Blood Tubes Research Articles

Abstract 1965: Multimodal analysis of circulating cell-free RNA (ccfRNA), cell-free DNA (ccfDNA) and genomic DNA (gDNA) from blood samples collected in PAXgene Blood ccfDNA Tubes

Abstract INTRODUCTION While circulating cell-free DNA (ccfDNA) from blood is widely used as an analyte in liquid biopsy cancer research applications, circulating cell-free RNA (ccfRNA) has only recently gained relevance for biomarker studies. Combined insights from both analytes promise to increase the understanding of molecular processes in tumors. However, challenges in the preanalytical workflow, such as choosing blood collection tubes and extraction methods suitable for multimodal analysis must still be overcome. We investigated multimodal extraction and analyses of ccfRNA, ccfDNA and gDNA from one blood sample collected using the PAXgene® Blood ccfDNA Tube*. METHODS Paired whole blood samples were collected from healthy consented donors into PAXgene Blood ccfDNA Tubes (PreAnalytiX®), BD Vacutainer® K2EDTA tubes (BD), Cell-Free DNA BCT® (Streck®), RNA Complete BCT™ (Streck) and LBgard® Blood Tubes (Biomatrica®). Plasma was generated by double centrifugation immediately after blood collection or after storage. Cell-free nucleic acids were extracted manually using the QIAamp® Circulating Nucleic Acid Kit (QIAGEN), the exoRNeasy Serum/Plasma Kit (QIAGEN®) and the miRNeasy Serum/Plasma Kit (QIAGEN). gDNA was extracted from the residual cellular fraction using the QIAamp DNA Blood Mini Kit (QIAGEN). QIAGEN qPCR assays were used to analyze ccfRNA. An internally validated qPCR assay was used for analysis of ccfDNA. RESULTS Quantitative PCR analysis of extracted ccfRNA revealed similar yields of miRNA, mRNA and ccfDNA targets from PAXgene and EDTA plasma. RNA detection sensitivity was lower for blood collected and stored in tubes containing formaldehyde-releasing formulations (Streck and Biomatrica) as indicated by higher CT values. Likewise, for gDNA, yield and purity were reduced by storage in Streck and Biomatrica tubes compared to blood stored in PAXgene tubes. Besides differences in yield, extraction of ccfRNA from plasma using the three tested isolation kits showed high sample reproducibility and precision for all collection tubes. CONCLUSION This study demonstrates the non-crosslinking technology of the PAXgene Blood ccfDNA Tubes enables the isolation and analyses of cell-free miRNA, mRNA, ccfDNA and cellular gDNA. These results are also in line with recent data reported indicating successful CTC enrichment and detection of RNA from blood stored in PAXgene Blood ccfDNA tubes. Taken together, the studies support a multimodal use of the PAXgene Blood ccfDNA Tube for in liquid biopsy research. The three tested ccfRNA isolation kits - the QIAamp Circulating Nucleic Acid, the exoRNeasy Serum/Plasma Kit and the miRNeasy Serum/Plasma Kit - enable high-quality isolation of ccfRNA. Other blood collection tubes for ccfDNA stabilization showed impaired extraction and detection efficiency for several mRNA and miRNA targets. *For Research Use Only. Not for use in diagnostic procedures. Citation Format: Andrea Ullius, Eric Provencher, Thorsten Voss. Multimodal analysis of circulating cell-free RNA (ccfRNA), cell-free DNA (ccfDNA) and genomic DNA (gDNA) from blood samples collected in PAXgene Blood ccfDNA Tubes [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1965.

Abstract 3110: In depth comparison of cell free DNA blood collection tubes

Abstract Liquid biopsy continues to gain traction as a minimally invasive method to monitor biomarkers associated with malignancy and metastasis such as cell-free DNA (cfDNA). However, cell-free DNAs are present in low quantities and are prone to rapid degradation and contamination, which presents challenges for the detection of rare cfDNA targets. Higher molecular weight genomic DNA from poorly stabilized leukocytes can also serve to contaminate plasma, compromising liquid biopsy assay results by increasing the total amount of extracted DNA while diluting rare cfDNA targets; leading to sample rejection or false negative results. Efficient and robust preservation of cfDNA and prevention of leukocyte lysis is thus essential for the accuracy of downstream tests, and several blood collection tubes (BCTs) designed to stabilize cfDNA are now commercially available. The work presented here evaluates LBgard® Blood Tubes (Biomatrica), the Streck Cell-Free DNA BCTs® (Streck), and PAXgene Blood ccfDNA Tubes (PreAnalytix) for analysis of cfDNA. Healthy donor blood was collected into collection tubes, and the samples were either stored for several days at a range of temperatures or subjected to various shipping conditions. Several metrics were evaluated, including hemolysis, plasma recovery, cfDNA preservation, contamination with genomic DNA (gDNA), and quantitation of cfDNA targets. Differences were observed between the various tubes evaluated, especially when tested under real-world stress shipping conditions. Data reports performance metrics and evaluations across the multiple formulations of BCTs and highlights the criticality of collection tube selection in biomarker investigation and study. Citation Format: Joel Desharnais, Jacob M. Vasquez, Katya J. Reshatoff, Quyen Bui, Han-Mei Chen, Billyana Tsvetanova, Jerry Lu, Amber N. Murray, Gina L. Costa. In depth comparison of cell free DNA blood collection tubes [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3110.

