LitCovid-sentences  

PMC:7402624 / 61473-75599 JSONTXT 10 Projects

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Id Subject Object Predicate Lexical cue
T355 0-21 Sentence denotes Materials and methods
T356 23-71 Sentence denotes Patients, subjects, and clinical data collection
T357 72-258 Sentence denotes Patients admitted to the Hospital of the University of Pennsylvania with a positive SARS-CoV2 PCR test were screened and approached for informed consent within 3 days of hospitalization.
T358 259-362 Sentence denotes Healthy donors (HD) were adults with no prior diagnosis of or recent symptoms consistent with COVID-19.
T359 363-453 Sentence denotes Normal reference ranges for HDs were the UPenn clinical laboratory values shaded in green.
T360 454-624 Sentence denotes Recovered COVID-19 subjects (RD) were adults with a prior positive COVID-19 PCR test by self-report who met the definition of recovery by the Centers for Disease Control.
T361 625-789 Sentence denotes HD and RD were recruited initially by word of mouth and subsequently through a centralized University of Pennsylvania resource website for COVID-19-related studies.
T362 790-839 Sentence denotes Peripheral blood was collected from all subjects.
T363 840-957 Sentence denotes For inpatients, clinical data were abstracted from the electronic medical record into standardized case report forms.
T364 958-1123 Sentence denotes ARDS was categorized in accordance with the Berlin definition reflecting each subject’s worst oxygenation level and with physician adjudication of chest radiographs.
T365 1124-1303 Sentence denotes APACHE III scoring was based on data collected in the first 24 hours of ICU admission or the first 24 hours of hospital admission for subjects admitted to general inpatient units.
T366 1304-1396 Sentence denotes Clinical laboratory data were abstracted from the date closest to research blood collection.
T367 1397-1441 Sentence denotes HD and RD completed a survey about symptoms.
T368 1442-1551 Sentence denotes After enrollment, the clinical team determined three patients to be COVID-negative and/or PCR false positive.
T369 1552-1606 Sentence denotes Two of these patients were classified as Immunotype 3.
T370 1607-1690 Sentence denotes In keeping with inclusion criteria, these subjects were maintained in the analysis.
T371 1691-1809 Sentence denotes The statistical significance reported in Fig. 6K did not change when analysis was repeated without the three patients.
T372 1810-1983 Sentence denotes All participants or their surrogates provided informed consent in accordance with protocols approved by the regional ethical research boards and the Declaration of Helsinki.
T373 1985-2002 Sentence denotes Sample processing
T374 2003-2077 Sentence denotes Peripheral blood was collected into sodium heparin tubes (BD, Cat#367874).
T375 2078-2145 Sentence denotes Tubes were spun (15 min, 3000 rpm, RT), plasma removed, and banked.
T376 2146-2336 Sentence denotes Remaining whole blood was diluted 1:1 with 1% RPMI (table S7) and layered into a SEPMATE tube (STEMCELL Technologies, Cat#85450) pre-loaded with lymphoprep (Alere Technologies, Cat#1114547).
T377 2337-2525 Sentence denotes SEPMATE tubes were spun (10 min, 1200xg, RT) and the PBMC layer collected, washed with 1% RPMI (10 min, 1600 rpm, RT) and treated with ACK lysis buffer (5 min, ThermoFisher, Cat#A1049201).
T378 2526-2605 Sentence denotes Samples were filtered with a 70 μm filter, counted, and aliquoted for staining.
T379 2607-2635 Sentence denotes Antibody panels and staining
T380 2636-2713 Sentence denotes Approximately 1-5×106 freshly isolated PBMCs were used per patient per stain.
T381 2714-2794 Sentence denotes See table S7 for buffer information and table S8 for antibody panel information.
T382 2795-2916 Sentence denotes PBMCs were stained with live/dead mix (100 μl, 10 min, RT), washed with FACS buffer, and spun down (1500 rpm, 5 min, RT).
T383 2917-3031 Sentence denotes PBMCs were incubated with 100 μl of Fc block (RT, 10 min) before a second wash (FACS buffer, 1500 rpm, 5 min, RT).
T384 3032-3133 Sentence denotes Pellet was resuspended in 25 μl of chemokine receptor staining mix, and incubated at 37°C for 20 min.
T385 3134-3270 Sentence denotes Following incubation, 25 μl of surface receptor staining mix was directly added and the PBMCs were incubated at RT for a further 45 min.
T386 3271-3444 Sentence denotes PBMCs were washed (FACS buffer, 1500 rpm, 5 min, RT) and stained with 50 μl of secondary antibody mix for 20 min at RT, then washed again (FACS buffer, 1500 rpm, 5 min, RT).
