The precision of ROTEM EXTEM is decreased in hypocoagulable blood: a prospective observational study

This was a prospective observational study performed at Skåne University Hospital, Lund, Sweden, with ethical approval from the Swedish Ethical Review Authority (Dnr 2010/482 and 2022–00,265-01). All participants gave signed informed consent before participation, and the manuscript was written in accordance with the STROBE-guidelines [9].

Patients were included during three separate periods: December 2018, June 2020 to June 2021, and February to April 2022. Both patients from the Intensive care unit (ICU) and patients subjected to neurosurgery were recruited during the first and second periods. The choice to include neurosurgery patients were only based on practical reasons. During the third period, only patients subjected to neurosurgery were recruited, and only in the third period were the blood samples modified in vitro. Patients undergoing elective neurosurgery were identified using the surgical planning software Orbit® (SYSteam Health & Care, Huskvarna, Sweden). As 3–5 patients underwent surgery at the same time, patients primarily subjected to intracranial tumour resections were selected. When two or more eligible intracranial tumour resections occurred on the same day, the patient whose surgery started first was included. In the case of two eligible surgeries starting simultaneously, the patient in the operating theatre with the lowest theatre number was included. On days when no intracranial tumour surgeries were scheduled, the patient whose surgery started first at the Department of Neurosurgery was included. Critically ill patients in the ICU were included at random, depending on staff being available to collect blood samples. Patients under the age of 18 and those unable to understand the information given in Swedish were excluded.

Collection and handling of blood samples

All blood samples were collected through an arterial line in 2.7-ml tubes containing 0.109-M citrate tubes (Becton, Dickinson and Company Vacutainer Systems, Franklin Lakes, NJ, USA). For neurosurgery patients, blood was sampled before surgery and within three hours after the closure of the dura mater. Blood samples from critically ill patients were collected at inclusion and only once.

During the third inclusion period, blood samples were both analysed at baseline and modified in vitro with the purpose of investigating the performance of the ROTEM® device with both strong and weak coagulation. For the in vitro modification of the experiment, 0.27 ml fibrinogen (Fibryga®, Octapharma, Lachen, Switzerland) 20 mg/ml was added to 2.7 ml citrated blood, and 1.5 ml Albumin (Alburex®, CSL Behring, Pennsylvania, USA) 5% was added to 1.5 ml citrated blood, as previously described [10, 11].

Rotational thromboelastometry

Each blood sample was simultaneously analysed in eight different channels: four in ROTEM® Delta (Pentapharm GmbH, Munich, Germany) and four in roTEG® (Pentapharm GmbH, Munich, Germany) with the same reagents being used as instructed from the manufacturer. The same reference values apply to the two devices and the results from the eight parallel ROTEM channels were used to calculate CV for the CT, CFT, alpha-angle and MCF parameters in each blood sample.

For classification of blood samples into hypo-, normo- and hypercoagulable states the manufacturers’ reference range was used. These were: CT (38–79 s), CFT (34–159 s), (alpha angle (63–83 degrees) and MCF (50–72 mm).

Before analyses, all samples were kept at rest in a pre-heated heating block at 37 °C for 20–30 min and analysed within three hours. The EXTEM assay was performed in accordance with the manufacturer’s instructions. This procedure was used for unmodified blood, blood spiked with fibrinogen, and blood diluted with albumin.

Data acquisition

Data from the measurements were retrieved from the databases of the ROTEM devices and exported to an Excel (Microsoft Office 365, Microsoft Corporation Redmond, WA, USA) database for further analyses. Baseline patient data were retrieved from medical records (Melior™, Cerner Corporation, North Kansas City, Missouri, USA) and Orbit®.

Statistics

The sample size was calculated based on the assumption that the CV for EXTEM CT in a hypocoagulable state would be 2% (units of percent) higher than the CV for EXTEM CT in a normocoagulable state, with a standard deviation of 4.5 (calculated from our previous unpublished results), a power of 95%, and a two-tailed alpha of 0.05. Given that the study was performed on three different cohorts (critically ill patients, neurosurgery patients and in vitro-modified samples), we expected that 1/4 of the samples would be classified as hypocoagulable, defined as a CT value above the normal reference range set by the manufacturer. Based on the above, the sample size was calculated to include a total of 216 blood samples (162 with normal CT values and 54 with high CT values). To allow for analysis failures and a lower ratio of hypocoagulable samples, the aim was to include 225 unique blood samples.

Values from analyses where the TEMograms were clearly incorrect were removed before calculating CVs. The data were exported to GraphPad Prism (version 9.3.1 for Windows, GraphPad Software, San Diego, California, USA) for statistical analysis.

The distribution of all variables was tested before choosing the appropriate statistical method. All non-parametric values were presented as medians with the interquartile ranges in parentheses and parametric values with mean and standard deviations. Numbers are presented with (%). The mean values of the eight channels for each variable and the corresponding CV were plotted in a scatterplot, and Spearman’s correlation was performed. The ROTEM variables were divided into groups based on whether the mean value of the eight channels for the variable was within, above, or below the manufacturers’ reference range. The CV for each measurement was calculated and compared between the groups. If the mean values for a variable could only be sorted into two groups, the Mann–Whitney U test was used for comparisons, and if the values could be divided into all three groups, a comparison between the groups was performed using the Kruskal–Wallis test with a post hoc Dunn’s test. To test whether the in vitro modification of the blood resulted in a hyper- or hypocoagulable state compared to baseline, Friedman’s test with a post hoc Dunn’s test was used. To test whether the values obtained from the four roTEG® channels were similar to those obtained from the four ROTEM®delta channels, XY plots and Bland–Altman plots were performed.

To address a potential problem with multiple testing, a modified Bonferroni correction was applied, and a p-value < 0.01 was considered significant.

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