High Rate of Passenger Lymphocyte Syndrome after ABO Minor Incompatible Lung Transplantation.

Rationale: Passenger lymphocyte syndrome (PLS) may complicate minor ABO mismatched lung transplantation (LuTX) via donor-derived red cell antibody-induced hemolysis.Objectives: To ascertain the incidence and specificity of PLS-relevant antibodies among the study population as well as the dynamics of hemolysis parameters and the transfusion requirement of patients with or without PLS.Methods: In this cohort study, 1,011 patients who received LuTX between January 2010 and June 2019 were studied retrospectively. Prospectively, 87 LuTX (July 2019 to June 2021) were analyzed. Postoperative ABO antibody and hemolytic marker determinations, transfusion requirement, and duration of postoperative hospital care were analyzed. Retrospectively, blood group A recipients of O grafts with PLS were compared with those without.Measurements and Main Results: PLS affected 18.18% (retrospective) and 30.77% (prospective) of A recipients receiving O grafts, 5.13% of B recipients of O grafts, and 20% of AB patients receiving O transplants. Anti-A and anti-A1 were the predominant PLS-inducing antibodies, followed by anti-B and anti-A,B. Significantly lower hemoglobin values (median, 7.4 vs. 8.3 g/dl; P = 0.0063) and an approximately twice as high percentage of patients requiring blood transfusions were seen in PLS. No significant differences in other laboratory markers, duration of hospital stay, or other complications after LuTX were registered.Conclusions: Minor ABO incompatible LuTX recipients are at considerable risk of developing clinically significant PLS. Post-transplant monitoring combining red cell serology and hemolysis marker determination appears advisable so as not to overlook hemolytic episodes that necessitate antigen-negative transfusion therapy.

The necessity of repeat testing for von Willebrand disease in adult patients with mild to moderate bleeding disorders.

BACKGROUND: In patients with mild-to-moderate bleeding disorders (MBD), von Willebrand disease (VWD) is diagnosed at von Willebrand factor (VWF) levels ≤50 IU/dL. Although VWF levels are unstable, repeated testing for VWD diagnosis is not necessarily advised in recent guidelines. OBJECTIVES: To analyze the relevance of repeated VWF testing to diagnose VWD in patients with MBD. METHODS: Data of 277 patients with MBD from the Vienna Bleeding Biobank with at least 2 separate assessments of VWF antigen (VWF:Ag) and activity (VWF:Act) were analyzed. RESULTS: In repeated VWF measurements, 36 patients (13.0%) had "changing" VWF levels (≤/>50 IU/dL), 27 (9.7%) had persistent levels ≤50 IU/dL ("pathologic"), and 214 (77.3%) had levels >50 IU/dL ("normal"). Of the 36 changing patients, 22 (61%) were diagnosed with VWD at baseline, whereas the others only met VWD diagnostic criteria at repeated measurements. Using logistic regression, we estimated a probability of change of 26.4% (95% CI, 12.5-47.4) at baseline VWF levels of 30 IU/dL, 50.8% (95% CI, 35.6-65.8) at 50 IU/dL, 18.8% (95% CI, 12.3-27.6) at 60 IU/dL, and 1.2% (95% CI, 0.3-4.9) at 80 IU/dL. Baseline VWF was a strong predictor for changing status (Χ2 = 49.9; P < .001), while age, sex, Vicenza score, and blood type O had limited added value (Χ2 = 5.1; P = .278). Baseline VWF:Ag or VWF:Act cutoffs of 80 IU/dL had negative predictive values of 98.1% and 99.1% for changing status, respectively. CONCLUSION: Our data emphasize an overlap between patients with VWD and MBD with bleeding disorder of unknown cause and underline the need for repeated VWF testing, especially in patients with VWF levels <80 IU/dL.

A novel EGFR inhibitor acts as potent tool for hypoxia-activated prodrug systems and exerts strong synergistic activity with VEGFR inhibition in vitro and in vivo.