T387 3445-3586 Sentence denotes Samples were fixed and permeabilized by incubating in 100 μl of Fix/Perm buffer (RT, 30 min) and washed in Perm Buffer (1800 rpm, 5 min, RT).
T388 3587-3654 Sentence denotes PBMCs were stained with 50μl of intracellular mix overnight at 4°C.
T389 3655-3770 Sentence denotes The following morning, samples were washed (Perm Buffer, 1800 rpm, 5 min, RT) and further fixed in 50 μl of 4% PFA.
T390 3771-3884 Sentence denotes Prior to acquisition, samples were diluted to 1% PFA and 10,000 counting beads added per sample (BD, Cat#335925).
T391 3885-3919 Sentence denotes Live/dead mix was prepared in PBS.
T392 3920-4058 Sentence denotes For the surface receptor and chemokine staining mix, antibodies were diluted in FACS buffer with 50% BD Brilliant Buffer (BD, Cat#566349).
T393 4059-4104 Sentence denotes Intracellular mix was diluted in Perm Buffer.
T394 4106-4120 Sentence denotes Flow cytometry
T395 4121-4176 Sentence denotes Samples were acquired on a 5 laser BD FACS Symphony A5.
T396 4177-4284 Sentence denotes Standardized SPHERO rainbow beads (Spherotech, Cat#RFP-30-5A) were used to track and adjust PMTs over time.
T397 4285-4360 Sentence denotes UltraComp eBeads (ThermoFisher, Cat#01-2222-42) were used for compensation.
T398 4361-4415 Sentence denotes Up to 2 × 106 live PBMC were acquired per each sample.
T399 4417-4424 Sentence denotes Luminex
T400 4425-4521 Sentence denotes PBMCs from patients were thawed and rested overnight at 37°C in complete RPMI (cRPMI, table S7).
T401 4522-4636 Sentence denotes 96-well flat bottom plates were coated with 1 μg/mL of anti-CD3 (UCHT1, #BE0231, BioXell) in PBS at 4°C overnight.
T402 4637-4722 Sentence denotes The next day, cells were collected and plated at 1 × 105/well in 100 μl in duplicate.
T403 4723-4836 Sentence denotes 2 μg/mL of anti-human CD28/CD49d was added to the wells containing plate-bound anti-CD3 (Clone L293, 347690, BD).
T404 4837-4967 Sentence denotes PBMCs were stimulated or left unstimulated for 16 hours, spun down (1200 rpm, 10 min) and 85 μL/well of supernatant was collected.
T405 4968-5090 Sentence denotes Plasma from matched subjects was thawed on ice, spun (3000 rpm, 1 min) to remove debris, and 85 μl collected in duplicate.
T406 5091-5238 Sentence denotes Luminex assay was run according to manufacturer’s instructions, using a custom human cytokine 31-plex panel (EMD Millipore Corporation, SPRCUS707).
T407 5239-5517 Sentence denotes The panel included: EGF, FGF-2, Eotaxin, sIL-2Ra, G-CSF, GM-CSF, IFN-α2, IFN-γ, IL-10, IL-12P40, IL-12P70, IL-13, IL-15, IL-17A, IL-1Ra, HGF, IL-1β, CXCL9/MIG, IL-2, IL-4, IL-5, IL-6, IL-7, CXCL8/IL-8, CXCL10/IP-10, CCL2/MCP-1, CCL3/MIP-1α, CCL4/MIP-1β, RANTES, TNF-α, and VEGF.
T408 5518-5611 Sentence denotes Assay plates were measured using a Luminex FlexMAP 3D instrument (Thermofisher, Cat#APX1342).
T409 5612-5705 Sentence denotes Data acquisition and analysis were done using xPONENT software www.luminexcorp.com/xponent/).
T410 5706-5764 Sentence denotes Data quality was examined based on the following criteria:
T411 5765-5879 Sentence denotes The standard curve for each analyte has a 5P R2 value > 0.95 with or without minor fitting using xPONENT software.
T412 5880-6071 Sentence denotes To pass assay technical quality control, the results for two controls in the kit needed to be within the 95% of CI (confidence interval) provided by the vendor for >25 of the tested analytes.
T413 6072-6147 Sentence denotes No further tests were done on samples with results out of range low (<OOR).
T414 6148-6322 Sentence denotes Samples with results that were out of range high (>OOR) or greater than the standard curve maximum value (SC max) were not tested at higher dilutions without further request.
