bacteria, exemplarily validated for Escherichia coli and Klebsiella spp. Methods Gram-negative isolates (E. coli and Klebsiella spp.) were either taken as single colonies from agar plates (n=136) or directly extracted and identified from positive blood cultures (n=42) using MALDI-TOF MS. Bacteria were incubated in glucose-supplemented Luria broths (LBs) each containing one antibiotic (ceftazidime, piperacillin, imipenem and ciprofloxacin), routinely used to classify Gram-negative bacteria in Germany. To determine susceptibility the dynamics of glucose utilization in bacterial suspensions were quantitatively measured in the presence or absence of antibiotics designated liquid-AST (L-AST). Results The L-AST can be run on clinical-chemistry analyzers and integrated into laboratory routines. It yields critical resistance information within 90-150 min downstream of a MS-based identification. The results showed a high concordance with routine susceptibility testing, with less than 1% very major errors (VME) and 3.51% major errors (ME) for 178 assessed isolates. Analysis of turnaround time (TAT) for 42 clinical samples indicated that L-AST results could be obtained 34 h earlier than the routine results. Conclusions As exemplified for E. coli and Klebsiella spp., L-AST provides substantial acceleration of susceptibility testing following MALDI-TOF MS identification. The assay is a simple and low-cost method that can be integrated into clinical laboratory to allow for 24/7 AST. This approach could improve antibiotic therapy.

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.

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.