D-Dimer test Exclude VTE with confidence in only 6 minutes*

EMBOL-1 study

Preliminary data from the EMBOL-1 study has demonstrated that the LumiraDx D-Dimer test provides accurate results that enable its use with a low pre-test probability score (PTP) for the exclusion of VTE at the point of care when patients are presenting with symptoms of VTE. The LumiraDx D-Dimer test performance when used in combination with a low PTP assessment (Wells ‘Unlikely VTE’ group) for direct capillary blood, venous blood and plasma resulted in 100% sensitivity and 100% NPV for direct capillary blood, plasma and venous blood samples. The LumiraDx D-Dimer test displays agreement with analysis in the same subject cohort using the VIDAS D-Dimer Exclusion II reference method.

*In conjunction with a PTP score

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Abstract

The EMBOL-1 Study (NCT04737954) is a prospective multicentre observational clinical study evaluating the use of the LumiraDx D-Dimer point of care test alongside a pre-test probability (PTP) assessment model (Wells Score) in patients presenting into emergency care centres with signs and symptoms of venous thromboembolism (VTE, comprising Pulmonary Embolism (PE) or Deep Vein Thrombosis (DVT)). This study was an interim data analysis. Data from capillary blood, venous blood and plasma samples were included in the evaluation. In 585 included subjects, 408 had a low PTP assessment (Wells ‘Unlikely VTE’ group). The LumiraDx D-Dimer test performance when used in combination with a low PTP assessment (Wells ‘Unlikely VTE’ group) for direct capillary blood (95% CI 72.2%-100.0%), venous blood (95% CI 74.1%-100%) and plasma (95% CI 74.1%-100%) resulted in 100% sensitivity and 100% NPV for direct capillary blood, plasma (95% CI 98.1%-100%) and venous blood samples (95% CI 98.3% - 100%). This was in agreement with analysis in the same subject cohort using the plasma sample on the laboratory reference test method (VIDAS Exclusion II D-dimer, sensitivity 100% (95% CI 74.1%-100%) and 100% NPV (95% CI 98.3% - 100%)).

Key summary points

A quantitative, point of care (POC) D-Dimer test could improve the effectiveness of the established diagnosis pathway of venous thromboembolism (VTE), thereby reducing the current rate of unnecessary referrals to secondary care along with associated cost and improve patient experience.

The primary aim of this study was to assess the performance of the new quantitative point of care LumiraDx D-Dimer test in the exclusion of VTE in a group of patients presenting with signs and symptoms VTE into emergency care. Fingerstick capillary blood, venous blood and plasma (citrate) were evaluated and compared to the laboratory reference test, VIDAS Exclusion II D-Dimer.

The quantitative LumiraDx D-Dimer test had excellent sensitivity (100%) in the assessment of VTE when used at 500 μg/L FEU cut-off and a low pre-test probability (PTP) score (Wells ‘Unlikely’). The NPV was 100% also across all matrices tested. Results were equivalent to those from plasma testing on the laboratory analyser

The LumiraDx D-Dimer test when used with a low (Wells ‘Unlikely’) PTP can be used at the point of care for the assessment of exclusion of VTE in symptomatic patients.

Introduction

D-Dimer testing is important when used with pre-test probability models to aid in the exclusion of venous thromboembolic events (VTEs), including deep venous thrombosis and pulmonary embolism^1,2. D-Dimer is produced upon activation of the coagulation system and is a plasmin-derived soluble degradation product of cross-linked fibrin^3,12.

With approximately 10 million cases reported globally every year, VTE is the third leading vascular disease⁴.

