Journal of Clinical Medicine

Research

17 pages, 2443 KiB

Open AccessArticle

Effects of Time-Interval since Blood Draw and of Anticoagulation on Platelet Testing (Count, Indices and Impedance Aggregometry): A Systematic Study with Blood from Healthy Volunteers

by Michael Hardy, Sarah Lessire, Sultan Kasikci, Justine Baudar, Maité Guldenpfennig, Adrien Collard, Jean-Michel Dogné, Bernard Chatelain, Hugues Jacqmin, Thomas Lecompte and François Mullier

J. Clin. Med. 2020, 9(8), 2515; https://doi.org/10.3390/jcm9082515 - 4 Aug 2020

Cited by 18 | Viewed by 3799

Abstract

Platelet count, indices (mean volume, young—immature platelet fraction) and aggregation are widely used laboratory parameters to investigate primary hemostasis. We performed a systematic, thorough evaluation of the influence of the time-interval since blood draw from 20 healthy individuals and of the anticoagulation of [...] Read more.

Platelet count, indices (mean volume, young—immature platelet fraction) and aggregation are widely used laboratory parameters to investigate primary hemostasis. We performed a systematic, thorough evaluation of the influence of the time-interval since blood draw from 20 healthy individuals and of the anticoagulation of collected blood on such parameters. Blood was anticoagulated with citrate, K₂-ethylenediaminetetraacetic acid (EDTA) and hirudin and analyzed 5, 30, 60, 120 and 180 min after blood draw. Multiple electrode aggregometry (MEA) was performed with either hirudin (half-diluted with NaCl) or citrate samples (half-diluted with NaCl or CaCl₂ 3 mM). Platelet count and indices (Sysmex XN-20) were rather stable over time with EDTA blood. MEA results were lower with citrate blood than with hirudin blood; supplementation with calcium was partially compensatory. MEA results were also lower when performed less than 30 or more than 120 min after blood draw. Platelet clumping, quantitatively estimated with microscope examination of blood smears, was more important in hirudin blood than citrate or EDTA blood and could explain some of the differences observed between preanalytical variables. The results stress once more the importance of preanalytical variables in hemostasis laboratory testing. Decision thresholds based on those tests are only applicable within specific preanalytical conditions. Full article

(This article belongs to the Special Issue Platelet Counting, Morphology Assessment and Functional Studies: Pitfalls, Uncertainties, Good Practice for Clinical Usefulness)

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14 pages, 1641 KiB

Open AccessFeature PaperArticle

A Morphometric Analysis of Platelet Dense Granules of Patients with Unexplained Bleeding: A New Entity of Delta-Microgranular Storage Pool Deficiency

by William T. Gunning III, Meera Raghavan, Edward P. Calomeni, James N. Turner, Bodri Roysam, Shantala Roysam, Mary R. Smith, Peter A. Kouides and Neil A. Lachant

J. Clin. Med. 2020, 9(6), 1734; https://doi.org/10.3390/jcm9061734 - 4 Jun 2020

Cited by 14 | Viewed by 4112

Abstract

One thousand and eighty patients, having prolonged bleeding times, frequent epistaxis, menorrhagia or easy bruising or other bleeding manifestations, and excluding those with von Willebrand’s disease, were evaluated for platelet dense granule deficiency. The mean diameter of platelet dense granules was determined for [...] Read more.

One thousand and eighty patients, having prolonged bleeding times, frequent epistaxis, menorrhagia or easy bruising or other bleeding manifestations, and excluding those with von Willebrand’s disease, were evaluated for platelet dense granule deficiency. The mean diameter of platelet dense granules was determined for all patients using image analysis. Four hundred and ninety-nine had “classic” dense (delta) granule storage pool deficiency (δ-SPD). Five hundred and eighty-one individuals (53.8%) were found to have a normal mean number of dense granules, but for some of these patients, the dense granules were smaller than for the controls. Of the patients having a normal number of dense granules, 165 (28.4%) were found to have significantly smaller granules than the platelets obtained from the control subjects. Their average granule diameter was 123.35 ± 0.86 nm, that is more than three standard deviations below the mean of the control data. Total δ-granule storage pool volumes (TDGV)/platelet were calculated using these measurements. Individuals with δ-SPD had half the number of granules (2.25 ± 0.04 DG/PL) and storage pool volume (3.88 ± 1.06 × 10⁶ nm³) when compared to our control data (4.64 ± 0.11 DG/PL; 10.79 × 10⁶ nm³ ± 0.42). Individuals having a bleeding history but a normal average of small dense granules had a calculated storage pool volume statistically different than controls and essentially the same storage pool volume as patients with δ-SPD. We have identified a sub-classification of δ-SPD that we have defined as micro-granular storage pool deficiency (δ-MGSPD). Full article