Abstract 2854: Multi-omic considerations: Exploring LBgard blood tubes for proteomic analysis

Abstract Expanding the number of -omic (genomic, proteomic, etc.) analyte classes, a single blood draw will serve to increase the scientific value of that sample by affording additional multi-variant analyses. The detection and monitoring of many chronic diseases and cancers requires multi-factorial/multi-analyte measurement. Specialized blood collection tubes (e.g., liquid biopsy tubes) are routinely used in the development of minimally invasive methods to monitor key cancer biomarkers in blood. The suitability of plasma isolated from liquid biopsy blood tubes for quantitative proteomics studies needs further exploration. Herein we performed proteomic analyses of whole blood collected and stored in LBgard Blood Tubes. In this study, matched blood samples from 3 healthy donors were collected in LBgard Blood Tubes and K3EDTA (EDTA) tubes, which is the standard blood collection tube used for plasma proteomic analyzes. Blood collected in LBgard Blood Tubes was stored for either 5 days or 7 days at ambient temperature. Following high-abundant plasma protein depletion, plasma proteins were analyzed in quantitative discovery proteomics by liquid chromatography tandem-mass spectrometry (TMT-LC-MS/MS). Over 800 unique proteins were identified by mass spectrometry in the plasma samples analyzed. Of those, >90% were found to be of similar abundance between EDTA Day 0 plasma samples and plasma isolated from whole blood collected in LBgard Blood Tubes. After storage of the LBgard Blood Tube samples for up to 7 days at ambient temperature, >85% of the proteins demonstrated similar abundance in both stored LBgard Blood samples and EDTA tube samples processed immediately after collection. Samples were also evaluated via ELISA to quantify the levels of proteins known to be cancer related. LBgard Blood Tubes prevented changes in the plasma concentrations of several protein biomarkers known to be sensitive to preanalytical variability in samples stored for 5 days at ambient temperature as compared to samples stored in EDTA tubes under the same conditions. Utilizing tandem-mass tag spectrometry and immunoassay methodologies, we have determined the compatibility of LBgard Blood Tube for determining proteomic profiles of blood samples. Additionally, blood collected in LBgard Blood Tubes was shown to have consistent concentrations of protein after storing the collected blood sample for several days, with enhanced protein detection as compared to EDTA tubes. Citation Format: Jacob M. Vasquez, Joel Desharnais. Multi-omic considerations: Exploring LBgard blood tubes for proteomic analysis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2854.

Abstract CT241: Trial in progress: IFx-Hu2.0 (plasmid DNA coding for Emm55 streptococcal antigen in a cationic polymer) phase I first in human study for unresectable stage III or stage IV cutaneous melanoma

Abstract Background: Anti-PD-1 and anti-CTLA4 immune-based therapeutics have vastly improved melanoma survival rates, but many patients still do not benefit from these therapies. IFx-Hu2.0 is a plasmid DNA encoding the streptococcal membrane protein, Emm55, contained within a cationic polymer. Preliminary correlative laboratory data from the first in human IFx-Hu2.0 phase I feasibility study for use as an intralesional melanoma therapy is reported. No clinical data will be presented. The melanoma tumor microenvironment, pre- and post-treatment, was analyzed on FFPE tissues collected from the first three patients with a panel consisting of CD3, CD8, FOXP3, PD1, PDL1, SOX10 and DAPI. The numbers of SOX10+ melanoma cells tended to decrease with treatment (Patient 1: 16.7% SOX10+ cells pre-treatment, 0.3% SOX10+ cells post treatment; Patient 2: 47.1% pre/6.0% post; Patient 3: 49.0% pre/52.7% post). Multi-parameter analysis revealed that when the numbers of SOX10+ cells decreased, there were an increased number of T cells in the tumor environment. This observation also held true for the whole section for patient 2. In this patient, there were 0.3% CD3+CD8+ T cells prior to treatment but this number increased to 15.2% after treatment. Interestingly, the numbers of FOXP3 regulatory T cells also increased in this patient suggesting the formation of an immune response. Taken together, IFx-Hu2.0 is proposed to create an immune response in melanoma patients and is being tested in a first in human trial. Methods: Up to six patients (>18 years) with unresectable stage III or IV cutaneous melanoma will have cutaneous melanoma lesions injected with IFx-Hu2.0 to test the safety, tolerability, and feasibility of intralesional IFx-Hu2.0 injection. The subjects must have at least one lesion available for injection and another biopsiable lesion. This trial is unique as a cutaneous lesion as small as 3 mm may be injected. To be eligible, subjects should have been considered for anti-PD-1 therapy, TVEC and/or BRAF inhibition as indicated in the FDA labels for those agents. Small brain metastases (1 cm) are permitted assuming the subjects receive concurrent radiation at a site distant from intralesional injection with a greater than 3-month life expectancy. These patients may not be receiving any other systemic therapies, may not have uncontrolled hepatitis/HIV infection, and may not have a history of immunosuppression as is seen in patients who have undergone organ transplantation. For correlative studies, 4 tubes of peripheral blood will be collected at the beginning of the study and at the dose limiting toxicity (DLT) assessment visit 28 days later. Trial subjects will have the option of continued dosing every 3 weeks if they have a response to therapy. Success of the study is defined as being able to treat 5/6 subjects without DLT at 28 days. Three of the six planned trial subjects have been enrolled as of January 2020 (Clinical trial registry number: NCT03655756). Citation Format: Joseph Markowitz, Andrew Brohl, Amod A. Sarnaik, Zeynep Eroglu, Deanryan B. De Aquino, Nikhil Khushalani, Ahmad Tarhini, Vernon K. Sondak, Shari Pilon-Thomas. Trial in progress: IFx-Hu2.0 (plasmid DNA coding for Emm55 streptococcal antigen in a cationic polymer) phase I first in human study for unresectable stage III or stage IV cutaneous melanoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT241.

