TITLE: Thromboelastometry measurements in severe and non-severe COVID-19 patients 
AUTHORS: Rodrigo  Aires, Alexandre A. de S. M. Soares, Ana Paula M. Gomides, Andre M. Nicola, Andrea Teixeira-Carvalho, Dayde Lane M. da Silva, Eliana T. de Góis, Flávia D. Xavier, Francielle P. Martins, Gabriela P. J. Santos, Heide Luise Schulte, Isabelle S. Luz, Laila S. Espíndola, Laurence R. do Amaral, Liza F. Felicori, Luciana A. Naves, Maíra R. M. de Carvalho, Matheus de S. Gomes, Otávio T. Nóbrega, Patrícia Albuquerque, Wagner Fontes, Ciro M. Gomes, Patrícia S. Kurizky, Cleandro P. Albuquerque, Olindo A. Martins-Filho, Licia Maria H. da Mota

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>In patients with severe forms of COVID-19, thromboelastometry has been reported to display a hypercoagulant pattern. However, an algorithm to differentiate severe COVID-19 patients from nonsevere patients and healthy controls based on thromboelastometry parameters has not been developed. Forty-one patients over 18 years of age with positive qRT-PCR for SARS-CoV-2 were classified according to the severity of the disease: nonsevere (NS, n=20) or severe (S, n=21). A healthy control (HC, n=9) group was also examined. Blood samples from all participants were tested by</span><a href="" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;"> extrinsic (EXTEM), intrinsic (INTEM), non-activated (NATEM) and functional assessment of fibrinogen (FIBTEM) assays of thromboelastometry. </span></a><span>The thrombodynamic potential index (TPI) was also calculated. Severe COVID-19 patients exhibited a thromboelastometry profile with clear hypercoagulability, which was significantly different from the NS and HC groups. Nonsevere COVID-19 cases showed a trend to thrombotic pole. The NATEM test suggested that nonsevere and severe COVID-19 patients presented endogenous coagulation activation (reduced clotting time and clot formation time). TPI data were significantly different between the NS and S groups. The maximum clot firmness profile obtained by FIBTEM showed moderate/elevated accuracy to differentiate severe patients from NS and HC. A decision tree algorithm based on the FIBTEM-MCF profile was proposed to differentiate S from HC and NS. Thromboelastometric parameters are a useful tool to differentiate the coagulation profile of nonsevere and severe COVID-19 patients for therapeutic intervention purposes.</span></div></div>

[STEPS]
?. [Quality control]
1)    Quality control must be performed weekly, using standardized system controls ROTROL N and ROTROL P.
?. [Quality control]
2)    Daily maintenance: outer surface, cup holder, pipette and filter
?. [Quality control]
3)    Weekly maintenance: replace pipette filter
?. [Quality control]
4)    Quarterly maintenance: device temperature (between 36oC and 38oC) and CCD chip (amplitude, centre and variance)
?. [Instrument preparation]
1)    All reagents must be taken from refrigerator prior to use
?. [Instrument preparation]
2)    The tests must be started only after target temperature are achieved
?. [Instrument preparation]
3)    Take the cup with the pin in it from the storage box
?. [Instrument preparation]
4)    Push the pin in the cup onto the axis chosen for the measurement.
?. [Instrument preparation]
5)    Place the cup with its opening facing upwards into the appropriate preheated cupholder.
?. [Instrument preparation]
6)    Place the cup holder onto the temperature-controlled work area again.
?. [Instrument preparation]
7)    Push and fix the cup in the cup holder using the MC Rod.
?. [EXTEM exam]
1)    Reagents: r ex-tem® and star-tem®
?. [EXTEM exam]
2)    Pipetting steps are dysplayed on the screen
?. [INTEM exam]
1)    Reagents: in-tem® and star-tem®
?. [INTEM exam]
2)    Pipetting steps are dysplayed on the screen
?. [FIBTEM exam]
1)    Reagents: r ex-tem, star-tem® and fib-tem®
?. [FIBTEM exam]
2)    Pipetting steps are dysplayed on the screen
?. [NATEM exam]
1)    Reagents: star-tem®
?. [NATEM exam]
2)    Pipetting steps are dysplayed on the screen
?. [Inclusion criteria ]
COVID-19 patients treated during the pandemic in midwestern Brazil (Hospital Regional da Asa Norte and Hospital Universitário de Brasília, Brasilia, DF, Brazil (between August 1st and September 30th, 2020) were included. Confirmatory diagnosis was based on positive SARS-CoV-2 infection results in oropharyngeal swabs by quantitative real-time polymerase chain reaction (qRT-PCR).