Small-molecule EGFR inhibitors have distinctly improved the overall survival especially in EGFR-mutated lung cancer. However, their use is often limited by severe adverse effects and rapid resistance development. To overcome these limitations, a hypoxia-activatable Co(III)-based prodrug (KP2334) was recently synthesized releasing the new EGFR inhibitor KP2187 in a highly tumor-specific manner only in hypoxic areas of the tumor. However, the chemical modifications in KP2187 necessary for cobalt chelation could potentially interfere with its EGFR-binding ability. Consequently, in this study, the biological activity and EGFR inhibition potential of KP2187 was compared to clinically approved EGFR inhibitors. In general, the activity as well as EGFR binding (shown in docking studies) was very similar to erlotinib and gefitinib (while other EGFR-inhibitory drugs behaved different) indicating no interference of the chelating moiety with the EGFR binding. Moreover, KP2187 significantly inhibited cancer cell proliferation as well as EGFR pathway activation in vitro and in vivo. Finally, KP2187 proved to be highly synergistic with VEGFR inhibitors such as sunitinib. This indicates that KP2187-releasing hypoxia-activated prodrug systems are promising candidates to overcome the clinically observed enhanced toxicity of EGFR-VEGFR inhibitor combination therapies.

Potentials and pitfalls of ChatGPT and natural-language artificial intelligence models for the understanding of laboratory medicine test results. An assessment by the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM) Working Group on Artificial Intelligence (WG-AI).

OBJECTIVES: ChatGPT, a tool based on natural language processing (NLP), is on everyone's mind, and several potential applications in healthcare have been already proposed. However, since the ability of this tool to interpret laboratory test results has not yet been tested, the EFLM Working group on Artificial Intelligence (WG-AI) has set itself the task of closing this gap with a systematic approach. METHODS: WG-AI members generated 10 simulated laboratory reports of common parameters, which were then passed to ChatGPT for interpretation, according to reference intervals (RI) and units, using an optimized prompt. The results were subsequently evaluated independently by all WG-AI members with respect to relevance, correctness, helpfulness and safety. RESULTS: ChatGPT recognized all laboratory tests, it could detect if they deviated from the RI and gave a test-by-test as well as an overall interpretation. The interpretations were rather superficial, not always correct, and, only in some cases, judged coherently. The magnitude of the deviation from the RI seldom plays a role in the interpretation of laboratory tests, and artificial intelligence (AI) did not make any meaningful suggestion regarding follow-up diagnostics or further procedures in general. CONCLUSIONS: ChatGPT in its current form, being not specifically trained on medical data or laboratory data in particular, may only be considered a tool capable of interpreting a laboratory report on a test-by-test basis at best, but not on the interpretation of an overall diagnostic picture. Future generations of similar AIs with medical ground truth training data might surely revolutionize current processes in healthcare, despite this implementation is not ready yet.

INTERCEPT Pathogen Reduction in Platelet Concentrates, in Contrast to Gamma Irradiation, Induces the Formation of trans-Arachidonic Acids and Affects Eicosanoid Release during Storage.

Pathogen inactivation techniques for blood products have been implemented to optimize clinically safe blood components supply. The INTERCEPT system uses amotosalen together with ultraviolet light wavelength A (UVA) irradiation. Irradiation-induced inactivation of nucleic acids may actually be accompanied by modifications of chemically reactive polyunsaturated fatty acids known to be important mediators of platelet functions. Thus, here, we investigated eicosanoids and the related fatty acids released upon treatment and during storage of platelet concentrates for 7 days, complemented by the analysis of functional and metabolic consequences of these treatments. Metabolic and functional issues like glucose consumption, lactate formation, platelet aggregation, and clot firmness hardly differed between the two treatment groups. In contrast to gamma irradiation, here, we demonstrated that INTERCEPT treatment immediately caused new formation of trans-arachidonic acid isoforms, while 11-hydroxyeicosatetraenoic acid (11-HETE) and 15-HETE were increased and two hydroperoxyoctadecadienoic acid (HpODE) isoforms decreased. During further storage, these alterations remained stable, while the release of 12-lipoxygenase (12-LOX) products such as 12-HETE and 12-hydroxyeicosapentaenoic acid (12-HEPE) was further attenuated. In vitro synthesis of trans-arachidonic acid isoforms suggested that thiol radicals formed by UVA treatment may be responsible for the INTERCEPT-specific effects observed in platelet concentrates. It is reasonable to assume that UVA-induced molecules may have specific biological effects which need to be further investigated.