T415 6324-6370 Sentence denotes Intracellular stain after CD3/CD28 stimulation
T416 6371-6484 Sentence denotes 96-well flat bottom plates were coated with 1μg/mL of anti-CD3 (UCHT1, #BE0231, BioXell) in PBS at 4°C overnight.
T417 6485-6595 Sentence denotes The next day, cells were collected and plated at 1 × 105/well in 100 μl with 1/1000 of GolgiPlug (BD #555029).
T418 6596-6708 Sentence denotes 2μg/mL of anti-human CD28/CD49d was added to the wells containing plate-bound anti-CD3 (Clone L293, 347690, BD).
T419 6709-6853 Sentence denotes GolgiPlug-treated PBMCs were stimulated or left unstimulated for 16 hours, spun down (1200 rpm, 10 min) and were stained for intracellular IFNɣ.
T420 6855-6903 Sentence denotes Longitudinal analysis D0-D7 and patient grouping
T421 6904-7191 Sentence denotes To identify subjects where the frequency of specific immune cell populations increased, decreased or stayed stable over time (day 0 to day 7), where data was available we used a previously published dataset to establish a standard range of fold change over time in a healthy cohort (44).
T422 7192-7384 Sentence denotes A fold change greater than the mean fold change ± 2 standard deviations was considered an increase, less than this range was considered a decrease, and within this range was considered stable.
T423 7385-7499 Sentence denotes Where this data was not available, a fold change from day 0 to day 7 of between 0.5 and 1.5 was considered stable.
T424 7500-7579 Sentence denotes A fold change <0.5 was considered decreased, and >1.5 was considered increased.
T425 7580-7830 Sentence denotes In order to eliminate redundant tests and maximize statistical power, the pairwise statistical tests shown in Fig. 5G were performed using fold-change as a continuous metric, irrespective of the discrete up/stable/down classification described above.
T426 7831-8063 Sentence denotes Similarly, in fig. S9G, pairwise association tests between changes in UMAP Component coordinates and clinical data were performed using each difference value as a continuous metric, irrespective of the up/stable/down classification.
T427 8065-8108 Sentence denotes Correlation plots and heatmap visualization
T428 8109-8223 Sentence denotes Pairwise correlations between variables were calculated and visualized as a correlogram using R function corrplot.
T429 8224-8412 Sentence denotes Spearman's Rank Correlation coefficient (ρ) was indicated by square size and heat scale; significance indicated by: *p < 0.05, **p < 0.01, and ***p < 0.001; black box indicates FDR < 0.05.
T430 8413-8508 Sentence denotes Heatmaps were created to visualize variable values using R function pheatmap or complexheatmap.
T431 8510-8520 Sentence denotes Statistics
T432 8521-8694 Sentence denotes Due to the heterogeneity of clinical and flow cytometric data, non-parametric tests of association were preferentially used throughout this study unless otherwise specified.
T433 8695-8977 Sentence denotes Correlation coefficients between ordered features (including discrete ordinal, continuous scale, or a mixture of the two) were quantified by the Spearman rank correlation coefficient and significance was assessed by the corresponding non-parametric methods (null hypothesis: ρ = 0).
T434 8978-9178 Sentence denotes Tests of association between mixed continuous versus non-ordered categorical variables were performed by unpaired Wilcoxon test (for n = 2 categories) or by Kruskal-Wallis test (for n > 2 categories).
T435 9179-9255 Sentence denotes Association between categorical variables was assessed by Fisher-exact test.
T436 9256-9457 Sentence denotes For association testing illustrated in heatmaps, categorical variables with more than 2 categories (e.g., ABO blood type) were transformed into binary dummy variables for each category versus the rest.
T437 9458-9572 Sentence denotes All tests were performed two-sided, using a nominal significance threshold of P < 0.05 unless otherwise specified.
T438 9573-9765 Sentence denotes When appropriate to adjust for multiple hypothesis testing, false discovery rate (FDR) correction was performed using the Benjamini-Hochberg procedure at the FDR < 0.05 significance threshold.
T439 9766-10055 Sentence denotes Joint statistical modeling to adjust for confounding of demographic factors (age, sex, and race) when testing for association of UMAP Components 1 and 2 with the NIH Ordinal Severity Scale was performed using ordinal logistic regression provided by the polr function of the R package MASS.
T440 10056-10138 Sentence denotes Statistical analysis of flow cytometry data was performed using R package rstatix.
T441 10139-10230 Sentence denotes Other details, if any, for each experiment are provided within the relevant figure legends.