Warming blood products for transfusion to neonates: In vitro assessments

Blood products may be transfused into neonates at temperatures at or below room temperature. The benefits and risks of warming blood to 37°C are not defined in this population or with the equipment used in neonates. Physiologic warming might enhance product effectiveness or decrease transfusion-associated hypothermia. We utilized an in vitro model of neonatal transfusions, with a syringe pump, blood tubing, and 24-gauge catheter and compared current practice (cold products) vs an inline blood warmer. Transfusions were performed rapidly (30 minutes) and slower (120 minutes) to model emergent vs routine situations. We tested red blood cells, fresh-frozen plasma, apheresis platelets (PLTs), and cold-stored low-titer group O whole blood. We used infrared detectors and inline probes to measure temperatures at the origin and at the simulated patient. We assessed warmer-induced damage by measuring plasma hemoglobin and hematocrit (seeking hemolysis), fibrinogen (seeking activation of coagulation), and PLT count and TEG-MA (seeking PLT destruction or dysfunction). The cold-stored products were 4.2 ± 1.0°C (mean ± SD) at the origin and 21.5 ± 0.1°C at the patient. With the inline warmer, products were 37.8 ± 0.6°C at the warmer and 32.6 ± 1.7°C at the patient during a 30-minute infusion, but were 34.5 ± 2.1 with a foil sheath covering the terminal tubing. We found no warmer-induced damage using any metric. In simulated neonatal intensive care unit (NICU) transfusions, an inline blood warmer can deliver blood products at near-physiologic temperatures with no detected damage. We suggest in vivo testing of warmed NICU transfusions, assessing product effectiveness and hypothermia risk reduction.

Shared changes in angiogenic factors across gastrointestinal vascular conditions: A pilot study.

BACKGROUNDNeovascularisation is common to a variety of gastrointestinal (GI) disorders with differing aetiologies and presentations; usually affecting adults above 60 years. Shared angiogenic factors modulated by disease specific elements could be a common denominator and represent novel diagnostic and therapeutic targets. As yet, assessment of angiogenic factors across several GI vascular disorders associated with recurrent bleeding and anaemia has not been reported.AIMTo assess serum levels of angiogenic factors in several intestinal vascular disorders.METHODSA case control study was performed in Tallaght University Hospital in patients with endoscopically proven small bowel angiodysplasia (SBA), portal hypertensive gastropathy (PHG), gastric antral vascular ectasia (GAVE) and non-bleeding, non-anaemic controls. Using enzyme-linked immunosorbent assay, concentrations of Angiopoietin 1 (Ang-1), Ang-2 and vascular endothelial growth factor (VEGF) were measured from 2 serum tubes of blood following informed consent. The relative expression of Ang-1 and Ang-2 and Ang-1/2 ratio was calculated and compared between groups. Statistical analysis was applied using a t-test, and a P value of < 0.05 was considered significant.RESULTSTo date 44 samples were tested: 10 SBA, 11 PHG, 8 GAVE and 15 controls. Mean age 60 (range 20-85) years and 20 (45%) were males. Controls were significantly younger (49 years vs 66 years, P = 0.0005). There was no difference in VEGF levels between the groups (P = 0.6). SBA, PHG and GAVE Ang-1 levels were similar and were significantly lower than controls, (P = 0.0002, 95%CI: 241 to 701). Ang-2 levels were statistically higher in PHG and GAVE groups compared to controls (P = 0.01, 95%CI: 77.8 to 668) and as a result, also had a lower Ang-1/2 ratios compared to controls. While SBA Ang-2 levels were higher than controls, this did not reach statistical significance. Neither age nor haemoglobin level, which was similar between disease groups, could explain the difference. In addition, the median Ang-1/Ang-2 ratio for all patients was found to be significantly lower compared to controls, 8 vs 28 respectively, P = 0.001, 95%CI: -27.55 to -7.12.CONCLUSIONOur novel pilot study suggests common alterations in Ang-1 and Ang-2 levels across several GI vascular disorders. Differences in Ang-1/Ang-2 ratios among vascular disorders compared to controls suggest disease-specific modulation.

Towards standardization of thrombin generation assays: Inventory of thrombin generation methods based on results of an International Society of Thrombosis and Haemostasis Scientific Standardization Committee survey

BackgroundThrombin generation (TG) is a better determinant of the overall function of the hemostatic system than the routinely used clotting time‐based assays. Nowadays, TG is widely used in hemostasis research teams, for both clinical and basic research. However, there is significant variability between laboratories regarding preanalytics, reagents, TG protocol, analysis interpretation, and normalization. ObjectivesTo document the extent of variation in the methodology of TG, we aim to collect all the methods that are being used to measure TG in a survey. MethodsWe organized a questionnaire through the Standardization committee for Lupus Anticoagulant/Antiphospholipid Antibodies of the International Society of Thrombosis and Haemostasis Scientific Standardization committee. The questionnaire consisted of 51 questions regarding the different aspects of TG: type of users, methods, sample type, analysis, and interpretation of results, normalization, and quality control. ResultsOf the 240 surveys that were started, 108 were completed (45%). However, not all questions were in scope for all laboratories. One‐half of the laboratories were research laboratories and half diagnostic laboratories. The most used TG assay was the calibrated automated thrombinography‐based assay (56%). There was a divergence regarding several aspects of the TG assay: type of needle for blood collection, blood tubes, centrifugation, sample storage and thawing, reagents, sample dilution, calibration, reference ranges, data normalization, and so on. ConclusionsThere is an important variation in the methods used for measuring TG. A standardized protocol and data normalization should lead to a better reproducibility and for comparing data from different laboratories.