Assay validity of point-of-care platelet function tests in thrombocytopenic blood samples.

Introduction: Point-of-care (POC) platelet function tests are faster and easier to perform than in-depth assessment by flow cytometry. At low platelet counts, however, POC tests are prone to assess platelet function incorrectly. Lower limits of platelet count required to obtain valid test results were defined and a testing method to facilitate comparability between different tests was established. Materials and methods: We assessed platelet function in whole blood samples of healthy volunteers at decreasing platelet counts (> 100, 80-100, 50-80, 30-50 and < 30 x109/L) using two POC tests: impedance aggregometry and in-vitro bleeding time. Flow cytometry served as the gold standard. The number of platelets needed to reach 50% of the maximum function (ED50) and the lower reference limit (EDref) were calculated to define limits of test validity. Results: The minimal platelet count required for reliable test results was 100 x109/L for impedance aggregometry and in-vitro bleeding time but only 30 x109/L for flow cytometry. Comparison of ED50 and EDref showed significantly lower values for flow cytometry than either POC test (P value < 0.05) but no difference between POC tests nor between the used platelet agonists within a test method. Conclusion: Calculating the ED50 and EDref provides an effective way to compare values from different platelet function assays. Flow cytometry enables correct platelet function testing as long as platelet count is > 30 x109/L whereas impedance aggregometry and in-vitro bleeding time are inconsistent unless platelet count is > 100 x109/L.

Prolonged Exposure to Oxaliplatin during HIPEC Improves Effectiveness in a Preclinical Micrometastasis Model.

Cytoreductive surgery combined with hyperthermic intraperitoneal chemotherapy (HIPEC) was considered a promising treatment for patients with peritoneal metastasis from colorectal cancer. However, the recently published randomized controlled PRODIGE 7 trial failed to demonstrate survival benefits through the addition of short-term oxaliplatin-based HIPEC. Constituting a complex multifactorial treatment, we investigated HIPEC in a preclinical model concerning the elimination of minimal tumor residues, thereby aiming to better understand the size of effects and respective clinical trial results. Patient samples of peritoneal perfusates obtained during HIPEC treatments and oxaliplatin-containing solutions at clinically relevant dosages, conforming with established HIPEC protocols, were assessed regarding their ability to eliminate modelled ~100 µm thickness cancer cell layers. Impedance-based real-time cell analysis and classical end-point assays were used. Flow cytometry was employed to determine the effect of different HIPEC drug solvents on tumor cell properties. Effectiveness of peritoneal perfusate patient samples and defined oxaliplatin-containing solutions proved limited but reproducible. HIPEC simulations for 30 min reduced the normalized cell index below 50% with peritoneal perfusates from merely 3 out of 9 patients within 72 h, indicating full-thickness cytotoxic effects. Instead, prolonging HIPEC to 1 h enhanced these effects and comprised 7 patients' samples, while continuous drug exposure invariably resulted in complete cell death. Further, frequently used drug diluents caused approximately 25% cell size reduction within 30 min. Prolonging oxaliplatin exposure improved effectiveness of HIPEC to eliminate micrometastases in our preclinical model. Accordingly, insufficient penetration depth, short exposure time, and the physicochemical impact of drug solvents may constitute critical factors.

Thrombomodulin in patients with mild to moderate bleeding tendency.

