Immature platelets and platelet reactivity in patients with acute ST-segment Elevation Myocardial Infarction using whole blood flow cytometry with SYTO-13 staining.

BACKGROUND: Reduced effect of antiplatelet therapy has been reported in patients with ST-segment elevation myocardial infarction (STEMI). Multiple factors may concur to explain this, including increased amount of highly reactive immature platelets. OBJECTIVES: To investigate the association between immature platelets and reactivity determined with multicolour flow cytometry using the SYTO-13 dye in STEMI patients. METHODS: We conducted an observational study of 59 patients with acute STEMI. Blood samples were obtained within 24 h after admission and after loading doses of dual antiplatelet therapy. For comparison, samples were obtained from 50 healthy individuals. Immature platelets and platelet reactivity were investigated using multicolour flow cytometry including the SYTO-13 dye that binds to platelet RNA and thus provides a method for subdividing platelets into immature and mature platelets. Additionally, we assessed platelet aggregation, serum-thromboxane B2 levels and standard immature platelet markers. RESULTS: Immature platelets were more reactive than mature platelets in both STEMI patients and healthy individuals (p-values < 0.05). STEMI patients had lower platelet aggregation and thromboxane B2 levels than healthy individuals. We found a positive association between automatically determined immature platelet markers and CD63 expression on activated platelets (Spearman's rho: 0.27 to 0.58, p-values < 0.05). CONCLUSIONS: Our study shows that immature platelets identified with a multicolour flow cytometric method using the SYTO-13 dye are more reactive than mature platelets in patients with acute STEMI and in healthy individuals. The presence of immature platelets may be important for the overall platelet reactivity, which may have implications for the effect of antiplatelet therapy.

Performance on complex memory tests is associated with ß-amyloid in individuals at risk of developing Alzheimer's disease.

The pathophysiological development of Alzheimer's disease (AD) begins in the brain years before the onset of clinical symptoms. The accumulation of beta-amyloid (Aß) is thought to be the first cortical pathology to occur. Carrying one apolipoprotein E (APOE) ε4 allele increases the risk of developing AD at least 2-3 times and is associated with earlier Aß accumulation. Although it is difficult to identify Aß-related cognitive impairment in early AD with standard cognitive tests, more sensitive memory tests may be able to do this. We sought to examine associations between Aß and performance on three tests within three subdomains of memory, verbal, visual, and associative memory, to elucidate which of these tests were sensitive to Aß-related cognitive impairment in at-risk subjects. 55 APOE ε4 carriers underwent MRI, 11 C-Pittsburgh Compound B (PiB) PET, and cognitive testing. A composite cortical PiB SUVR cut-off score of 1.5 was used to categorise subjects as either APOE ε4 Aß+ or APOE ε4 Aß-. Correlations were carried out using cortical surface analysis. In the whole APOE ε4 group, we found significant correlations between Aß load and performance on verbal, visual, and associative memory tests in widespread cortical areas, the strongest association being with performance on associative memory tests. In the APOE ε4 Aß+ group, we found significant correlations between Aß load and performance of verbal and associative, but not visual, memory in localised cortical areas. Performance on verbal and associative memory tests provides sensitive markers of early Aß-related cognitive impairment in at-risk subjects.

Platelet Function and Maturity and Related microRNA Expression in Whole Blood in Patients with ST-Segment Elevation Myocardial Infarction.