(This article belongs to the Special Issue Platelet Counting, Morphology Assessment and Functional Studies: Pitfalls, Uncertainties, Good Practice for Clinical Usefulness)

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20 pages, 9793 KiB

Open AccessArticle

Diagnosis of Inherited Platelet Disorders on a Blood Smear

by Carlo Zaninetti and Andreas Greinacher

J. Clin. Med. 2020, 9(2), 539; https://doi.org/10.3390/jcm9020539 - 17 Feb 2020

Cited by 32 | Viewed by 15361

Abstract

Inherited platelet disorders (IPDs) are rare diseases featured by low platelet count and defective platelet function. Patients have variable bleeding diathesis and sometimes additional features that can be congenital or acquired. Identification of an IPD is desirable to avoid misdiagnosis of immune thrombocytopenia [...] Read more.

Inherited platelet disorders (IPDs) are rare diseases featured by low platelet count and defective platelet function. Patients have variable bleeding diathesis and sometimes additional features that can be congenital or acquired. Identification of an IPD is desirable to avoid misdiagnosis of immune thrombocytopenia and the use of improper treatments. Diagnostic tools include platelet function studies and genetic testing. The latter can be challenging as the correlation of its outcomes with phenotype is not easy. The immune-morphological evaluation of blood smears (by light- and immunofluorescence microscopy) represents a reliable method to phenotype subjects with suspected IPD. It is relatively cheap, not excessively time-consuming and applicable to shipped samples. In some forms, it can provide a diagnosis by itself, as for MYH9-RD, or in addition to other first-line tests as aggregometry or flow cytometry. In regard to genetic testing, it can guide specific sequencing. Since only minimal amounts of blood are needed for the preparation of blood smears, it can be used to characterize thrombocytopenia in pediatric patients and even newborns further. In principle, it is based on visualizing alterations in the distribution of proteins, which result from specific genetic mutations by using monoclonal antibodies. It can be applied to identify deficiencies in membrane proteins, disturbed distribution of cytoskeletal proteins, and alpha as well as delta granules. On the other hand, mutations associated with impaired signal transduction are difficult to identify by immunofluorescence of blood smears. This review summarizes technical aspects and the main diagnostic patterns achievable by this method. Full article

(This article belongs to the Special Issue Platelet Counting, Morphology Assessment and Functional Studies: Pitfalls, Uncertainties, Good Practice for Clinical Usefulness)

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12 pages, 786 KiB

Open AccessArticle

Reduction of Preoperative Waiting Time Before Urgent Surgery for Patients on P2Y₁₂ Inhibitors Using Multiple Electrode Aggregometry: A Retrospective Study

by Michaël Hardy, Camie Dupuis, Anne-Sophie Dincq, Hugues Jacqmin, Thomas Lecompte, François Mullier and Sarah Lessire

J. Clin. Med. 2020, 9(2), 424; https://doi.org/10.3390/jcm9020424 - 4 Feb 2020

Cited by 6 | Viewed by 3553

Abstract

P2Y₁₂ inhibitor discontinuation is essential before most surgical interventions to limit bleeding complications. Based on pharmacodynamic data, fixed discontinuation durations have been recommended. However, as platelet function recovery is highly variable among patients, a more individualized approach based on platelet function testing [...] Read more.