INTRODUCTION: A massive increase of soluble thrombomodulin (sTM) due to variants in the thrombomodulin gene (THBD) has recently been identified as a novel bleeding disorder. AIM: To investigate sTM levels and underlying genetic variants as a cause for haemostatic impairment and bleeding in a large number of patients with a mild to moderate bleeding disorder (MBD), including patients with bleeding of unknown cause (BUC). PATIENTS AND METHODS: In 507 MBD patients, sTM levels, thrombin generation and plasma clot formation were measured and compared to 90 age- and sex-matched healthy controls. In patients, genetic analysis of the THBD gene was performed. RESULTS: No difference in sTM levels between patients and controls was found overall (median ([IQR] 5.0 [3.8-6.3] vs. 5.1 [3.7-6.4] ng/ml, p = .762), and according to specific diagnoses of MBD or BUC, and high sTM levels (≥95th percentile of healthy controls) were not overrepresented in patients. Soluble TM levels had no impact on bleeding severity or global tests of haemostasis, including thrombin generation or plasma clot formation. In the THBD gene, no known pathogenic or novel disease-causing variants affecting sTM plasma levels were identified in our patient cohort. CONCLUSION: TM-associated coagulopathy appears to be rare, as it was not identified in our large cohort of patients with MBD. Soluble TM did not arise as a risk factor for bleeding or altered haemostasis in these patients.

SARS-CoV-2 in Solid Organ Transplant Recipients: A Structured Review of 2020.

BACKGROUND: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic is challenging health systems all over the world. Particularly high-risk groups show considerable mortality rates after infection. In 2020, a huge number of case reports, case series, and consecutively various systematic reviews have been published reporting on morbidity and mortality risk connected with SARS-CoV-2 in solid organ transplant (SOT) recipients. However, this vast array of publications resulted in an increasing complexity of the field, overwhelming even for the expert reader. METHODS: We performed a structured literature review comprising electronic databases, transplant journals, and literature from previous systematic reviews covering the entire year 2020. From 164 included articles, we identified 3451 cases of SARS-CoV-2-infected SOT recipients. RESULTS: Infections resulted in a hospitalization rate of 84% and 24% intensive care unit admissions in the included patients. Whereas 53.6% of patients were reported to have recovered, cross-sectional overall mortality reported after coronavirus disease 2019 (COVID-19) was at 21.1%. Synoptic data concerning immunosuppressive medication attested to the reduction or withdrawal of antimetabolites (81.9%) and calcineurin inhibitors (48.9%) as a frequent adjustment. In contrast, steroids were reported to be increased in 46.8% of SOT recipients. CONCLUSIONS: COVID-19 in SOT recipients is associated with high morbidity and mortality worldwide. Conforming with current guidelines, modifications of immunosuppressive therapies mostly comprised a reduction or withdrawal of antimetabolites and calcineurin inhibitors, while frequently maintaining or even increasing steroids. Here, we provide an accessible overview to the topic and synoptic estimates of expectable outcomes regarding in-hospital mortality of SOT recipients with COVID-19.

Systematic Analysis of the Transcriptome Profiles and Co-Expression Networks of Tumour Endothelial Cells Identifies Several Tumour-Associated Modules and Potential Therapeutic Targets in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is the sixth most common cancer and the third most common cause of cancer-related death, with tumour associated liver endothelial cells being thought to be major drivers in HCC progression. This study aims to compare the gene expression profiles of tumour endothelial cells from the liver with endothelial cells from non-tumour liver tissue, to identify perturbed biologic functions, co-expression modules, and potentially drugable hub genes that could give rise to novel therapeutic targets and strategies. Gene Set Variation Analysis (GSVA) showed that cell growth-related pathways were upregulated, whereas apoptosis induction, immune and inflammatory-related pathways were downregulated in tumour endothelial cells. Weighted Gene Co-expression Network Analysis (WGCNA) identified several modules strongly associated to tumour endothelial cells or angiogenic activated endothelial cells with high endoglin (ENG) expression. In tumour cells, upregulated modules were associated with cell growth, cell proliferation, and DNA-replication, whereas downregulated modules were involved in immune functions, particularly complement activation. In ENG+ cells, upregulated modules were associated with cell adhesion and endothelial functions. One downregulated module was associated with immune system-related functions. Querying the STRING database revealed known functional-interaction networks underlying the modules. Several possible hub genes were identified, of which some (for example FEN1, BIRC5, NEK2, CDKN3, and TTK) are potentially druggable as determined by querying the Drug Gene Interaction database. In summary, our study provides a detailed picture of the transcriptomic differences between tumour and non-tumour endothelium in the liver on a co-expression network level, indicates several potential therapeutic targets and presents an analysis workflow that can be easily adapted to other projects.