Testing Antemortem Blood for Ethanol Concentration from a Blood Kit in a Refrigerator Fire

The stability of ethanol in antemortem blood stored under various conditions has been widely studied. Antemortem blood samples stored at refrigerated temperature, at room temperature, and at elevated temperatures tend to decrease in ethanol concentration with storage. It appears that the stability of ethanol in blood exposed to temperatures greater than 38°C has not been evaluated. The case presented here involves comparison of breath test results with subsequent analysis of blood drawn at the time of breath testing. However, the blood tubes were in a refrigerator fire followed by refrigerated storage for 5months prior to analysis by headspace gas chromatography. The subject's breath was tested twice using an Intoxilyzer 8000. The subject's blood was tested in duplicate using an Agilent headspace gas chromatograph. The measured breath ethanol concentration was 0.103g/210L and 0.092g/210L. The measured blood ethanol concentration was 0.0932g/dL for both samples analyzed. Although the mean blood test result was slightly lower than the mean breath test result, the mean breath test result was within the estimated uncertainty of the mean blood test result. Even under the extreme conditions of the blood kit being in a refrigerator fire, the measured blood ethanol content agreed well with the paired breath ethanol test.

Point-of-Care Analysis of Neutrophil Phenotypes: A First Step Toward Immuno-Based Precision Medicine in the Trauma ICU.

The amount of tissue damage and the amplitude of the immune response after trauma are related to the development of infectious complications later on. Changes in the neutrophil compartment can be used as read out of the amplitude of the immune response after trauma. The study aim was to test whether 24/7 point-of-care analysis of neutrophil marker expression by automated flow cytometry can be achieved after trauma. A prospective cohort study was performed. Polytrauma patients who developed infectious complications were compared with polytrauma patients who did not develop infectious complications. The study was performed in a level 1 trauma center. All trauma patients presented in the trauma bay were included. An extra blood tube was drawn from all patients. Thereafter, a member of the trauma team placed the blood tube in the fully automated flow cytometer, which was located in the corner of the trauma room. Next, a modified and tailored protocol for this study was automatically performed. The trauma team was able to successfully start the point-of-care automated flow cytometry analysis in 156 of 164 patients, resulting in a 95% success rate. Polytrauma patients who developed infectious complications had a significantly higher %CD16dim/CD62Lbright neutrophils compared with polytrauma patients who did not develop infectious complications (p = 0.002). Area under the curve value for %CD16dim/CD62Lbright neutrophils is 0.90 (0.83-0.97). This study showed the feasibility of the implementation of a fully automated point-of-care flow cytometry system for the characterization of the cellular innate immune response in trauma patients. This study supports the concept that the assessment of CD16dim/CD62Lbright neutrophils can be used for early detection of patients at risk for infectious complications. Furthermore, this can be used as first step toward immuno-based precision medicine of polytrauma patients at the ICU.

Importance of Preanalytical Factors in Measuring Cr and Co Levels in Human Whole Blood: Contamination Control, Proper Sample Collection and Long-Term Storage Stability.

A number of errors with potentially significant consequences may be introduced at various points in the analytical process, which result in skewed, erroneous analytical results. Precautionary procedures such as contamination control, following established sample collection protocols, and having a complete understanding of the long-term stability of the elements of interest can minimize or eliminate these errors. Contamination control is critical in the quantification of Cr and Co in human whole blood. Cr and Co levels in most biological samples are low, but these elements occur naturally in the environment and are often found in commercial and consumer products, which increases the risk of contamination. In this paper, we demonstrated that lot screening process in which we pre-screen a sub-set of manufactured lots used in collecting, analyzing and storing blood samples is a critical step in controlling Cr and Co contamination. Stainless steel needles are often utilized in blood collection but are considered as a potential source of introducing metal contamination to the patient sample. We conducted two studies to determine if there is a possibility of Cr or Co leaching into the human whole blood from the needles during blood collection. We analyzed blood collected from 100 donors and blood collected in vitro in the laboratory from designated vessel containing spiked blood with higher levels of Cr and Co. Two blood tubes were consecutively collected through one needle. In both studies, Cr and Co concentration levels in the two consecutively collected tubes were compared. Based on the results from donor and in vitro blood collection studies, we concluded that there was no Cr and Co leaching from the limited sets of stainless steel needles used in these studies. Furthermore, we demonstrated that Cr and Co human whole blood samples are stable for 1 year stored at temperatures of -70, -20 and 4°C and 6 months at room temperature.

Ameliorative Effects of Honey, Propolis, Pollen, and Royal Jelly Mixture against Chronic Toxicity of Sumithion Insecticide in White Albino Rats.

