Patients
This study was approved by the Institutional Review Board of Columbia University Irving Medical Center (CUIMC) (Protocol Number AAAT0680). Data were obtained for patients who were either admitted to the hospital or seen in the Emergency Department from April 14, 2020 through May 31, 2020 (i.e., before the identification of and routine testing for novel variants in the USA), and were evaluated for SARS-CoV-2 by RT-PCR, serology. As part of routine care, hemostasis was evaluated on STAR Evolution and STAR Max analyzers (Diagnostica Stago, Parsippany, NJ), hematology testing by Sysmex XN900 (Lincolnshire, IL), and chemistry testing by Roche Cobas c502 (Indianapolis, IN). Laboratory values, including antithrombin (AT), prothrombin time (PT)/international normalized ratio (INR), activated partial thromboplastin time aPTT, fibrinogen, d-dimer, white blood cell count (WBC), absolute neutrophil count (ANC), absolute lymphocyte count (ALC), absolute monocyte count (AMC), hemoglobin, red blood cell count (RBC), RBC distribution width (RDW), reticulocyte count, platelet count, IL-6, lactate dehydrogenase (LDH), lactic acid, procalcitonin, troponin, blood urea nitrogen (BUN), creatinine, glucose, total-, direct-, and indirect bilirubin, aspartate amino transferase (AST), alanine amino transferase (ALT), albumin, total protein, ferritin, C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), creatine kinase (CK), triglycerides, and blood type, were collected. Laboratory data were obtained from the Clinical Data Warehouse at CUIMC after approval from the Tripartite Request Assessment Committee. Clinical and demographic data, including name, medical record number (MRN), sex, date of birth, age, race, ethnicity, weight, body mass index, comorbidities (hypertension, diabetes mellitus, coronary artery disease, renal disease, hyperlipidemia, liver disease, lung disease), intubation/ventilator requirement, continuous veno-venous hemofiltration (CVVH) requirement, radiographically-confirmed thrombotic complications (deep vein thrombosis, pulmonary embolism, stroke), clotting of CVVH, hospitalization course (admission date, date of Emergency Department presentation, discharge date), mortality and date of death) were collected manually by reviewing the electronic medical record. Data were collected retrospectively for patients treated at two New York-Presbyterian Hospital campuses (CUIMC and The Allen hospital). Residual platelet poor plasma samples were collected for subsequent analyses.
Sample processing and metabolite extraction: Plasma samples were extracted via a modified Folch method (chloroform/methanol/water 8:4:3), which completely inactivates other coronaviruses, such as MERS-CoV.74 Briefly, 20 µL of plasma were diluted in 130 µl of LC-MS grade water, 600 µl of ice-cold chloroform/methanol (2:1) was added, and the samples vortexed for 10 seconds. Samples were then incubated at 4ºC for 5 minutes, quickly vortexed (5 seconds), and centrifuged at 14,000 x g for 10 minutes at 4ºC. The top (i.e., aqueous) phase was transferred to a new tube for metabolomics analysis.
Ultra-High-Pressure Liquid Chromatography-Mass Spectrometry metabolomics and lipidomics
Analyses were performed using a Vanquish UHPLC coupled online to a Q Exactive mass spectrometer (Thermo Fisher, Bremen, Germany). Samples were analyzed using a 5 and 17 min gradient as described.53,54,75 Solvents were supplemented with 0.1% formic acid for positive mode runs and 1 mM ammonium acetate for negative mode runs. MS acquisition, data analysis and elaboration was performed as described. 53,54,75
Metabolomics: UHPLC-MS metabolomics analyses were performed as described in method53,54,75 and application papers,1,76 using a Vanquish UHPLC system coupled online to a high-resolution Q Exactive mass spectrometer (Thermo Fisher, Bremen, Germany). Samples were resolved over a Kinetex C18 column (2.1x150 mm, 1.7 µm; Phenomenex, Torrance, CA, USA) at 45°C. A volume of 10 ul of sample extracts was injected into the UHPLC-MS. Each sample was injected and run four times with two different chromatographic and MS conditions as follows: 1) using a 5 minute gradient at 450 µL/minute from 5–95% B (A: water/0.1% formic acid; B:acetonitrile/0.1% formic acid) and the MS was operated in positive mode and 2) using a 5 minute gradient at 450 µL/minute from 5–95% B (A: 5% acetonitrile, 95%water/1 mM ammonium acetate; B:95%acetonitrile/5% water, 1 mM ammonium acetate) and the MS was operated in negative ion mode. The UHPLC system was coupled online with a Q Exactive (Thermo, San Jose, CA, USA) scanning in Full MS mode at 70,000 resolution in the 60–900 m/z range, 4 kV spray voltage, 15 sheath gas and 5 auxiliary gas, operated in negative or positive ion mode (separate runs). These chromatographic and MS conditions were applied for both relative and targeted quantitative metabolomics measurements, with the differences that for the latter targeted quantitative post hoc analyses were performed on the basis of the stable isotope-labeled internal standards used as a reference quantitative measurement, as detailed below.