Elevated levels of tissue factor pathway inhibitor in patients with mild to moderate bleeding tendency.

High levels of tissue factor pathway inhibitor (TFPI), caused by a longer TFPIα half-life after binding to a factor V splice variant and variants in the F5 gene, were recently identified in 2 families with an as-yet-unexplained bleeding tendency. This study aimed to investigate free TFPIα in a well-characterized cohort of 620 patients with mild to moderate bleeding tendencies and its association to genetic alterations in the F5 gene. TFPIα levels were higher in patients with bleeding compared with healthy controls (median [interquartile range], 8.2 [5.5-11.7] vs 7.8 [4.3-11.1]; P = .026). A higher proportion of patients had free TFPIα levels more than or equal to the 95th percentile compared with healthy controls (odds ratio [OR] [95% confidence interval (CI)], 2.82 [0.98-8.13]). This was pronounced in the subgroup of patients in whom no bleeding disorder could be identified (bleeding of unknown cause [BUC; n = 420]; OR [95% CI], 3.03 [1.02-8.98]) and in platelet function defects (PFDs) (n = 121; OR [95% CI], 3.47 [1.09-11.08]). An increase in free TFPIα was associated with a mild delay in thrombin generation (prolonged lag time and time to peak), but not with alterations in routinely used global clotting tests. We could neither identify new or known genetic variations in the F5 gene that are associated with free TFPIα levels, nor an influence of the single-nucleotide variant rs10800453 on free TFPIα levels in our patient cohort. An imbalance of natural coagulation inhibitors such as TFPIα could be an underlying cause or contributor for unexplained bleeding, which is most probably multifactorial in a majority of patients.

Joining European Scientific Forces to Face Pandemics.

Despite the international guidelines on the containment of the coronavirus disease 2019 (COVID-19) pandemic, the European scientific community was not sufficiently prepared to coordinate scientific efforts. To improve preparedness for future pandemics, we have initiated a network of nine European-funded Cooperation in Science and Technology (COST) Actions that can help facilitate inter-, multi-, and trans-disciplinary communication and collaboration.

The assessment of platelet function by thromboelastometry as a point-of-care test to guide Intercept-treated platelet support in hemato-oncological patients and hematopoietic stem cell transplantation recipients.

BACKGROUND: Pathogen inactivation (PI) techniques for platelet concentrates (PCs) are one of the latest innovations to improve blood safety and reduce the risk of transfusion-transmitted infections (TTIs). An impaired function and in vivo recovery of platelets as well as an increased PC demand are concerns regarding these techniques. The intent of this study was to evaluate the hemostatic effect of PCs treated with the Intercept™ System by thromboelastometry (TEM) and to assess the clinical validity of its results in comparison to post-transfusion increase (PTI) and corrected count increment (CCI). STUDY-DESIGN AND METHODS: This prospective-observational study included 47 patients (m:f = 25:22; median age: 54 years [21-70]) of our Bone Marrow Transplantation unit with hemato-oncological malignancies transfused with Intercept™-treated PCs. Serial TEM measurements were performed just before and 1 hour after PC transfusion and were analyzed for their correlation with PTI and CCI as well as for clinical variables. RESULTS: The majority of our patients had received a hematopoietic stem cell transplantation (HSCT) (n = 41; 87%). In median 9 (1-50) PCs were transfused. Serial TEM, PTI, and CCI measurements were available for 150 transfusion episodes. The median platelet dose transfused was 2.65 × 1011 /unit (1.8-6). The median CCI was 9.250 (0-28.000). We observed a significant improvement in TEM parameters (p < 0.05) after transfusion of PI PCs, which did not mandatory correlate with the 1-hour PTI and CCI. CONCLUSION: Serial TEM measurements indicate the hemostatic effect of Intercept™-treated PCs. The 1-hour PTI and CCI may not appropriately reflect the in vivo function of platelets after PI PC transfusion.