P2Y₁₂ inhibitor discontinuation is essential before most surgical interventions to limit bleeding complications. Based on pharmacodynamic data, fixed discontinuation durations have been recommended. However, as platelet function recovery is highly variable among patients, a more individualized approach based on platelet function testing (PFT) has been proposed. The aim of this retrospective single-centre study was to determine whether PFT using whole blood adenosine diphosphate–multiple electrode aggregometry (ADP–MEA) was associated with a safe reduction of preoperative waiting time. Preoperative ADP–MEA was performed for 29 patients on P2Y₁₂ inhibitors. Among those, 17 patients underwent a coronary artery bypass graft. Twenty one were operated with an ADP–MEA ≥ 19 U (quantification of the area under the aggregation curve), and the waiting time was shorter by 1.6 days (median 1.8 days, IQR 0.5–2.9), by comparison with the current recommendations (five days for clopidogrel and ticagrelor, seven days for prasugrel). Platelet function recovery was indeed highly variable among individuals. With the 19 U threshold, high residual platelet inhibition was associated with perioperative platelet transfusion. These results suggest that preoperative PFT with ADP–MEA could help reduce waiting time before urgent surgery for patients on P2Y₁₂ inhibitors. Full article

(This article belongs to the Special Issue Platelet Counting, Morphology Assessment and Functional Studies: Pitfalls, Uncertainties, Good Practice for Clinical Usefulness)

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8 pages, 1706 KiB

Open AccessCommunication

Head-to-Head Comparison of Consensus-Recommended Platelet Function Tests to Assess P2Y₁₂ Inhibition—Insights for Multi-Center Trials

by Jean-Christophe Bélanger, Fabio Luiz Bandeira Ferreira, Mélanie Welman, Rahma Boulahya, Jean-François Tanguay, Derek Y.F. So and Marie Lordkipanidzé

J. Clin. Med. 2020, 9(2), 332; https://doi.org/10.3390/jcm9020332 - 24 Jan 2020

Cited by 3 | Viewed by 3811

Abstract

The vasodilator-associated stimulated phosphoprotein (VASP) phosphorylation level is a highly specific method to assess P2Y12 receptor inhibition. Traditionally, VASP phosphorylation is analyzed by flow cytometry, which is laborious and restricted to specialized laboratories. Recently, a simple ELISA kit has been commercialized. The primary [...] Read more.

The vasodilator-associated stimulated phosphoprotein (VASP) phosphorylation level is a highly specific method to assess P2Y12 receptor inhibition. Traditionally, VASP phosphorylation is analyzed by flow cytometry, which is laborious and restricted to specialized laboratories. Recently, a simple ELISA kit has been commercialized. The primary objective of this study was to compare the performance of VASP assessment by ELISA and flow cytometry in relation to functional platelet aggregation testing by Multiplate^® whole-blood aggregometry. Blood from 24 healthy volunteers was incubated with increasing concentration of a P2Y12 receptor inhibitor (AR-C 66096). Platelet function testing was carried out simultaneously by Multiplate^® aggregometry and by VASP assessment through ELISA and flow cytometry. As expected, increasing concentrations of the P2Y12 receptor inhibitor induced a proportional inhibition of platelet aggregation and P2Y12 receptor activation across the modalities. Platelet reactivity index values of both ELISA- and flow cytometry-based VASP assessment methods correlated strongly (r = 0.87, p < 0.0001) and showed minimal bias (1.05%). Correlation with Multiplate^® was slightly higher for the flow cytometry-based VASP assay (r = 0.79, p < 0.0001) than for the ELISA-based assay (r = 0.69, p < 0.0001). Intraclass correlation (ICC) was moderate for all the assays tested (ICC between 0.62 and 0.84). However, categorization into low, optimal, or high platelet reactivity based on these assays was strongly concordant (κ between 0.86 and 0.92). In conclusion, the consensus-recommended assays with their standardized cut-offs should not be used interchangeably in multi-center clinical studies but, rather, they should be standardized throughout sites. Full article

(This article belongs to the Special Issue Platelet Counting, Morphology Assessment and Functional Studies: Pitfalls, Uncertainties, Good Practice for Clinical Usefulness)

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13 pages, 2262 KiB

Open AccessArticle

Rivaroxaban Effects Illustrate the Underestimated Importance of Activated Platelets in Thrombin Generation Assessed by Calibrated Automated Thrombography

by Stephanie Makhoul, Marina Panova-Noeva, Véronique Regnault, Wolfram Ruf, Philip Wenzel and Jeremy Lagrange

J. Clin. Med. 2019, 8(11), 1990; https://doi.org/10.3390/jcm8111990 - 15 Nov 2019

Cited by 11 | Viewed by 3624

Abstract

Background: The direct oral anticoagulant rivaroxaban inhibiting specifically activated factor X (FXa) causes delayed thrombin generation (TG) as measured by calibrated automated thrombography (CAT). The implications of these changes for assessing bleeding or residual prothrombotic risks of patients are unclear in the absence [...] Read more.