BACKGROUND: Reduced effect of antiplatelet therapy has been reported in patients with ST-segment elevation myocardial infarction (STEMI). MicroRNAs (miRs) may influence platelet function and maturity, and subsequently the effect of antiplatelet therapy. OBJECTIVES: We aimed to explore the association between miR expression and platelet function and maturity in patients with acute STEMI and healthy individuals. METHODS: We performed an observational study of STEMI patients admitted directly to primary percutaneous coronary intervention. Patients were treated with antiplatelet therapy according to guidelines. Within 24 hours after admission, blood samples were obtained to measure: the expression of 10 candidate miRs, platelet function markers using advanced flow cytometry, platelet aggregation, serum thromboxane B2, and platelet maturity markers. Furthermore, blood samples from healthy individuals were obtained to determine the normal variation. RESULTS: In total, 61 STEMI patients and 50 healthy individuals were included. STEMI patients had higher expression of miR-21-5p, miR-26b-5p, and miR-223-3p and lower expression of miR-150-5p, miR423-5p, and miR-1180-3p than healthy individuals. In STEMI patients, the expression of miR-26b-5p showed the most consistent association with platelet function (all p-values <0.05, Spearman's rho ranging from 0.27 to 0.41), while the expression of miR-150-5p and miR-223-3p showed negative associations with platelet function. No association between miR expression and platelet maturity markers was observed. CONCLUSION: In patients with STEMI, the expression of six miRs was significantly different from healthy individuals. The expression of miR-26b-5p may affect platelet function in acute STEMI patients and potentially influence the effect of antiplatelet therapy.

Short-term biological variation of plasma uracil in a Caucasian healthy population.

OBJECTIVES: Plasma uracil is a new biomarker to assess the activity of dihydropyrimidine dehydrogenase before cancer treatment with fluoropyrimidine drugs. Knowledge on the biological variation of plasma uracil is important to assess the applicability of plasma uracil as a biomarker of drug tolerance and efficacy. METHODS: A total of 33 apparently healthy individuals were submitted to sequential blood draws for three days. On the second day, blood draws were performed every third hour for 12 h. Plasma uracil was quantified by LC-MS/MS. The within-subject (CVI) and between-subject (CVG) biological variation estimates were calculated using linear mixed-effects models. RESULTS: The overall median value of plasma uracil was 10.6 ng/mL (range 5.6-23.1 ng/mL). The CVI and CVG were 13.5 and 22.1%, respectively. Plasma uracil remained stable during the day, and there was no day-to-day variation observed. No differences in biological variation components were found between sex and no correlation to age was found. Four samples were calculated to be required to estimate the homeostatic set-point ±15% with 95% confidence. CONCLUSIONS: Plasma uracil is subject to tight homeostatic regulation without semidiurnal and day-to-day variation, however between-subject variation exists. This emphasizes plasma uracil as a well-suited biomarker for evaluation of dihydropyrimidine dehydrogenase activity, but four samples are required to establish the homeostatic set-point in a patient.

Impact of age-dependent red blood cell parameters on α-globin gene genotyping in children.

When screening for α-thalassemia in children, adult cut-offs for mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH) are generally applied to guide genetic evaluation. However, the normal ranges for MCV and MCH are lower in children than in adults, so we hypothesized that using age-matched cut-offs could lead to a more rational diagnostic strategy. The aim of this study was to evaluate if age-matched cut-offs could be applied advantageously. Data on children referred to a hemoglobin fractionation at the Department of Clinical Biochemistry, Aarhus University Hospital between 2016-2021 were identified in the laboratory information system. α-globin gene (HBA1/HBA2) genotyping was performed using multiplex gap-polymerase chain reaction. A total of 387 children were identified. HBA1/HBA2-genotyping was performed in 207 children (53%), and α-thalassemia was diagnosed in 47 children (23%) with -α3.7/αα being the predominant genotype (13%). We found that 23 children had MCV and MCH levels in the normal age-matched range, and two of these children (9%) were α+ thalassemia carriers with three functional α-globin genes. Using age-specific cut-off levels resulted in a reduction of 23 (11%) genotypes performed. In conclusion, applying age-matched cut-offs for MCV and MCH when screening children for α-thalassemia lead to 11% fewer genotypes performed while 9% carriers of α+ thalassemia (of the medically innocuous genotype -α3.7/αα) would have been overlooked.

Expression of microRNA Predicts Cardiovascular Events in Patients with Stable Coronary Artery Disease.