Across six European countries, just under half a million DVTs and 300,000 PEs occur every year. Venous thromboembolism is associated with substantial morbidity and mortality. Although the 30 day mortality rate after pulmonary embolism is decreasing, about 20% of patients with pulmonary embolism still die before diagnosis or shortly thereafter⁴ . Early diagnosis of VTE is important to decrease its associated morbidity and mortality rates, as there are effective therapies available, such as anticoagulant and mechanical treatments, to stop the progression of VTE5 . Imaging methods, such as ultrasound for DVT and computerised tomography (CT and CTPA) scans for PE, are widely accepted tools for diagnosing VTE^6,7. However, referrals to secondary care are required for these tools which are costly and time consuming and this slows down diagnosis and treatment² . Signs and symptoms alone have poor sensitivity and specificity for diagnosis⁴ . In those with suspected VTE, prevalence of the disease is only about 20%⁴ , so imaging/ultrasound is needed to avoid performing expensive radiological tests. In the outpatient setting, clinical pre-test probability (PTP) using validated scoring systems such as Wells or Geneva are used³ to guide further testing. For those with ‘low/ intermediate’ or ‘Unlikely VTE’, a D-Dimer test is the next step where a negative result (usually 500 μg/L FEU) enables rule out of VTE^1,3. Patients with high PTP scores (Likely VTE) are subsequently referred to secondary care for further analysis; using PTP scores in combination with D-Dimer testing has been reported to lead to more specific rule out of VTE. Indeed, it was reported that with additional D-Dimer testing VTE could be ruled out in another 20-40% of patients that were initially identified to be at risk of developing VTE using Wells score alone^6,8,9. As a consequence, several guidelines have recommended the use of Wells score in combination with quantitative D-Dimer testing to aid the diagnosis of VTE^5,7,10,11. As well as using a general cut-off, age adjusted D-Dimer levels are recommended in some guidelines^10,11,13. For the exclusion of pulmonary embolism/ deep vein thrombosis, D-Dimer assays need to be clinically validated and cut-off levels verified in clinical studies to avoid inappropriate use of this biomarker in routine care¹².

Point of care quantitative D-Dimer tests offer the ability to rapidly assess patients with symptoms in acute care and primary care settings. Quantitative D-Dimer point of care tests are recommended that meet the CLSI requirements of ≥98% NPV and sensitivity of ≥97% ^7,14.

This study (EMBOL-1) evaluates the clinical sensitivity and specificity of the point of care (POC) quantitative LumiraDx D-Dimer test in combination with Wells scoring to rule out VTE using fingerstick blood, compared to venous and plasma on the same instrument. The results were compared to those obtained with plasma in the laboratory using the VIDAS D-Dimer Exclusion II reference method. The data represents an interim analysis of the EMBOL study with prospectively, consecutively recruited subjects between March 2021 and June 2022 in 10 clinical sites.

Methods

Study design

The EMBOL 1 study (NCT04737954) is a prospective observational study to evaluate the clinical performance of the LumiraDx D-Dimer test. The study was conducted at multiple sites in the UK and Germany between March 2021 and June 2022. The study sites were in the Emergency Departments of the following hospitals: Manchester Royal Infirmary, Manchester; Edinburgh Royal Infirmary, Edinburgh; St George’s Hospital, London; University College London Hospital, London; Royal London/ St Barts, London; Homerton Hospital, London; Addenbrookes Hospital, Cambridge; South Warwickshire Hospital, Warwick; St John’s Hospital, Edinburgh; University Medical Centre-Hamburg Eppendorf, Germany.

Participants were of any sex and ≥18 years old, presenting with the symptoms of a thromboembolic event. Exclusion criteria included suspected sinus or cerebral venous thromboembolism; end-stage renal failure on haemodialysis; current anticoagulant therapy with dalteparin (Fragmin) or low-molecular-weight heparin (LMWH); previous participation in this study; documented life expectancy of <30 days; haemodynamic instability; anticoagulant therapy with direct-acting anticoagulants (DOACs), warfarin or heparin within the last 30 days; patient being deemed medically unfit to participate.