Background: The direct oral anticoagulant rivaroxaban inhibiting specifically activated factor X (FXa) causes delayed thrombin generation (TG) as measured by calibrated automated thrombography (CAT). The implications of these changes for assessing bleeding or residual prothrombotic risks of patients are unclear in the absence of a better understanding of the underlying mechanism. Methods: We compared platelet rich plasma (PRP) without or with prior collagen-induced platelet aggregation (agPRP) in the CAT assay to better characterize TG in the presence of rivaroxaban. Results: In the presence of rivaroxaban, TG curves in agPRP showed a distinct profile with a rapidly ascending phase followed with a protracted phase. Inhibition of tissue factor pathway inhibitor amplified the first phase of the curve which was also modulated by procoagulant phospholipids. Inhibition of FXIIa-dependent FXI activation revealed that aggregated platelets influenced the first phase by a combination of extrinsic and intrinsic coagulation pathway initiations. Thrombin-dependent amplification of TG (even prior collagen activation) was responsible for the second phase of the TG curve. Conclusions: AgPRP fully includes platelet ability to support TG and reveal distinct TG phases in the presence of direct FXa inhibitors highlighting its potential use in an anticoagulated setting. Full article

(This article belongs to the Special Issue Platelet Counting, Morphology Assessment and Functional Studies: Pitfalls, Uncertainties, Good Practice for Clinical Usefulness)

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Review

Jump to: Research, Other

17 pages, 2089 KiB

Open AccessReview

Advances in Platelet Function Testing—Light Transmission Aggregometry and Beyond

by Jessica Le Blanc, François Mullier, Caroline Vayne and Marie Lordkipanidzé

J. Clin. Med. 2020, 9(8), 2636; https://doi.org/10.3390/jcm9082636 - 13 Aug 2020

Cited by 44 | Viewed by 6421

Abstract

Platelet function testing is essential for the diagnosis of hemostasis disorders. While there are many methods used to test platelet function for research purposes, standardization is often lacking, limiting their use in clinical practice. Light transmission aggregometry has been the gold standard for [...] Read more.

Platelet function testing is essential for the diagnosis of hemostasis disorders. While there are many methods used to test platelet function for research purposes, standardization is often lacking, limiting their use in clinical practice. Light transmission aggregometry has been the gold standard for over 60 years, with inherent challenges of working with live dynamic cells in specialized laboratories with independent protocols. In recent years, standardization efforts have brought forward fully automated systems that could lead to more widespread use. Additionally, new technical approaches appear promising for the future of specialized hematology laboratories. This review presents developments in platelet function testing for clinical applications. Full article

(This article belongs to the Special Issue Platelet Counting, Morphology Assessment and Functional Studies: Pitfalls, Uncertainties, Good Practice for Clinical Usefulness)

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23 pages, 2986 KiB

Open AccessReview

Platelet δ-Storage Pool Disease: An Update

by Arnaud Dupuis, Jean-Claude Bordet, Anita Eckly and Christian Gachet

J. Clin. Med. 2020, 9(8), 2508; https://doi.org/10.3390/jcm9082508 - 4 Aug 2020

Cited by 38 | Viewed by 6367

Abstract

Platelet dense-granules are small organelles specific to the platelet lineage that contain small molecules (calcium, adenyl nucleotides, serotonin) and are essential for the activation of blood platelets prior to their aggregation in the event of a vascular injury. Delta-storage pool diseases (δ-SPDs) are [...] Read more.

Platelet dense-granules are small organelles specific to the platelet lineage that contain small molecules (calcium, adenyl nucleotides, serotonin) and are essential for the activation of blood platelets prior to their aggregation in the event of a vascular injury. Delta-storage pool diseases (δ-SPDs) are platelet pathologies leading to hemorrhagic syndromes of variable severity and related to a qualitative (content) or quantitative (numerical) deficiency in dense-granules. These pathologies appear in a syndromic or non-syndromic form. The syndromic forms (Chediak–Higashi disease, Hermansky–Pudlak syndromes), whose causative genes are known, associate immune deficiencies and/or oculocutaneous albinism with a platelet function disorder (PFD). The non-syndromic forms correspond to an isolated PFD, but the genes responsible for the pathology are not yet known. The diagnosis of these pathologies is complex and poorly standardized. It is based on orientation tests performed by light transmission aggregometry or flow cytometry, which are supplemented by complementary tests based on the quantification of platelet dense-granules by electron microscopy using the whole platelet mount technique and the direct determination of granule contents (ADP/ATP and serotonin). The objective of this review is to present the state of our knowledge concerning platelet dense-granules and the tools available for the diagnosis of different forms of δ-SPD. Full article