BACKGROUND: New biomarkers are warranted to identify patients with coronary artery disease (CAD) at high risk of recurrent cardiovascular events. It has been reported that the expression of microRNAs (miRs) may influence the development of CAD. OBJECTIVES: We aimed to investigate whether the expression of selected candidate miRs is a predictor of cardiovascular events in a cohort of stable CAD patients. METHODS: We performed a single-center prospective study of 749 stable CAD patients with a median follow-up of 2.8 years. We investigated the expression of nine candidate miRs and their relation to cardiovascular events in this cohort. The primary endpoint was the composite of nonfatal myocardial infarction (MI), stent thrombosis (ST), ischemic stroke, and cardiovascular death. The composite of nonfatal MI and ST was analyzed as a secondary endpoint. Furthermore, nonfatal MI, ST, ischemic stroke, and all-cause mortality were analyzed as individual endpoints. RESULTS: Employing receiver operating characteristic curves, it was shown that compared with traditional cardiovascular risk factors alone, combining the expression of miR-223-3p with existing traditional cardiovascular risk factors increased the predictive value of ST (area under the curve: 0.88 vs. 0.77, p = 0.04), the primary composite endpoint (0.65 vs. 0.61, p = 0.049), and the secondary endpoint of the composite of nonfatal MI and ST (0.68 vs. 0.62, p = 0.04). CONCLUSION: Among patients with CAD, adding miR-223-3p expression to traditional cardiovascular risk factors may improve prediction of cardiovascular events, particularly ST. Clinical trials confirming these findings are warranted.

Advanced Flow Cytometry Using the SYTO-13 Dye for the Assessment of Platelet Reactivity and Maturity in Whole Blood.

Newly produced immature platelets are larger, contain higher amounts of residual RNA, and are more reactive than mature platelets. Flow cytometry using the SYTO-13 dye is a method for the subdivision of immature platelets from mature platelets based on the labelling of intracellular platelet RNA, enabling the simultaneous investigation of the reactivity of each platelet population. This method provides detailed information on several aspects of platelet physiology using a combination of platelet surface markers and agonists. Currently, no standardized protocol exists across laboratories. Here, we describe a flow cytometry protocol in detail to investigate platelet reactivity and its relation to platelet maturity. We analyzed 20 healthy individuals with the protocol and compared the platelet subpopulation with the highest SYTO-13 labelling (in the first quintile, "SYTO-high") corresponding to the most immature platelets (highest RNA content) with the platelet subpopulation with the lowest SYTO-13 labelling (in the fifth quintile, "SYTO-low") corresponding to the mature platelets with the lowest RNA content. SYTO-high platelets had overall significantly increased platelet reactivity compared with that of SYTO-low platelets. The presented method may be a valuable research tool for the analysis of platelet reactivity and its relation to platelet maturity.

Arterial and venous blood sampling is equally applicable for coagulation and fibrinolysis analyses.

OBJECTIVES: No consensus exists upon whether arterial and venous blood samples are equivalent when it comes to coagulation analyses. We therefore conducted a comparative cohort study to clarify if arteriovenous differences affect analyses of primary and secondary hemostasis as well as fibrinolysis. METHODS: Simultaneous paired blood samplings were obtained from a cannula in the radial artery and an antecubital venipuncture in 100 patients immediately before or one day after thoracic surgery. Analyses of platelet count and aggregation, International Normalized Ratio (INR), activated partial thromboplastin time (APTT), antithrombin, thrombin time, fibrinogen, D-dimer, rotational thromboelastometry (ROTEM), thrombin generation, prothrombin fragment 1 + 2, and an in-house dynamic fibrin clot formation and lysis assay were performed. RESULTS: No differences were found between arterial and venous samples for the far majority of parameters. The only differences were found in INR, median (IQR): venous, 1.1 (0.2) vs. arterial, 1.1 (0.2) (p<0.002) and in prothrombin fragment 1 + 2: venous, 289 (209) pmol/L vs. arterial, 279 (191) pmol/L (p<0.002). CONCLUSIONS: The sampling site does not affect the majority of coagulation analyses. Small differences were found for two parameters. Due to numerically very discrete differences, they are of no clinical relevance. In conclusion, the present data suggest that both samples obtained from arterial and venous blood may be applied for analyses of coagulation and fibrinolysis.

Flow Cytometric Assessment of Changes in Platelet Reactivity after Acute Coronary Syndrome: A Systematic Review.