Diagnostic high-throughput sequencing of 2396 patients with bleeding, thrombotic, and platelet disorders.

A targeted high-throughput sequencing (HTS) panel test for clinical diagnostics requires careful consideration of the inclusion of appropriate diagnostic-grade genes, the ability to detect multiple types of genomic variation with high levels of analytic sensitivity and reproducibility, and variant interpretation by a multidisciplinary team (MDT) in the context of the clinical phenotype. We have sequenced 2396 index patients using the ThromboGenomics HTS panel test of diagnostic-grade genes known to harbor variants associated with rare bleeding, thrombotic, or platelet disorders (BTPDs). The molecular diagnostic rate was determined by the clinical phenotype, with an overall rate of 49.2% for all thrombotic, coagulation, platelet count, and function disorder patients and a rate of 3.2% for patients with unexplained bleeding disorders characterized by normal hemostasis test results. The MDT classified 745 unique variants, including copy number variants (CNVs) and intronic variants, as pathogenic, likely pathogenic, or variants of uncertain significance. Half of these variants (50.9%) are novel and 41 unique variants were identified in 7 genes recently found to be implicated in BTPDs. Inspection of canonical hemostasis pathways identified 29 patients with evidence of oligogenic inheritance. A molecular diagnosis has been reported for 894 index patients providing evidence that introducing an HTS genetic test is a valuable addition to laboratory diagnostics in patients with a high likelihood of having an inherited BTPD.

Time from venipuncture to cell isolation: Impact on granulocyte-reactive antibody testing.

BACKGROUND AND OBJECTIVES: Classical neutrophil-reactive antibody testing depends on the quick isolation of neutrophils from freshly taken whole blood. To allow a better logistic preparation before testing, the influence of time interval between venipuncture and cell isolation has been evaluated in this study. MATERIALS AND METHODS: Neutrophils and whole leukocytes were isolated from EDTA whole blood immediately (T0) as well as 4, 8 and 24 h after blood donation (T4, T8 and T24). These cells were tested against reference sera containing antibodies against HNA-1b, -2, -3a and HLA class I using granulocyte aggregation test (GAT), microscopic granulocyte immunofluorescence test (GIFT) and flow-cytometric white blood cell immunofluorescence test (Flow-GIFT/WIFT). RESULTS: GAT was the most error-prone test displaying overall weaker aggregation strengths already at T4 (overall accuracy OA = 0.72, κ = 0.58). GIFT results showed good agreement at T4 (OA = 0.86, κ = 0.79) and remained stable until T8, while test results were slightly impaired at T24 (OA = 0.71, κ = 0.55). Flow-GIFT/WIFT was identified as the most robust screening method, remaining stable even at T24. Calculated ratios (sample/negative control) decreased non-significantly and remained highly above the cut-off in all samples. CONCLUSION: Acceptable time limits for cell isolation are different for each screening method investigated. For GAT, cell isolation should be performed within 4 h, while GIFT tolerates a neutrophil isolation delay of 8 h. Flow-GIFT/WIFT isolation can be performed even after 24 h without impairment of the results. Using the latter test as a stand-alone pre-screening test, whole blood can be used from donors who are not directly accessible.

Systematic Review of Variations in Hyperthermic Intraperitoneal Chemotherapy (HIPEC) for Peritoneal Metastasis from Colorectal Cancer.

BACKGROUND: Cytoreductive surgery (CRS), followed by hyperthermic intraperitoneal chemotherapy (HIPEC), combines radical surgery with abdominal heated chemotherapy, constituting a multimodal treatment approach. Since clear standards for HIPEC conduct in colorectal carcinoma (CRC) are lacking, we aimed to provide a comprehensive structured survey. Data sources and study eligibility criteria: A systematic literature search was performed in PubMed, with keywords "HIPEC" and "colorectal cancer", according to established guidelines. Articles were systematically screened, selecting 87 publications complemented by 48 publications identified through extended search for subsequent synthesis and evaluation, extracting inter alia details on used drugs, dosage, temperature, exposure times, and carrier solutions. RESULTS: Compiled publications contained 171 reports on HIPEC conduct foremost with mitomycin C and oxaliplatin, but also other drugs and drug combinations, comprising at least 60 different procedures. We hence provide an overview of interconnections between HIPEC protocols, used drugs and carrier solutions as well as their volumes. In addition, HIPEC temperatures and dosing benchmarks, as well as an estimate of in vivo resulting drug concentrations are demonstrated. CONCLUSIONS AND IMPLICATIONS: Owing to recent developments, HIPEC conduct and practices need to be reassessed. Unfortunately, imprecise and lacking reporting is frequent, which is why minimal information requirements should be established for HIPEC and the introduction of final drug concentrations for comparability reasons seems sensible.