(This article belongs to the Special Issue Platelet Counting, Morphology Assessment and Functional Studies: Pitfalls, Uncertainties, Good Practice for Clinical Usefulness)

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15 pages, 1607 KiB

Open AccessReview

Platelet Functions During Extracorporeal Membrane Oxygenation. Platelet–Leukocyte Aggregates Analyzed by Flow Cytometry as a Promising Tool to Monitor Platelet Activation

by Alexandre Mansour, Mikael Roussel, Pascale Gaussem, Fabienne Nédelec-Gac, Adeline Pontis, Erwan Flécher, Christilla Bachelot-Loza and Isabelle Gouin-Thibault

J. Clin. Med. 2020, 9(8), 2361; https://doi.org/10.3390/jcm9082361 - 23 Jul 2020

Cited by 13 | Viewed by 5576

Abstract

Extracorporeal membrane oxygenation (ECMO) is an extracorporeal circulation used to manage patients with severe circulatory or respiratory failure. It is associated with both high bleeding and thrombosis risks, mainly as a result of biomaterial/blood interface phenomena, high shear stress, and complex inflammatory response [...] Read more.

Extracorporeal membrane oxygenation (ECMO) is an extracorporeal circulation used to manage patients with severe circulatory or respiratory failure. It is associated with both high bleeding and thrombosis risks, mainly as a result of biomaterial/blood interface phenomena, high shear stress, and complex inflammatory response involving the activation of coagulation and complement systems, endothelial cells, leukocytes, and platelets. Besides their critical role in hemostasis, platelets are important players in inflammatory reactions, especially due to their ability to bind and activate leukocytes. Hence, we reviewed studies on platelet function of ECMO patients. Moreover, we addressed the issue of platelet–leukocyte aggregates (PLAs), which is a key step in both platelet and leukocyte activation, and deserves to be investigated in these patients. A reduced expression of GPIb and GPVI was found under ECMO therapy, due to the shedding processes. However, defective platelet aggregation is inconsistently reported and is still not clearly defined. Due to the high susceptibility of PLAs to pre-analytical conditions, defining and strictly adhering to a rigorous laboratory methodology is essential for reliable and reproducible results, especially in the setting of complex inflammatory situations like ECMO. We provide results on sample preparation and flow cytometric whole blood evaluation of circulating PLAs. Full article

(This article belongs to the Special Issue Platelet Counting, Morphology Assessment and Functional Studies: Pitfalls, Uncertainties, Good Practice for Clinical Usefulness)

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12 pages, 975 KiB

Open AccessReview

Management of Bleeding Events Associated with Antiplatelet Therapy: Evidence, Uncertainties and Pitfalls

by Anne Godier, Pierre Albaladejo and the French Working Group on Perioperative Haemostasis (GIHP) Group

J. Clin. Med. 2020, 9(7), 2318; https://doi.org/10.3390/jcm9072318 - 21 Jul 2020

Cited by 18 | Viewed by 8774

Abstract

Bleeding complications are common in patients treated with antiplatelet agents (APA), but their management relies on poor evidence. Therefore, practical guidelines and guidance documents are mainly based on expert opinion. The French Working Group on Perioperative Haemostasis provided proposals in 2018 to enhance [...] Read more.