Increased platelet activity is an important predictor for recurrent cardiovascular events in patients with acute coronary syndromes (ACS). Flow cytometry is an advanced method for evaluation of platelet activity. We aimed to summarize the current literature on dynamic changes in platelet activity analyzed by flow cytometry in patients with ACS. Employing the guidelines of Preferred Report Items for Systematic Reviews and Meta-Analyses (PRISMA), we searched PubMed and Embase on October 26, 2021, and identified studies measuring platelet activity with flow cytometry in ACS patients in the acute phase (baseline) and at follow-up in a more stable phase. In the 12 included studies, fibrinogen receptor, α-granule secretion, platelet reactivity index, monocyte-platelet aggregates, neutrophil-platelet aggregates, and reticulated platelets were measured. The fibrinogen receptor and α-granule secretion were either unchanged or lower during follow-up measurements than in the acute phase. Platelet reactivity index showed inconsistent results. Values of monocyte-platelet aggregates and neutrophil-platelet aggregates were lower at follow-up than at baseline (p-values <0.05). Reticulated platelets were either unchanged (p-value >0.64) or lower at 1 to 2 months follow-up (p-value 0.04), and also lower at 5 months to 1-year follow-up (p-value >0.005) compared with baseline. Overall, flow cytometric analyses of platelet function in ACS patients showed that platelet activity was lower at follow-up than at baseline. However, in some patients, platelet activity remained unchanged from baseline to follow-up, possibly indicating a sustained high platelet activity that may increase the risk of recurrent cardiovascular events.

Association of whole blood microRNA expression with platelet function and turnover in patients with coronary artery disease.

INTRODUCTION: The risk of recurrent cardiovascular events in patients with coronary artery disease (CAD) is determined by multiple factors including platelet function and turnover. MicroRNAs (miRs) may regulate both platelet function and turnover. We aimed to identify candidate miRs associating with platelet function and turnover in a cohort of stable CAD patients. Furthermore, we retrieved information on binding targets of the candidate miRs to obtain a more comprehensive biological insight into miR regulation of platelet function and turnover. METHODS: Based on existing literature and a pilot study, we identified nine candidate miRs. Subsequently, we investigated the expression of the candidate miRs in whole blood and their relation to platelet function and turnover in 749 CAD patients. Platelet function was analysed using impedance aggregometry, optical aggregometry and serum thromboxane B2 measurements. Platelet turnover markers (immature platelet count, immature platelet fraction and mean platelet volume) were measured using monochromatic automated flow cytometry. RESULTS: Expression of miR-93-5p, miR-126-3p, miR-150-5p, miR-423-3p and miR-1180-3p showed negative correlations with platelet function (p-values from <0.0001 to 0.0006, rho from -0.13 to -0.36). In addition, expression of miR-423-3p showed negative correlation with platelet turnover markers (p-values from 0.001 to 0.004, rho from -0.11 to -0.12). CONCLUSIONS: We identified several novel miRs that may regulate platelet function and turnover, thereby contributing to the increased risk of recurrent cardiovascular events in CAD patients.

The Role of Plasminogen Activator Inhibitor Type 1 (PAI-1) in Placenta-Mediated Pregnancy Complications: A Systematic Review.

Plasminogen activator inhibitor type 1 (PAI-1) is a main inhibitor of fibrinolysis. The PAI-1 gene (SERPINE1) harbors genetic variants with the potential of modifying plasma levels of PAI-1. A delicate balance exists between the coagulation and fibrinolytic system, and changes in PAI-1 have been suggested to compromise establishment of a successful pregnancy. Therefore, this systematic review investigated the association between genetic variants and/or plasma levels of PAI-1 and placenta-mediated pregnancy complications. An extensive literature search was conducted in PubMed, Embase, and Web of Science on the 29th of April 2021. All studies underwent quality rating according to The Study Quality Assessment Tools checklist provided by National Heart, Lung and Blood Institute. A total of 71 studies were included, among which 60 studies investigated PAI-1 genotypes and 11 studies measured PAI-1 plasma levels. In 32 out of 59 studies, no association was found between the PAI-1 4G/5G polymorphism (rs1799768) and placenta-mediated pregnancy complications, which was stated as no significant difference in the genotype distribution comparing women with and without placenta-mediated pregnancy complications or no significantly increased odds of placenta-mediated pregnancy complications carrying the 4G/4G or 4G/5G genotype. Eight out of 11 studies reported significantly higher PAI-1 plasma levels in preeclamptic women than in women without preeclampsia. In conclusion, no clear evidence indicates that PAI-1 polymorphisms are associated with placenta-mediated pregnancy complications, and the possible association between high PAI-1 plasma levels and preeclampsia needs further investigations. Thus, investigation of PAI-1 genotypes and PAI-1 plasma levels does not currently seem to have a place in daily clinical practice managing placenta-mediated pregnancy complications.