Sumithion (Fenitrothion) (SUM) is an organophosphorus insecticide used to combat a wide variety of plant pests. Exposure to SUM causes significant toxicity to the brain, liver, kidney, and reproductive organs through, for example, binding to DNA, and it induces DNA damage, which ends with oxidative stress. Therefore, the present study aimed to examine the protective role of bee products: a mixture of honey, propolis, palm pollen, and royal jelly (HPPJ) against SUM-induced toxicity. Twenty-four male albino rats (Rattus norvegicus) were classified into four groups, each containing six rats: control (corn oil), SUM (85 mg/kg; 1/20 LD50), HPPJ, and SUM + HPPJ once daily for 28 consecutive days. Blood samples were gently collected in sterilized ethylenediaminetetraacetic acid (EDTA) tubes for blood picture analyses and tubes without anticoagulant for serum isolation. Serum was used for assays of enzymatic and biochemical characteristics. The results revealed that SUM increased the weights of the liver, kidney, and brain as well as the enzymatic activity of glutathione peroxidase (GP), serum superoxide dismutase (SOD), and glutathione-S-transferase (GST). Additionally, SUM significantly increased the activity of lactate dehydrogenase (LDH), alkaline phosphatase (ALP), and γ-glutamyltransferase (γ-GT) and glucose, uric acid, and creatinine contents, while decreasing the acetylcholine esterase (AChE) activity and total lipids and total protein content. Furthermore, because of the inclusion of phenolic, flavonoids, terpenoids, and sugars, the HPPJ mixture counteracted the hematological, renal, and hepatic toxicity of SUM exposure.

Abstract IA17: Comprehensive genomic profiling of cell-free DNA enables treatment response monitoring

Abstract Liquid biopsies have advanced significantly over the past decade but are still not routinely used to guide patient care. Part of the reason is that prospective clinical trials will be needed to demonstrate clinical utility, but detection sensitivity also needs to improve. For instance, liquid biopsies are not yet capable of discerning which patients need surgery after neoadjuvant treatment or which patients need adjuvant treatment right after surgery. They are also not yet sensitive enough to determine whether it is safe to stop therapy, as BCR-ABL testing has shown for patients with CML. We reason that 1,000-fold higher detection sensitivity (10−6) will be required to provide sufficient dynamic range to guide treatment decisions in most patients. One way to improve detection sensitivity is to track more mutations per patient. This is because when tumor fraction is low in the bloodstream, only some mutations will be drawn in a blood tube, and the sampling of mutations will be random. Yet, each patient’s cancer is unique, with mutations scattered throughout the genome, and existing technologies are not well suited for tracking large numbers of distinct mutations. Conventional sequencing has an error rate of 10−3 and methods that repeatedly sequence each fragment, or model noise in normal samples, can achieve 10−4 to 10−5 but are not robust to spontaneous errors. Duplex sequencing is the most accurate method for mutation detection (10−6) but requires extremely deep sequencing to form a consensus of sense strands of each cfDNA duplex, which makes it highly inefficient and costly. In my talk, I will first describe our team’s efforts to thoroughly optimize duplex sequencing for accurate tracking of large mutation fingerprints. I will show that detection limits up to 100-fold better than ddPCR can be achieved in serial dilution series, and that this enables treatment response monitoring in patients treated for early-stage breast cancer. I will then describe a new method to improve the efficiency of duplex sequencing using mutation enrichment. I will show that it is possible to track tens of thousands of distinct mutations in cfDNA using up to 1000-fold less sequencing, and that this enables 1000-fold better detection limit than ddPCR. In all, we believe that the improved dynamic range, afforded by the higher accuracy and efficiency in cfDNA sequencing, could make treatment response monitoring feasible for more cancer patients. Citation Format: Viktor Adalsteinsson. Comprehensive genomic profiling of cell-free DNA enables treatment response monitoring [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr IA17.

Abstract B51: Performance assessment of total RNA sequencing of human biofluids and extracellular vesicles

Abstract RNA profiling has emerged as a powerful tool to investigate the biomarker potential of human biofluids. Despite the interest in extracellular nucleic acids, RNA sequencing methods to quantify the total RNA content outside cells are rare. Therefore, we evaluated the performance of the SMARTer Stranded Total RNA-Seq method in human platelet-rich plasma, platelet-free plasma, urine, conditioned medium, and extracellular vesicles (EVs) from these biofluids. We found the method to be accurate, precise, compatible with low-input volumes, and able to quantify a few thousand genes. We picked up distinct classes of RNA molecules, including mRNA, lncRNA, circRNA, miscRNA, and pseudogenes. Notably, the read distribution and gene content drastically differed among biofluids. We also observed differences between EDTA and citrate platelet-free plasma, which are identical biofluids collected in different blood tubes and prepared with a slightly different centrifugation protocol. In conclusion, we are the first to show that the SMARTer method can be used for unbiased charting of the complete transcriptome of a wide range of biofluids and their extracellular vesicles. The advantage of the SMARTer Stranded Total RNA-Seq method is its potential to process low amounts of input material. Indeed, collecting samples is often the bottleneck in fundamental, (pre)clinical, or translational research projects, and being able to disseminate large amounts of information from only 200 μL (or less) can substantially impact research progress. Citation Format: Celine Everaert, Hetty Helsmoortel, Anneleen Decock, Eva Hulstaert, Ruben Van Paemel, Kimberly Verniers, Justine Nuytens, Jasper Anckaert, Nele Nijs, Joeri Tulkens, Bert Dhondt, An Hendrix, Pieter Mestdagh, Jo Vandesompele. Performance assessment of total RNA sequencing of human biofluids and extracellular vesicles [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr B51.