From each patient a Wells Score assessment was completed (Likely or Unlikely PTP categorisation)7 . Following this, using a finger-stick method, two 20 µL capillary blood samples were obtained for direct testing on the LumiraDx D-Dimer test. Two additional 20 µL capillary blood samples were obtained for immediate testing on the LumiraDx D-Dimer test using a transfer tube application method.

A venous blood sample, <20 mL, was also drawn from each patient into tubes containing an anticoagulant (citrate). Two 20 µL samples from the drawn blood were immediately tested on the LumiraDx D-Dimer test. The remaining venous blood samples were processed immediately (within 1 hour) to plasma using centrifugation, frozen at -80 °C and transported to the LumiraDx laboratory (Stirling, UK). At the laboratory site, plasma samples were thawed and tested in duplicate within 1 hour of thawing, on the reference test VIDAS D-Dimer Exclusion II and on the LumiraDx D-Dimer test.

Test details

The LumiraDx Platform test comprises the Instrument and LumiraDx D-Dimer Test Strips which can be used with capillary blood (direct blood drop or transfer tube application) venous blood (citrate tube), or plasma samples (citrate)15. D-Dimer molecules in the sample form sandwich complexes with the fluorescent latex particles and magnetic beads on the test strip; a magnetic field is applied to retain such complexes while an air wash removes any residual sample and unbound latex particles. The fluorescence emitted by the latex particles that are bound in the complexes is detected and quantified by the spectrometer in the LumiraDx Instrument. The test provides a quantitative result in 6 minutes.

The testing device also has built-in quality control features, which include haematocrit analysis to check sample is within 25-55%, correction to Fibrinogen Equivalent Units (FEU) regardless of the sample type (capillary/venous blood or plasma), a sample volume check, automated test strip positioning and test strip expiration checks.

Statistical analysis

The performance of the LumiraDx D-Dimer test in excluding the presence of a VTE in symptomatic patients was assessed in the following analyses:

1. LumiraDx D-Dimer cut-offs of 500 µg/L FEU and 533 µg/L FEU in combination with Wells Score (PTP) – applied to all sample matrices.
2. PTP (Unlikely) subset + LumiraDx D-Dimer cut-off of 500 µg/L FEU – applied to all sample matrices.
3. PTP (Unlikely) subset + VIDAS Exclusion II D-Dimer cut-off of 500 µg/L FEU – plasma only.

The sensitivity, specificity, and negative and positive predictive values, with their 95% Wilson Score confidence intervals were calculated for each sample type and application method for ruling out VTE. Software used for the analysis was Microsoft® Excel® for Microsoft 365 MSO (16.0.14326.20900) 64-bit and Analyse-it for Microsoft Excel 5.40.2 build 7187.28957. Concurrent validation of the analysis was performed by a 2nd operator in Python 3.9.7 on JupyterLab 3.2.6.

Ethical approval

Ethics approval was granted by North West - Greater Manchester South Research Ethics Committee (UK) under REC reference 19/NW/0070. Approval in Germany was obtained from the Ethics Committee of the Ärztekammer (Ref Number: 00013476).

Results

Study participants

684 participants were prospectively consented onto the EMBOL-1 trial. Between March 2021 and June 2022. Ninety-nine subjects were excluded. Figure 1 presents the Inclusion Decision Flow Chart and reasons for exclusion. In the final included dataset, 585 subjects had a valid PTP assessment score and 54 VTE events were confirmed (all PTP Group). The mean age of participants was 51 years (range 18–97), and 59% were female. Participants were presenting in hospital with suspected VTE. Samples were collected between March 2021 and June 2022.

Of these subjects, 408 were classified as ‘VTE Unlikely’ by the Wells PTP score. Some subjects did not have a full set of sample matrices collected (e.g., fingerstick test failure, but venous tube / plasma collected and tested). Therefore, included subjects per matrix varies as valid data was included where available (Figure 1).