Impact of centrifugation time and pneumatic tube transport on plasma concentrations of direct oral anticoagulants.

INTRODUCTION: Rapid results are needed when plasma concentrations of direct oral anticoagulants (DOACs) are required in acute clinical settings. We evaluated the impact of centrifugation time and pneumatic tube transport on DOAC plasma concentrations with the overall aim of reducing turnaround time. METHODS: Blood samples were spiked with rivaroxaban, apixaban or dabigatran in a low and a high concentration prior to centrifugation for 25 minutes (3163 g) or 5 minutes (3000 g) (n = 20 for each DOAC). Both samples spiked with DOACs (n = 20 for each DOAC) and patient samples (n = 25 in total) were transported manually or by pneumatic tube system samples. RESULTS: For samples spiked with DOAC, statistically significant differences in DOAC plasma concentrations were found between centrifugation times for rivaroxaban in low (P < .05) and high (P < .05) concentrations. Relative bias was below 9% for all DOACs. Statistically significant differences were found between modes of transportation for rivaroxaban (P < .01) and dabigatran (P < .01) in high concentrations. Relative bias was 4%-23% for all DOACs. For patient samples, no statistically significant differences were found between modes of transportation, and relative bias was below 12% for all DOACs. CONCLUSION: Minor, clinically insignificant, differences regarding centrifugation times were found in DOAC plasma concentrations. Importantly, no significant differences were found according to transportation modes for samples collected from patients. Although statistically significant differences were found depending on mode of transportation of spiked samples, relative bias was clinically acceptable. Thus, reduced centrifugation time and pneumatic tube transport should be considered to reduce turnaround time for rapid measurement of DOAC plasma concentrations.

MicroRNA as Biomarkers for Platelet Function and Maturity in Patients with Cardiovascular Disease.

Patients with cardiovascular disease (CVD) are at increased risk of suffering myocardial infarction. Platelets are key players in thrombus formation and, therefore, antiplatelet therapy is crucial in the treatment and prevention of CVD. MicroRNAs (miRs) may hold the potential as biomarkers for platelet function and maturity. This systematic review was conducted using the guidelines of Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA). To identify studies investigating the association between miRs and platelet function and maturity in patients with CVD, PubMed and Embase were searched on October 13 and December 13, 2020 without time boundaries. Risk of bias was evaluated using a standardized quality assessment tool. Of the 16 included studies, 6 studies were rated "good" and 10 studies were rated "fair." In total, 45 miRs correlated significantly with platelet function or maturity (rho ranging from -0.68 to 0.38, all p < 0.05) or differed significantly between patients with high platelet reactivity and patients with low platelet reactivity (p-values ranging from 0.0001 to 0.05). Only four miRs were investigated in more than two studies, namely miR-223, miR-126, miR-21 and miR-150. Only one study reported on the association between miRs and platelet maturity. In conclusion, a total of 45 miRs were associated with platelet function or maturity in patients with CVD, with miR-223 and miR-126 being the most frequently investigated. However, the majority of the miRs were only investigated in one study. More data are needed on the potential use of miRs as biomarkers for platelet function and maturity in CVD patients.

Platelet function assessed by ROTEM®platelet in patients receiving antiplatelet therapy during cardiac and vascular surgery.