P1611PRE-ANALYTICAL CONSIDERATIONS IN STUDYING CIRCULATING MICRORNA EXPRESSION: COMPARISON BETWEEN PAIRED EDTA PLASMA, EDTA WHOLE BLOOD AND PAXGENE BLOOD RNA TUBES

Abstract Background and Aims microRNA (miRNA) dysregulations have been related to pathological processes, including kidney disease. Relative stability in blood makes miRNAs attractive biomarkers. The current recommendation is to use fresh EDTA plasma samples (i.e. processed within 30 min. from sampling) to study circulating miRNA. However, cumbersome logistics might preclude broad implementation. Therefore, we investigated the potential of whole blood EDTA and PAXgene blood RNA tubes as alternative sources to study circulating microRNA expression profiling. Method Paired EDTA plasma, EDTA whole blood and PAXgene blood RNA tubes were obtained from 10 healthy adults (50% male). EDTA plasma samples were processed within 30 min. after sampling and immediately stored at -80°C. EDTA whole blood tubes and PAXgene tubes were kept at room temperature for 48 hours after sampling. Subsequently, the content of the EDTA whole blood samples was transferred to a 15 mL Falcon tube and stored at -80°C. PAXgene tubes were transferred to -20°C following the manufacturer’s protocol. Within 1 month of storage, all samples were thawed and miRNA was extracted using the Qiagen miRNeasy serum/plasma kit and subjected to RNA-sequencing (Oxford Genomics Centre). Based on the raw data, a count table was created using the online tool miRDeep* for the identification of both novel and known microRNAs. Subsequent downstream bio-informatic analyses approaches consisted of 1) unsupervised hierarchical clustering with principal component analysis (PCA); 2) calculation of differential miRNA expression using generalized linear models with differences considered significant if the false discovery rate-adjusted p-value was inferior to 10%. Results Initial assessment of the count table showed significant differences in the number of detected microRNAs. A median of 220 different microRNAs was detected in EDTA plasma samples versus 661 in PaxGene samples (p &amp;lt; 0.05) and 490 in EDTA whole blood samples (p &amp;lt; 0.05) (Figure 1A). We also found fewer novel miRNAs in EDTA plasma samples than in PAXgene samples (p &amp;lt; 0.001) and EDTA whole blood samples (p &amp;lt; 0.05). Low count microRNAs, defined as below 10 reads in more than 20% of the samples, were more abundant in Paxgene samples versus EDTA plasma samples (p = 0.0039), but this difference was not significant when comparing EDTA whole blood samples with EDTA plasma samples (Figure 1B). PCA analysis (Figure 1C) showed a clear separation of samples according to the blood collection method, strongly suggesting that the blood collection method predominantly determines the miRNA expression profile. Conclusion Bio-informatic analyses demonstrated different miRNA expression profiles according to three different blood collection methods, underpinning the importance of a standardized method for the collection of blood aimed at studying circulating miRNAs. As such, this study has important implications for the design of novel studies aiming to investigate circulating miRNAs.

P1092THE ENVIRONMENTAL IMPACT OF DISPOSABLES IN A NEW CONFIGURATION OF HEMODIALYSIS (HD) SYSTEM

Abstract Background and Aims A new configuration of Artis Dialysis System, namely Artis Physio Plus (AP+), has been recently released for chronic HD market. The AP+ Dialysis System features 3 stationary ultrafilters integrated in the hydraulic flow path and designed to retain bacteria and endotoxins, in order to provide fluid suitable for infusion to be used for on-line hemodiafiltration (HDF), hemofiltration (HF) or on-line bolus, priming and rinse-back also in HD. With this new fully internal filtration process, no external single-use filter is needed on the infusion lines, resulting in a simplified configuration of the extra-corporeal circuit (EC). HD has a considerable environmental impact because it uses large volumes of water, energy and medical supplies, and hence a significant amount of waste is produced. The aim of this study was to provide a comparison of the plastic waste amount between the previous Artis Dialysis System configuration (i.e., Artis Physio, AP) and the new one (AP+). Method The weight of the EC components (i.e., the blood tubing set, the related infusion lines for HD or HDF/HF and the dialyzer) after their discharge from dialysis machine was measured. In particular, the EC components for AP includes the Ultra Prime Line for HD (Fig. 1a) and Ultra HDF Line for HDF/HF in Pre or Post dilution (Fig. 2a). The EC components for AP+ includes the ULTRALINE HD for HD (Fig. 1b) and ULTRALINE HDF for HDF/HF in Pre or Post dilution (Fig. 2b). For simplicity, the same blood tubing set (ArtiSet PrePost) has been assumed for all treatment types. The dialyzers used were Revaclear 400 for HD and Polyflux 210H for HDF/HF. Other plastic waste, like concentrates and disinfectant cartridges, were not considered since their consumption is the same for AP and AP+. Results Table 1 shows the weight of the EC components before (i.e., components dry) and after dialysis (i.e., after patient rinse-back and automatic draining by AP/AP+). Table 2 reports an estimation of the total weekly amount of plastic waste for a single Artis Dialysis System performing 3 shifts a day with a mix of different dialysis treatments (i.e., 60% of HD, 30% of HDF Post, 10% of HDF/HF Pre). The results show a significant decrease of the disposable waste weight. Conclusion The new AP+ infusion lines, dealing with the ready to use infusion fluid delivered from the machine, are much simpler: this leads not only to a significant usability improvement, but also to a better shipment packing factor for the goods, a better storage in the dialysis unit, and a reduction of plastic waste. In particular, a weight decrease of 10.2% is achieved considering the EC items used on a single Artis Dialysis System during a week. Higher reduction of plastic waste will be achieved in clinics running higher number of HDF treatments. This contributes to have a more eco-friendly dialysis and to reduce the expenditures for the hospitals, both in terms of goods delivery and of waste disposal.