Lipidomics: Samples were resolved as described,4–6, 45 over an ACQUITY HSS T3 column (2.1 x 150 mm, 1.8 µm particle size (Waters, MA, USA) using an aqueous phase (A) of 25% acetonitrile and 5 mM ammonium acetate and a mobile phase (B) of 50% isopropanol, 45% acetonitrile and 5 mM ammonium acetate. Samples were eluted from the column using either the solvent gradient: 0–1 min 25% B and 0.3 mL/min; 1–2 min 25–50% B and 0.3 mL/min, 2–8 min 50–90% B and 0.3 mL/min, 8–10 min 90–99% B and 0.3 mL/min, 10–14 min hold at 99% B and 0.3 mL/min, 14-14.1 min 99 − 25% B 1and 0.3 mL/min, 14.1–16.9 min hold at 25% B and 0.4 mL/min, 16.9–17 min hold at 25% B and resume flow of 0.3 mL/min. isocratic elution of 5% B flowed at 250 µl/min and 25ºC or a gradient from 0–5% B over 0.5 min; 5–95% B over 0.6 min, hold at 95% B for 1.65 min; 95 − 5% B over 0.25 min; hold at 5% B for 2 min, flowed at 450 µl/min and 35ºC53. The Q Exactive mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA) was operated independently in positive or negative ion mode, scanning in Full MS mode (2 µscans) from 150 to 1500 m/z at 70,000 resolution, with 4 kV spray voltage, 45 shealth gas, 15 auxiliary gas.
MS2 analyses for untargeted metabolomics For discovery mode untargeted metabolomics, dd-MS2 was performed at 17,500 resolution, AGC target = 1e5, maximum IT = 50 ms, and stepped NCE of 25, 35 for positive mode, and 20, 24, and 28 for negative mode, as described in Stefeanoni et al. Haematologica 2020,77 and applied to similar samples (i.e., stored RBCs) in D’Alessandro et al. Haematologica 202057.
Quality control and data processing
Calibration was performed prior to analysis using the Pierce™ Positive and Negative Ion Calibration Solutions (Thermo Fisher Scientific). Acquired data was then converted from .raw to .mzXML file format using Mass Matrix (Cleveland, OH, USA). Samples were analyzed in randomized order with a technical mixture (generated by mixing 5 ul of all samples tested in this study) injected every 10 runs to qualify instrument performance. This technical mixture was also injected three times per polarity mode and analyzed with the parameters above, except CID fragmentation was included for unknown compound identification (10 ppm error for both positive and negative ion mode searches for intact mass, 50 ppm error tolerance for fragments in MS2 analyses – further details about the database searched below).