?. [Exclusion criteria]
under 18 years old.pregnancy.thrombophilia or previous thromboembolic events.previous use of anticoagulants.previous use of antiplatelet drugssurgical procedures in the last 4 weeks.hereditary coagulopathies andpsychiatric diseases that impaired the understanding of the informed consent form.
?. [Blood sampling]
Citrated whole blood is collected of each research participant (enough to complete standard coagulation tests tubes:4 ml) and kept at room temperature.  When not possible to make measurements immediately, the samples must be preheated for 5–10 min before measurement in the sample preheating station of the ROTEM® delta.
?. [Patient inclusion]
Patients with confirmed COVID-19 infection by SARS-CoV-2 PCR were offered participation in the study.Participation in the study was not necessary for receving the proper disease treatment.Those who decided to participate were informed about the study, the possibility to abandon it at any time and received all information for a proper Informed Consent, which was sent by electronic form.
?. [Patient classification]
Patients were classified according to diseae severity as follows:Severe: patients needing hospitalization due to at least one of the following: dyspnea (respiratory rate >30 respiratory incursions per minute), SpO2 Non-severe: patients not necessitating hospitalization
?. [Sample calculation]
The sample size calculation was carried out based on a previous study of NATEM curves in septic patients and healthy controls (Adamzik, 2010).The G*Power software version 3.1.9.6 was used:Test family: t tests
Statistical test: Means: Difference between two independent means (two groups)
Type of power analysis: A priori: Compute required sample size - given alfa, power, and effect size
Tail: Two
alfa err prob: 0,05
power (1-beta err prob): 0,95,
effect size: d = 1.664101
Determine:
 - n1=n2
 - Mean group 1: 229
 - Mean group 2: 259
 - SD group 1: 19
 - SD group 2: 17
yielded a minimal sample of 11 patients in each group.
No data was excluded.
?. [ Statistics]
GraphPad Prism version 8.0.0 for Windows software (GraphPad Software, San Diego, California USA, www.graphpad.com) was used for the descriptive statistical analysis.
 A Normal distribution was tested with the Shapiro-Wilk test.
 Categorical variables were described as absolute and relative frequencies and analyzed with the Chi-square or Fisher exact test.
 Continuous variables were described as the mean ± SD.
 Multiple comparisons among groups were performed using one-way ANOVA followed by Tukey’s test for pairwise comparisons.
 Two-tailed Student’s t test was used for comparisons between nonsevere and severe patients.
 In all cases, p values < 0.05 indicated statistical significance.
ROC curves were constructed using MedCalc software, Version 7.3.0.0 (Ostend, Belgium, URL https://www.medcalc.org/), to define the cutoff values and estimate the global accuracy based on the area under the ROC curve (AUC).
 Performance indices expressed as percentages (sensitivity and specificity) were obtained for each thromboelastometric parameter in all ROTEM tests.
 TG-ROC curves were assembled to confirm the selected cutoffs.
Decision trees were built using WEKA software (Waikato Environment for Knowledge Analysis, version 3.6.11, University of Waikato, New Zealand, URL https://www.cs.waika to.ac.nz/ml/weka/) to classify COVID patients and healthy controls based on selected thromboelastometric parameters.
 Leave-one-out cross-validation (LOOCV) was applied to estimate the classification accuracy and test the generalizability of the model.
The graphics (bar diagrams, scatter charts and decision trees) were generated using Microsoft Office Package version 2012 and GraphPad Prism software, Version 8.0
?. [Parameters analyzed]
CT: clotting time, expressed in seconds
 ALPHA: alpha angle, expressed in o
 CFT: clot formation time, expressend in seconds
 MCF: Maximum clot firmness, expressed in mm
 ML: maximum lysis, expressed in %
 TPI: thrombodynamic potential index: calculated as [ (100 x MCF) / (100 - MCF) ] / CFT