Combined Cell-free DNA and RNA Profiling of the Androgen Receptor: Clinical Utility of a Novel Multianalyte Liquid Biopsy Assay for Metastatic Prostate Cancer

BackgroundThe androgen receptor (AR) remains a critical driver in metastatic castration-resistant prostate cancer (mCRPC). Profiling AR aberrations in both circulating DNA and RNA may identify key predictive and/or prognostic biomarkers in the context of contemporary systemic therapy. ObjectiveTo profile AR aberrations in circulating nucleic acids and correlate with clinical outcomes. Design, setting, and participantsWe prospectively enrolled 67 mCRPC patients commencing AR pathway inhibitors (ARPIs; n = 41) or taxane chemotherapy (n = 26). Using a first-in-class next-generation sequencing-based assay, we performed integrated cell-free DNA (cfDNA) and cell-free RNA (cfRNA) profiling from a single 10 ml blood tube. Outcome measurements and statistical analysisKaplan-Meier survival estimates and multivariable Cox regression analyses were used to assess associations between clinical outcomes and the following AR aberrations: copy number variation, splice variants (AR-V7 and AR-V9) and somatic mutations. Results and limitationsCell-free DNA and cfRNA were successfully sequenced in 67 (100%) and 59 (88%) patients, respectively. Thirty-six (54%) patients had one or more AR aberrations. AR gain and cumulative number of AR aberrations were independently associated with clinical/radiographic progression-free survival (PFS; hazard ratio [HR] 3.2, p = 0.01 and HR 3.0 for 0 vs ≥2, p = 0.04) and overall survival (HR 2.8, p = 0.04 and HR 2.9 for 0 vs ≥2, p = 0.03). Notably, concurrent AR gain and AR splice variant expression (AR gain/AR-V+) was associated with shorter prostate-specific antigen PFS on both ARPIs (HR 6.7, p = 0.009) and chemotherapy (HR 3.9, p = 0.04). Importantly, key findings were validated in an independent cohort of mCRPC patients (n = 40), including shorter OS in AR gain/AR-V+ disease (HR 3.3, p = 0.02). Limitations include sample size and follow-up period. ConclusionsWe demonstrate the utility of a novel, multianalyte liquid biopsy assay capable of simultaneously detecting AR alterations in cfDNA and cfRNA. Concurrent profiling of cfDNA and cfRNA may provide vital insights into disease biology and resistance mechanisms in mCRPC. Patient summaryIn this study of men with advanced prostate cancer, DNA and RNA abnormalities in the androgen receptor detected in blood were associated with poor outcomes on available drug treatments. This information could be used to better guide treatment of advanced prostate cancer.

Analytical validation of digital cytometry (iSort) for leukocyte enumeration using stored blood.

3542 Background: Blood leukocyte enumeration is a cornerstone for clinical diagnosis and immune monitoring of diverse cancers and immunotherapies. Existing methods rely on intact/living cells and can thus be limiting due to handling time constraints and need for predefined antibody panels. While indirect cytometry methods including digital cytometry can overcome this limitation on archival specimens, their clinical performance has not been extensively characterized. We developed iSort, a novel transcriptome deconvolution method based on CIBERSORTx. Here, we comprehensively evaluated iSort and validated it against established diagnostic standards. Methods: We recruited 36 healthy adult blood donors and characterized their blood leukocyte profiles. We used several established clinical cytometry methods requiring intact cells in a CLIA laboratory including Complete Blood Count [CBC] and 6-color TBNK [TBNK]. We also immunophenotyped leukocytes by a research flow cytometry panel (FACS) and by mass cytometry (CyTOF). We then used these techniques as standards for validating leukocyte populations enumerated by iSort. iSort was performed on whole blood through deconvolution of 22 subsets from RNA-Seq. We assessed iSort’s analytical detection performance by spiking purified lymphocyte subsets into lymphodepleted human blood and by simulating blood mixtures using defined leukocyte mixtures within latin square designs. We assessed iSort concordance with an FDA approved IVD assay (TBNK) comparing distinct RNA-Seq library preparation chemistries. Results: iSort was highly correlated with TBNK/CBC across CD4 T, CD8 T, B cells, NK cells, monocytes, and neutrophils (r≥0.95). When comparing correlations to TBNK/CBC, we found no significant differences between iSort, CyTOF, and FACS, nor between RNA-Seq library chemistries. iSort demonstrated high linearity at low abundance levels (0.1 - 1%, r = 0.99, B-cells spiked into lymphodepleted blood samples after Rituximab) and at higher abundance levels (0.5 - 90%, r &gt; 0.99) across lymphoid and myeloid subsets. iSort also showed high reproducibility among triplicate blood tubes for each population (median CV = 11%). Conclusions: iSort digital cytometry achieves highly accurate and robust leukocyte enumeration for diverse hematopoietic subsets. Given its favorable performance against existing clinical standards that require intact/living cells, iSort is a promising approach for the development of immunotherapy biomarkers.

A comprehensive testing algorithm for the diagnosis of Fabry disease in males and females