Patients undergoing coronary artery bypass graft (CABG) surgery or carotid endarterectomy (CEA) continue antiplatelet therapy perioperatively, which may increase bleeding risk. We aimed to investigate whether Rotational thromboelastometry (ROTEM®) platelet, a newly marketed platelet function analysis, would detect antiplatelet therapy in CABG and CEA patients; whether detection of reduced platelet function was associated with increased bleeding; and whether ex vivo desmopressin increased platelet function. We included 20 CABG patients continuing aspirin and 20 CEA patients continuing clopidogrel (n = 1) or clopidogrel and aspirin (n = 19). Platelet function was analyzed with ROTEM®platelet and light transmission aggregometry (LTA). According to the lower reference limit, ROTEM®platelet managed to detect aspirin, but clopidogrel detection was inadequate compared to LTA. Using a previously published cut-off for bleeding risk, 6 (30%) patients receiving aspirin and 4 (21%) patients receiving both clopidogrel and aspirin demonstrated platelet function below this cut-off. One of the four CEA patients below the cut-off died from intracerebral hemorrhage postoperatively. CABG patients below (n = 6) and above (n = 14) the cut-off did not differ in chest tube output (median [range]: 373 ml [250-900] vs. 368 ml [195-820]). Ex vivo addition of desmopressin did not increase platelet function. In conclusion, ROTEM®platelet does reveal aspirin treatment whereas clopidogrel treatment is most often overlooked. Due to low bleeding in the study population, it was not possible to conclude on the association with bleeding risk.

Asymmetric amyloid deposition in preclinical Alzheimer's disease: A PET study.

Introduction: The typical spatial pattern of amyloid-ß (Aß) in diagnosed Alzheimer's disease (AD) is that of a symmetrical hemispheric distribution. However, Aß may be asymmetrically distributed in early stages of AD. Aß distribution on PET has previously been explored in MCI and AD, but it has yet to be directly investigated in preclinical AD (pAD). We examined how Aß was distributed in individuals with pAD and MCI using 11C-Pittsburgh Compound B (PiB) PET. Methods: In this PET study, 79 subjects were retrospectively enrolled, including 34 controls, 24 pAD, and 21 MCI. All subjects underwent APOE genotyping, 11C-PiB PET, MRI, and cognitive testing. We explored differences in Aß load, Aß lateralisation, and Aß distribution, as well as associations between Aß distribution and cognition. Results: The Aß asymmetry index (AI) differed between groups, with pAD having the highest Aß AI as compared to both controls and MCI. There was no clear Aß lateralisation in pAD, but there was a non-significant trend towards Aß being more left-lateralised in MCI. There were no correlations between the cognitive scores and Aß AI or Aß lateralisation in pAD or MCI. Conclusion: The distribution of Aß is most asymmetrical in pAD, as Aß first starts accumulating, and it then becomes less asymmetrical in MCI, when Aß has spread further, suggesting that more pronounced asymmetrical Aß distribution may be a distinguishing factor in pAD. Longitudinal studies examining the distribution of Aß across the AD continuum are needed.

Genetic Variants in the Protein S ( PROS1 ) Gene and Protein S Deficiency in a Danish Population.

Protein S (PS) deficiency is a risk factor for venous thromboembolism (VTE) and can be caused by variants of the gene encoding PS ( PROS1 ). This study aimed to evaluate the clinical value of molecular analysis of the PROS1 gene in PS-deficient participants. We performed Sanger sequencing of the coding region of the PROS1 gene and multiplex ligation-dependent probe amplification to exclude large structural rearrangements. Free PS was measured by a particle-enhanced immunoassay, while PS activity was assessed by a clotting method. A total of 87 PS-deficient participants and family members were included. In 22 index participants, we identified 13 PROS1 coding variants. Five variants were novel. In 21 index participants, no coding sequence variants or structural rearrangements were identified. The free PS level was lower in index participants carrying a PROS1 variant compared with index participants with no variant (0.51 [0.32-0.61] vs. 0.62 [0.57-0.73] × 10 3 IU/L; p < 0.05). The p.(Thr78Met) variant was associated with only slightly decreased free PS levels (0.59 [0.53-0.66] × 10 3 IU/L) compared with the p.(Glu390Lys) variant (0.27 [0.24-0.37] × 10 3 IU/L, p < 0.01). The frequency of VTE in participants with a coding PROS1 variant was 43 and 17% in the group with normal PROS1 gene ( p = 0.05). In conclusion, we report 13 PROS1 coding variants including five novel variants. PS levels differ by PROS1 variant and the frequency of VTE was higher when a coding PROS1 variant was present. Hence, molecular analysis of the PROS1 gene may add clinical value in the diagnostic work-up of PS deficiency.