Metabolite assignment and relative quantitation
Metabolite assignments, isotopologue distributions, and correction for expected natural abundances of deuterium, 13C, and 15N isotopes were performed using MAVEN (Princeton, NJ, USA),78 against an in house library of deuterated lipid standards (SPLASH® LIPIDOMIX® Mass Spec Standard, Avanti Lipids) and in house libraries of 3,000 unlabeled (MSMLS, IROATech, Bolton, MA, USA; IroaTech ; product A2574 by ApexBio; standard compounds for central carbon and nitrogen pathways from SIGMA Aldrich, St Louis, MO, USA) and labeled standards (see below for the latter). Discovery mode analysis was performed with standard workflows using Compound Discoverer 2.1 SP1 (Thermo Fisher Scientific, San Jose, CA). From these analyses, metabolite IDs or unique chemical formulae were determined from high-resolution accurate intact mass, isotopic patterns, identification of eventual adducts (e.g., Na + or K+, etc.) and MS2 fragmentation spectra against the KEGG pathway, HMDB, ChEBI, and ChEMBL databases. Additional untargeted lipidomics analyses were performed with the software LipidSearch (Thermo Fisher, Bremen, Germany).
Simultaneous thrombin and plasmin generation assay (STPGA)
Simultaneous evaluation of thrombin and plasmin generation (TG and PG, respectively) was performed as described previously79. Briefly, plasma samples were mixed with either thrombin specific substrate, Z-Gly-Gly-Arg-AMC (Bachem, Bubendorf, Switzerland) or plasmin specific substrate, Boc-Glu-Lys-Lys-AMC (Bubendorf, Switzerland) and 16 nM of thrombomodulin (PeproTech, Rocky Hill, NJ, USA). The reaction was initiated by adding an activator solution that yielded a final concentration of 1 pM tissue factor (Diagnostica Stago, Parsippany, NJ, USA), 0.7 µg/mL of tissue plasminogen activator (Sigma-Aldrich, St. Louis, MO, USA) and 16 mM CaCl2. Sample wells supplemented with buffer (150 mM NaCl and 20 mM HEPES) and AMC fluorophore instead of activator solution were used for background and calibrator measurements respectively. Calculation of thrombin and plasmin concentration was performed as described previously 80.
VWF, FVIII and ADAMTS13 activity and antigen quantitation: The antigen and activity measurement of VWF and ADAMTS13 was performed by using commercial ELISA kits. VWF antigen and collagen binding activity levels were measured by using Human Von Willebrand Factor ELISA Kit (ab168548, Abcam, Cambridge, UK) and TECHNOZYM® vWF:CBA ELISA Kit (5450301, Technoclone, Vienna, Austria) respectively. ADAMTS13 antigen and activity levels were measured by using Human ADAMTS13 ELISA Kit (ab234559, Abcam) and TECHNOZYM® ADAMTS13 Activity ELISA (5450701, Technoclone) respectively. FVIII antigen levels were measured by using Human Factor VIII total antigen assay ELISA kit (HFVIIIKT-TOT, Molecular Innovations, Novi, MI, USA). All assays were performed following manufacturer’s recommendations with additional dilution of plasma samples as required.
Statistical Analysis
Graphs and statistical analyses (either t-test or repeated measures ANOVA) were prepared with GraphPad Prism 5.0 (GraphPad Software, Inc, La Jolla, CA), GENE E (Broad Institute, Cambridge, MA, USA), and MetaboAnalyst 4.0.81 In MetaboAnalyst, relative quant data (but not for abs quant), raw values for integrated peak areas for each metabolite were normalized on a pool of day 0 controls and auto-scaled for each species independently prior to margining all the data for multivariate analysis. Analyses through MetaboAnalyst included principal component analysis, partial least square discriminant analysis, hierarchical clustering analyses (including time-series repeated measures and two-way ANOVA analyses), calculation of receiver operating characteristic (ROC) curves, correlation analyses (Spearman) and machine learning analyses (random forest, support-vector machine – SVM).
Disclosure of Conflict of Interest
Though unrelated to the contents of this manuscript, the authors declare that AD, and TN are founders of Omix Technologies Inc and Altis Biosciences LLC. AD and SLS are consultants for Hemanext Inc. SLS is also a consultant for Tioma, Inc. and TCIP, Inc., and the Executive Director of the Worldwide Initiative for Rh Disease Eradication (WIRhE). AD is a consultant for FORMA LLC. All the other authors disclose that no conflict of interest exist.