PurposeSuccessful diagnosis of Fabry disease is often delayed or missed in patients, especially females, due to clinical heterogeneity and a lack of disease awareness. We present our experience testing for Fabry disease in high risk populations and discuss the relative sensitivities of α-galactosidase A (α-Gal A) enzyme activity in blood, plasma lyso-globotriaosylceramide (lyso-Gb3) biomarker, and GLA gene sequencing as diagnostic tests for Fabry disease in both males and females. MethodsPatients with a clinical suspicion of Fabry disease were evaluated with enzyme analysis, biomarker analysis, and GLA sequencing. All three assays were performed from a single tube of EDTA blood. α-Gal A activity was determined in dried blood spots using a fluorometric assay, plasma lyso-Gb3 by UPLC-MS/MS, and GLA analysis by Sanger sequencing. ResultsPeripheral blood samples were received from 94 males and 200 females, of which 29% of males and 22% of females had a positive family history of Fabry disease. A likely pathogenic or pathogenic variant was identified in 87 (30%) patients (50 males, 37 females), confirming a diagnosis of Fabry disease. Of the remaining patients, 178 (61%) were determined to be unaffected based on normal enzyme activity (males) or normal lyso-Gb3 and negative sequencing results (females). A VUS was identified in 29 (10%) patients. The positive and negative predictive value of plasma lyso-Gb3 was 100% and 97% in males and 100% and 99% in females, respectively. This compares with 84% and 100% in males, and 58% and 50% in females for α-Gal A activity testing, respectively. ConclusionsPlasma lyso-Gb3 has high sensitivity and specificity for Fabry disease in males and females, and provides supportive diagnostic information when gene sequencing results are negative or inconclusive. α-Gal A activity in dried blood spots (DBS) has high sensitivity, but lower specificity for Fabry disease in males, as not all males with low α-Gal A activities were confirmed to have Fabry disease. Therefore, reflexing to gene sequencing and plasma lyso-Gb3 is useful for disease confirmation in males. For females, we found that first tier testing consisting of GLA sequencing and plasma lyso-Gb3 analysis provided the greatest sensitivity and specificity. Enzyme testing has lower sensitivity in females and is therefore less useful as a first-tier test. Enzyme analysis in females may still be helpful as a second-tier test in cases where molecular testing and plasma lyso-Gb3 analysis are uninformative and in vitro enzyme activity is low. SummarySex-specific testing algorithms that prioritize tests with high specificity and sensitivity offer an effective means of identifying individuals with Fabry disease.

LO36: Reducing emergency department bloodwork and eliminating waste

Background: Patients presenting to the Emergency Department (ED) may be subjected to unnecessary bloodwork. This leads to excessive work for front-line nurses, physicians and laboratory staff, contributing to increased ED length of stay (LOS), patient discomfort, and health care costs. Aim Statement: By January 1, 2020, we will reduce the number of targeted blood tests (AST, GGT, aPTT and CK) by 40% in the Mount Sinai ED, as measured by the percent per 1000 ED visits of AST to ALT, GGT to ALT, aPTT to INR and CK to troponin. Measures &amp; Design: This was a prospective time series quality improvement study. Using the Model for Improvement, we engaged front-line ED staff, as well as stakeholders from Consultant, Laboratory and Information Services. Data was analyzed using run chart rules. Intervention: a) Removed rarely used tests from electronic nursing order sets b) Uncoupled order panels c) Developed six presentation-based medical directives with appropriate blood testing. d) Staff education Family of measures Outcomes: percent of targeted uncoupled test per 1000 ED visits for each of AST to ALT, GGT to ALT, aPTT to INR, and CK to troponin; Total number of blood tests ordered per 1000 ED visits Process: number of “separate and hold” tubes; number of blood tubes used in the ED; proportion of staff attending education Balancing: volume of blood drawn; LOS Evaluation/Results: Outcome: Estimated relative reduction in proportion of all uncoupled tests per 1000 ED visits by: • 33% AST/ALT • 52% GGT/ALT • 50% CK/troponin •18% aPTT/INR Total number of lab tests per 1000 ED visits decreased by 7.7% (5742 to 5331). Evidence of special cause variation on all outcomes. Process measures: 1. 100% reduction in weekly “Separate and Hold” tubes (56 to 0). 2. Monthly total of blood tubes used in the ED decreased by 2.8% (11620 to 11300) 3. Attendance pending. Balancing measures: Monthly average volume of blood drawn decreased by 1.4L(2%) from 50.4L to 49.0L; LOS pending Discussion/Impact: A multi-pronged intervention resulted in a decrease in blood testing in the ED. We achieved the sub-aim of reducing targeted blood tests and are on track to achieve the overall aim of total lab reduction in the ED by April 2020. Final interventions to be implemented in the coming months include changes to the ED paper record and replacement of the paper add-on order process with an electronic ordering tool. Complete data will be available by April 2020. This intervention is scalable and has the potential to reduce costs and preventable harm to patients.

Pre‐analytical factors affecting the establishment of a single tube assay for multiparameter liquid biopsy detection in melanoma patients

The combination of liquid biomarkers from a single blood tube can provide more comprehensive information on tumor development and progression in cancer patients compared to single analysis. Here, we evaluated whether a combined analysis of circulating tumor cells (CTCs), circulating tumor DNA (ctDNA), and circulating cell‐free microRNA (miRNA) in total plasma and extracellular vesicles (EV) from the same blood sample is feasible and how the results are influenced by the choice of different blood tubes. Peripheral blood from 20 stage IV melanoma patients and five healthy donors (HD) was collected in EDTA, Streck, and Transfix tubes. Peripheral blood mononuclear cell fraction was used for CTC analysis, whereas plasma and EV fractions were used for ctDNA mutation and miRNA analysis. Mutations in cell‐free circulating DNA were detected in 67% of patients, with no significant difference between the tubes. CTC was detected in only EDTA blood and only in 15% of patients. miRNA NGS (next‐generation sequencing) results were highly influenced by the collection tubes and could only be performed from EDTA and Streck tubes due to hemolysis in Transfix tubes. No overlap of significantly differentially expressed miRNA (patients versus HD) could be found between the tubes in total plasma, whereas eight miRNA were commonly differentially regulated in the EV fraction. In summary, high‐quality CTCs, ctDNA, and miRNA data from a single blood tube can be obtained. However, the choice of blood collection tubes is a critical pre‐analytical variable.