Implementation of the new EU IVD regulation - urgent initiatives are needed to avert impending crisis.

Laboratory medicine in the European Union is at the dawn of a regulatory revolution as it reaches the end of the transition from IVDD 98/79/EC (https://eur-lex.eur-opa.eu/legal-content/EN/TXT/?uri=CELEX%3A31998L0079&qid=1628781352814) to IVDR 2017/746 https://eur-lex.europa.eu/eli/reg/2017/746. Without amendments and contingency plans, implementation of the IVDR in May 2022 will lead the healthcare sector into uncharted waters due to unpreparedness of the EU regulatory infrastructure. Prospective risk analyses were not made by the European Commission, and if nothing happens it can be anticipated that the consequences will impact all stakeholders of the medical test pipeline, may seriously harm patients and may prevent caregivers from making appropriate clinical decisions due to non-availability of medical tests. Finally, it also may discourage manufacturers and academia from developing specialty tests, thereby hampering innovation in medical diagnostic care. We hereby inform laboratory professionals about the imminent diagnostic collapse using testimonies from representative stakeholders of the diagnostic supply chain and from academia developing innovative in-house tests in domains of unmet clinical needs. Steps taken by the EFLM Task Force on European Regulatory Affairs, under the umbrella of the Biomedical Alliance in Europe, will be highlighted, as well as the search for solutions through dialogue with the European Commission. Although we recognize that the IVDR promotes positive goals such as increased clinical evidence, surveillance, and transparency, we need to ensure that the capabilities of the diagnostic sector are not damaged by infrastructural unpreparedness, while at the same time being forced to submit to a growing bureaucratic and unsupportive structure that will not support its "droit d'exister".

Whole blood platelet aggregation determined by the ROTEM platelet equipment; reference intervals and stability.

Point of care testing of residual effect of antiplatelet therapy in trauma patients or during major surgery may result in improved clinical management of significant bleeding. We included 121 healthy individuals (57 females and 64 males, aged 22-65 years) in order to establish reference intervals for platelet aggregation induced by adenosine diphosphate (ADPTEM, 10 µM), arachidonic acid (ARATEM, 0.42 mM) and thrombin activating peptide (TRAPTEM, 36 µM) employing the ROTEM platelet module. Further, the impact of citrate (3.2%) and hirudin (>15 µg/ml) as anticoagulants was evaluated. Finally, we investigated assay stability (15, 30, 60, and 120 min after blood sampling) (n = 8) and between-day variation (n = 5). We report reference intervals for 121 healthy individuals and reference intervals by gender. We observed significantly higher platelet aggregation in females than in males (all P-values < 0.05). No correlation between age and platelet aggregation was observed, except for the parameter TRAPTEM amplitude (A6), in which a decline in A6 was observed with increasing age (P = 0.03). We observed significantly lower levels of platelet aggregation in citrate tubes than in hirudin tubes (all P-values < 0.05), except from TRAPTEM maximum slope, where no significant difference was observed (P = 0.40).The stability was acceptable (≤20% deviation) for up to 120 min for ARATEM in citrate tubes, and up to 60 min for the ADPTEM and TRAPTEM assays in citrate tubes. In hirudin tubes we found ADPTEM and ARATEM assays to be stable for 60 min, while the stability of TRAPTEM in hirudin tubes was found to be stable for 30 min. Using citrate tubes, the between-day variation (mean coefficient of variation, CV) was 19-20% for ADPTEM, 19-26% for TRAPTEM, and 10% for ARATEM, whereas the mean CV was 11-13% for all three assays in hirudin tubes.In conclusion, we established combined and gender-specific reference intervals for three platelet aggregation assays in both citrate- and hirudin tubes. In citrate tubes, the stability of the ROTEM platelet assays was 60-120 min, while the stability in hirudin tubes was 30-60 min. The between-day variation was lowest for samples obtained in hirudin tubes.