Pharmacodynamics of Oxaliplatin-Derived Platinum Compounds During Hyperthermic Intraperitoneal Chemotherapy (HIPEC): An Emerging Aspect Supporting the Rational Design of Treatment Protocols.

BACKGROUND: Hyperthermic intraperitoneal chemotherapy (HIPEC) is used to treat peritoneal surface malignancies with application of cytostatic drugs such as oxaliplatin (OX) after cytoreductive surgery. Despite its increased use, evidence for optimal drug dosage, and notably duration of HIPEC, is scarce. METHODS: In this study, OX distribution was comprehensively assessed in nine patients during HIPEC (300 mg OX/m2 body surface area in Physioneal solution for 30 min). Oxaliplatin and its derivatives were measured in peritoneal perfusates over time by liquid chromatography coupled with mass spectrometry (LC-MS), and the resulting total platinum concentration in tissue was analyzed by atomic absorption spectrometry. Additionally, a novel impedance-based real-time cytotoxicity assay was used to evaluate the bioactivity of perfusates ex vivo. RESULTS: Compared with amounts of OX expected in peritoneal perfusates by calculation, only 10-15% of the parent drug could be detected by LC-MS during HIPEC. Notably, the study additionally detected platinum compounds consistent with OX transformation, accounting for a further fraction of the applied drug. The cytotoxic properties of perfusates remained unchanged during HIPEC, with only a slight but significant attenuation evidenced after 30 min. CONCLUSIONS: The bioactivity of peritoneal perfusates ex vivo is a useful parameter for evaluating the actual cytotoxic potential of OX and its derivatives used in HIPEC over time, overcoming important limitations and disadvantages associated with respective drug monitoring only. Ex vivo cytotoxicity assays may be a promising tool to aid guiding future standardization and harmonization of HIPEC protocols based on drug-mediated effects.

Preanalytical Conditions and DNA Isolation Methods Affect Telomere Length Quantification in Whole Blood.

Telomeres are located at chromosome ends and their length (TL) has been associated with aging and human diseases such as cancer. Whole blood DNA is frequently used for TL measurements but the influence of preanalytical conditions and DNA isolation methods on TL quantification has not been thoroughly investigated. To evaluate potential preanalytical as well as methodological bias on TL, anonymized leftover EDTA-whole blood samples were pooled according to leukocyte counts and were incubated with and without actinomycin D to induce apoptosis as a prototype of sample degradation. DNA was isolated from fresh blood pools and after freezing at -80°C. Commercially available kits using beads (Invitrogen), spin columns (Qiagen, Macherey-Nagel and 5prime) or precipitation (Stratec/Invisorb) and a published isopropanol precipitation protocol (IPP) were used for DNA isolation. TL was assessed by qPCR, and normalized to the single copy reference gene 36B4 using two established single-plex and a new multiplex protocol. We show that the method of DNA isolation significantly affected TL (e.g. 1.86-fold longer TL when comparing IPP vs. Invitrogen). Sample degradation led to an average TL decrease of 22% when using all except for one DNA isolation method (5prime). Preanalytical storage conditions did not affect TL with exception of samples that were isolated with the 5prime kit, where a 27% increase in TL was observed after freezing. Finally, performance of the multiplex qPCR protocol was comparable to the single-plex assays, but showed superior time- and cost-effectiveness and required > 80% less DNA. Findings of the current study highlight the need for standardization of whole blood processing and DNA isolation in clinical study settings to avoid preanalytical bias of TL quantification and show that multiplex assays may improve TL/SCG measurements.