Assay validation

Analysis of sensitivity, specificity, positive and negative predictive values (PPV, NPV) were calculated and are summarised for all subjects with PTP assessments using either 500 μg/L or 533 μg/L FEU LumiraDx D-Dimer result as cut-off for positive and negative results. All results and 95% CI are presented in Table 1. Two cut-off values were analysed, as 533 μg/L is the 90th centile previously published for the LumiraDx D-Dimer test15. In clinical use of D-Dimer, 500μg/L is commonly used1 , therefore the study evaluated differences between using these two cut-offs. Sensitivity for all matrices using the cut-off values plus Wells PTP Score was 100% (95% CI 91.9 %-100%) and NPV was 100% (95% CI 98.1%-100%) at 500 μg/L and at 533μg/L sensitivity was 100% (95% CI 91.9 %-100%) and NPV was 100% (95% CI 98.1%-100%). There was no significant difference detectable if 500 μg/L or 533 μg/L were used as cutoff with PTP assessment.

PTP assessments are commonly used with D-Dimer when low/ intermediate or ‘Unlikely VTE’ scores are determined. A further performance analysis was completed with the LumiraDx D-Dimer test was used in combination with a low PTP assessment (Wells ‘Unlikely VTE’ group) at the 500 μg/L cut-off. This was completed for direct capillary blood, capillary blood by transfer tube application, venous blood (citrate) and plasma (citrate) (Table 2).

The LumiraDx D-Dimer test sensitivity at 500 μg/L when used in combination with a low PTP assessment (Wells ‘Unlikely VTE’ group) for direct capillary blood (95% CI 72.2%-100.0%) and , capillary blood by transfer tube application (95% CI 67.6%-100.0%) was 100.0% and NPV was 100.0% (95% CI 98.1%-100.0%). Both direct capillary application to the test strip and transfer tube methods gave equivalent results (Table 2).

The LumiraDx D-Dimer test sensitivity at 500 μg/L when used in combination with a low PTP assessment (Wells ‘Unlikely VTE’ group) for venous blood (sodium citrate) was 100.0% (95% CI 74.1%-100.0%) and NPV was 100.0% (95% CI 98.3%-100.0%).

The LumiraDx D-Dimer test sensitivity at 500 μg/L when used in combination with a low PTP assessment (Wells ‘Unlikely VTE’ group) for plasma (sodium citrate) was 100.0% (95% CI 74.1%-100.0%) and NPV was 100.0% (95% CI 98.1%-100.0%).

Analysis of 500 μg/L and 533 μg/L as cut-off with and without PTP assessment (all subjects Wells score) indicated no significant difference. This together, with the independent analysis of the Unlikely PTP + D-Dimer, supports use of 500 μg/L as the cut-off with the Unlikely PTP assessment.

The results from the cut-off assessment (EMBOL-1 study interim analysis 2) are aligned with the CLSI recommendation for a D-Dimer test with PTP assessment model (low/ intermediate) of NPV ≥98% and sensitivity ≥97%¹⁴.

The same analysis was completed using the low PTP assessment (Wells ‘Unlikely VTE’ group) and VIDAS Exclusion II D-Dimer test with citrated plasma at the 500 μg/L cut-off. Results in agreement were found with the LumiraDx point of care test (Table 3).

Discussion

The LumiraDx D-Dimer point of care test is a quantitative test that has been previously shown to have accurate results compared to the laboratory reference test and also demonstrated to be easy to use by users in POC settings15. The study results show that this novel quantitative POC test provides accurate results that enable its use with a low pre-test probability score (PTP) for the exclusion of VTE at the point of care when patients are presenting with symptoms of VTE, and a quantitative D-Dimer test result is quickly needed. When utilized in line with the diagnostic guidance, this could facilitate more accurate referrals for imaging diagnostics of VTE and has the potential to save costs due to unnecessary referrals and lab D-Dimer tests.