T442 10232-10285 Sentence denotes High dimensional data analysis of flow cytometry data
T443 10286-10363 Sentence denotes viSNE and FlowSOM analysis were performed on Cytobank (https://cytobank.org).
T444 10364-10593 Sentence denotes B cells, non-naïve CD4 T cells, and non-naïve CD8 T cells were analyzed separately. viSNE analysis was performed using equal sampling of 1000 cells from each FCS file, with 5000 iterations, a perplexity of 30, and a theta of 0.5.
T445 10594-10793 Sentence denotes For B cells, the following markers were used to generate the viSNE maps: CD45RA, IgD, CXCR5, CD138, Eomes, TCF-1, CD38, CD95, CCR7, CD21, KI67, CD27, CX3CR1, CD39, T-bet, HLA-DR, CD16, CD19 and CD20.
T446 10794-10970 Sentence denotes For non-naïve CD4 and CD8 T cells, the following markers were used: CD45RA, PD1, CXCR5, TCF-1, CD38, CD95, Eomes, CCR7, KI67, CD16, CD27, CX3CR1, CD39, CD20, T-bet, and HLA-DR.
T447 10971-11044 Sentence denotes Resulting viSNE maps were fed into the FlowSOM clustering algorithm (59).
T448 11045-11170 Sentence denotes For each cell subset, a new self-organizing map (SOM) was generated using hierarchical consensus clustering on the tSNE axes.
T449 11171-11277 Sentence denotes For each SOM, 225 clusters and 10 or 15 metaclusters were identified for B cells and T cells respectively.
T450 11278-11538 Sentence denotes To group individuals based on B cell landscape, pairwise Earth Mover’s Distance (EMD) value was calculated on the B cell tSNE axes for all COVID-19 day 0 patients, healthy donors, and recovered donors using the emdist package in R as previously described (60).
T451 11539-11616 Sentence denotes Resulting scores were hierarchically clustered using the hclust package in R.
T452 11618-11634 Sentence denotes Batch correction
T453 11635-11723 Sentence denotes During the sample acquisition period, the flow panel was changed to remove one antibody.
T454 11724-11855 Sentence denotes Batch correction was performed for samples acquired before and after this change to remove potential bias from downstream analysis.
T455 11856-12130 Sentence denotes Because the primary flow features were expressed as a fraction of the parent population (falling in the 0-to-1 interval) a variance stabilizing transform (logit) was first applied to each data value, prior to re-centering the second panel to have the same mean as the first.
T456 12131-12242 Sentence denotes After mean-centering, data were transformed back to the original fraction of parent scale by inverse transform.
T457 12243-12355 Sentence denotes This procedure was applied separately to all 553 flow features annotated in the main text and supplemental data.
T458 12356-12480 Sentence denotes Notably, this procedure avoids any batch-corrected feature values artificially falling outside of the original 0 to 1 range.
T459 12481-12634 Sentence denotes Following batch correction, neither UMAP Component 1 nor Component 2 had a statistically significant difference between panels by unpaired Wilcoxon test.
T460 12636-12717 Sentence denotes Visualizing variation of flow cytometric features across the UMAP embedding space
T461 12718-12861 Sentence denotes A feature-weighted kernel density was computed across all COVID-19 patients, and was displayed as a contour plot (Fig. 6G and fig. S8, A to D).
T462 12862-13248 Sentence denotes Whereas traditional kernel density methods apply the same base kernel function to every point to visualize point density, here the base kernel function centered at each individual COVID-19 patient sample was instead weighted (multiplied) by the Z-transform (mean-centered and standard deviation-scaled) of the log-transformed input feature prior to computing the overall kernel density.
T463 13249-13461 Sentence denotes This weighting procedure facilitated visualization of the overall feature gradients (going from relatively low-to-high expression) across UMAP coordinates independent of the different range of each input feature.
T464 13462-13713 Sentence denotes A radially symmetric two-dimensional Gaussian was used as the base kernel function with a variance parameter equal to one-half, which was tuned to be sufficiently broad in order to smooth out local discontinuities and best visualize feature gradients.
T465 13715-13741 Sentence denotes Definition of immunotype 3
T466 13742-14126 Sentence denotes To define COVID-19 patients with low or absent immune responses, classified as immunotype 3, the intersection of the bottom 50% of 5 different flow parameters was used: PB as % of B cells, KI67+ as % of non-naïve CD4 T cells, KI67+ as % of non-naïve CD8 T cells, HLA-DR+CD38+ as % of non-naïve CD4 T cells, HLA-DR+CD38+ as % of non-naïve CD8 T cells—graphically displayed in fig. S10.