Evaluation of clinical risk factors to predict high on-treatment platelet reactivity and outcome in patients with stable coronary artery disease (PREDICT-STABLE).

OBJECTIVES: This study was designed to identify the multivariate effect of clinical risk factors on high on-treatment platelet reactivity (HPR) and 12 months major adverse events (MACE) under treatment with aspirin and clopidogrel in patients undergoing non-urgent percutaneous coronary intervention (PCI). METHODS: 739 consecutive patients with stable coronary artery disease (CAD) undergoing PCI were recruited. On-treatment platelet aggregation was tested by light transmittance aggregometry. Clinical risk factors and MACE during one-year follow-up were recorded. An independent population of 591 patients served as validation cohort. RESULTS: Degree of on-treatment platelet aggregation was influenced by different clinical risk factors. In multivariate regression analysis older age, diabetes mellitus, elevated BMI, renal function and left ventricular ejection fraction were independent predictors of HPR. After weighing these variables according to their estimates in multivariate regression model, we developed a score to predict HPR in stable CAD patients undergoing elective PCI (PREDICT-STABLE Score, ranging 0-9). Patients with a high score were significantly more likely to develop MACE within one year of follow-up, 3.4% (score 0-3), 6.3% (score 4-6) and 10.3% (score 7-9); odds ratio 3.23, P=0.02 for score 7-9 vs. 0-3. This association was confirmed in the validation cohort. CONCLUSIONS: Variability of on-treatment platelet function and associated outcome is mainly influenced by clinical risk variables. Identification of high risk patients (e.g. with high PREDICT-STABLE score) might help to identify risk groups that benefit from more intensified antiplatelet regimen. Additional clinical risk factor assessment rather than isolated platelet function-guided approaches should be investigated in future to evaluate personalized antiplatelet therapy in stable CAD-patients.

Comparison of whole blood RNA preservation tubes and novel generation RNA extraction kits for analysis of mRNA and MiRNA profiles.

BACKGROUND: Whole blood expression profiling is frequently performed using PAXgene (Qiagen) or Tempus (Life Technologies) tubes. Here, we compare 6 novel generation RNA isolation protocols with respect to RNA quantity, quality and recovery of mRNA and miRNA. METHODS: 3 PAXgene and 3 Tempus Tubes were collected from participants of the LIFE study with (n = 12) and without (n = 35) acute myocardial infarction (AMI). RNA was extracted with 4 manual protocols from Qiagen (PAXgene Blood miRNA Kit), Life Technologies (MagMAX for Stabilized Blood Tubes RNA Isolation Kit), and Norgen Biotek (Norgen Preserved Blood RNA Purification Kit I and Kit II), and 2 (semi-)automated protocols on the QIAsymphony (Qiagen) and MagMAX Express-96 Magnetic Particle Processor (Life Technologies). RNA quantity and quality was determined. For biological validation, RNA from 12 representative probands, extracted with all 6 kits (n = 72), was reverse transcribed and mRNAs (matrix metalloproteinase 9, arginase 1) and miRNAs (miR133a, miR1), shown to be altered by AMI, were analyzed. RESULTS: RNA yields were highest using the Norgen Kit I with Tempus Tubes and lowest using the Norgen Kit II with PAXgene. The disease status was the second major determinant of RNA yields (LIFE-AMI 11.2 vs. LIFE 6.7 µg, p<0.001) followed by the choice of blood collection tube. (Semi-)automation reduced overall RNA extraction time but did not generally reduce hands-on-time. RNA yields and quality were comparable between manual and automated extraction protocols. mRNA expression was not affected by collection tubes and RNA extraction kits but by RT/qPCR reagents with exception of the Norgen Kit II, which led to mRNA depletion. For miRNAs, expression differences related to collection tubes (miR30b), RNA isolation (Norgen Kit II), and RT/qRT reagents (miR133a) were observed. CONCLUSION: We demonstrate that novel generation RNA isolation kits significantly differed with respect to RNA recovery and affected miRNA but not mRNA expression profiles.