References

1. Kearon C, de Wit K, Parpia S, Schulman S, Spencer FA et al Diagnosis of deep vein thrombosis with D-dimer adjusted to clinical probability: prospective diagnostic management study. BMJ 2022 BMJ 2022;376:e067378.
2. Price C, Fay, M, Hopstaken, R. Point-of-Care Testing for D-Dimer in the Diagnosis of Venous Thromboembolism in Primary Care: A Narrative Review. Cardiol Ther 2021 10:27-40.
3. Weitz JI, Fredenburgh JL, Eikenboom, JW. A Test in Context: D-Dimer. JAMA 2017 70: (19) 2411-2420.
4. Di Nisio M, van Es N, Büller, HR. Deep vein thrombosis and pulmonary embolism. Lancet 2016 388: 3060-3073.
5. Konstantinides SV, Meyer G, Becattini C, Bueno H, Geersing G-J, Harjola V-P, et al. 2019 ESC guidelines for the diagnosis and management of acute pulmonary embolism developed in collaboration with the European Respiratory Society (ERS): The Task Force for the diagnosis and management of acute pulmonary embolism of the European Society of Cardiology (ESC). European Heart Journal. 2020;41(4):543-603.
6. Patel H, Sun H, Hussain AN, Vakde T. Advances in the diagnosis of venous thromboembolism: a literature review. Diagnostics. 2020;10(6).
7. NICE. Venous thromboembolic diseases: diagnosis, management and thrombophilia testing 2020 [Available from: https://www.nice.org.uk/guidance/NG158. 07/07/2022]
8. Ten Cate-Hoek AJ, Prins MH. Management studies using a combination of D-dimer test result and clinical probability to rule out venous thromboembolism: a systematic review. Journal of Thrombosis and Haemostasis. 2005;3(11):2465-70.
9. Geersing GJ, Zuithoff NPA, Kearon C, Anderson DR, ten Cate-Hoek AJ, Elf JL, et al. Exclusion of deep vein thrombosis using the Wells rule in clinically important subgroups: individual patient data meta-analysis. BMJ : British Medical Journal. 2014;348:g1340
10. Mazzolai L, Aboyans V, Ageno W, Agnelli G, Alatri A, Bauersachs R, et al. Diagnosis and management of acute deep vein thrombosis: a joint consensus document from the European Society of Cardiology working groups of aorta and peripheral vascular diseases and pulmonary circulation and right ventricular function. Eur Heart J. 2018;39(47):4208-18.
11. Raja AS, Greenberg JO, Qaseem A, Denberg TD, Fitterman N, Schuur JD. Evaluation of patients with suspected acute pulmonary embolism: best practice advice from the clinical guidelines committee of the american college of physicians. Ann Intern Med. 2015;163(9):701-11.
12. .Giannitsis, E, Mair, J, Christersson, C, Siegbahn, A, Huber, K et al Editor’s Choice-How to use D-dimer in acute cardiovascular care. Eur Heart Journal 2017; 6(1) 69-80.
13. De Pooter N, Brionne-François M, Smahi M, Abecassis L, Toulon P. Age-adjusted D-dimer cut-off levels to rule out venous thromboembolism in patients with non-high pre-test probability: Clinical performance and costeffectiveness analysis. Journal of Thrombosis and Haemostasis. 2021;19(5):1271-82
14. CLSI H59-A. A Quantitative D-Dimer for the Exclusion of Venous Thromboembolic Disease, approved guideline.
15. Ellis JE, Johnston TW, Craig D, Scribner A, Simon W, Kirstein J. Performance Evaluation of the Quantitative Pointof-Care LumiraDx D-Dimer Test. Cardiol Ther. 2021;10(2):547-59.

Figures and tables

Table 1

Clinical Performance of LumiraDx D-Dimer cut-offs of 500 µg/L FEU and 533 µg/L FEU in combination with PTP assessment (Wells) applied to all sample matrices.

S-COM-ART-02900 R1