Bleeding complications are common in patients treated with antiplatelet agents (APA), but their management relies on poor evidence. Therefore, practical guidelines and guidance documents are mainly based on expert opinion. The French Working Group on Perioperative Haemostasis provided proposals in 2018 to enhance clinical decisions regarding the management of APA-treated patients with a bleeding event. In light of these proposals, this review discusses the evidence and uncertainties of the management of patients with a bleeding event while on antiplatelet therapy. Platelet transfusion is the main option as an attempt to neutralise the effect of APA on primary haemostasis. Nevertheless, efficacy of platelet transfusion to mitigate clinical consequences of bleeding in patients treated with APA depends on the type of antiplatelet therapy, the time from the last intake, the mechanism (spontaneous versus traumatic) and site of bleeding and the criteria of efficacy (in vitro, in vivo). Specific antidotes for APA neutralisation are needed, especially for ticagrelor, but are not available yet. Despite the amount of information that platelet function tests are expected to give, little data support the clinical benefit of using such tests for the management of bleeding events in patients treated or potentially treated with APA. Full article

(This article belongs to the Special Issue Platelet Counting, Morphology Assessment and Functional Studies: Pitfalls, Uncertainties, Good Practice for Clinical Usefulness)

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19 pages, 1872 KiB

Open AccessReview

Pathophysiology and Diagnosis of Drug-Induced Immune Thrombocytopenia

by Caroline Vayne, Eve-Anne Guéry, Jérôme Rollin, Tatiana Baglo, Rachel Petermann and Yves Gruel

J. Clin. Med. 2020, 9(7), 2212; https://doi.org/10.3390/jcm9072212 - 13 Jul 2020

Cited by 74 | Viewed by 20842

Abstract

Drug-induced immune thrombocytopenia (DITP) is a life-threatening clinical syndrome that is under-recognized and difficult to diagnose. Many drugs can cause immune-mediated thrombocytopenia, but the most commonly implicated are abciximab, carbamazepine, ceftriaxone, eptifibatide, heparin, ibuprofen, mirtazapine, oxaliplatin, penicillin, quinine, quinidine, rifampicin, suramin, tirofiban, trimethoprim-sulfamethoxazole, [...] Read more.

Drug-induced immune thrombocytopenia (DITP) is a life-threatening clinical syndrome that is under-recognized and difficult to diagnose. Many drugs can cause immune-mediated thrombocytopenia, but the most commonly implicated are abciximab, carbamazepine, ceftriaxone, eptifibatide, heparin, ibuprofen, mirtazapine, oxaliplatin, penicillin, quinine, quinidine, rifampicin, suramin, tirofiban, trimethoprim-sulfamethoxazole, and vancomycin. Several different mechanisms have been identified in typical DITP, which is most commonly characterized by severe thrombocytopenia due to clearance and/or destruction of platelets sensitized by a drug-dependent antibody. Patients with typical DITP usually bleed when symptomatic, and biological confirmation of the diagnosis is often difficult because detection of drug-dependent antibodies (DDabs) in the patient’s serum or plasma is frequently not possible. This is in contrast to heparin-induced thrombocytopenia (HIT), which is a particular DITP caused in most cases by heparin-dependent antibodies specific for platelet factor 4, which can strongly activate platelets in vitro and in vivo, explaining why affected patients usually have thrombotic complications but do not bleed. In addition, laboratory tests are readily available to diagnose HIT, unlike the methods used to detect DDabs associated with other DITP that are mostly reserved for laboratories specialized in platelet immunology. Full article

(This article belongs to the Special Issue Platelet Counting, Morphology Assessment and Functional Studies: Pitfalls, Uncertainties, Good Practice for Clinical Usefulness)

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25 pages, 1974 KiB

Open AccessReview

Detection of Platelet-Activating Antibodies Associated with Heparin-Induced Thrombocytopenia

by Brigitte Tardy, Thomas Lecompte, François Mullier, Caroline Vayne and Claire Pouplard

J. Clin. Med. 2020, 9(4), 1226; https://doi.org/10.3390/jcm9041226 - 24 Apr 2020

Cited by 37 | Viewed by 11910

Abstract

Heparin-induced thrombocytopenia (HIT) is a prothrombotic immune drug reaction caused by platelet-activating antibodies that in most instances recognize platelet factor 4 (PF4)/polyanion complexes. Platelet activation assays (i.e., functional assays) are more specific than immunoassays, since they are able to discern clinically relevant heparin-induced [...] Read more.

Heparin-induced thrombocytopenia (HIT) is a prothrombotic immune drug reaction caused by platelet-activating antibodies that in most instances recognize platelet factor 4 (PF4)/polyanion complexes. Platelet activation assays (i.e., functional assays) are more specific than immunoassays, since they are able to discern clinically relevant heparin-induced antibodies. All functional assays used for HIT diagnosis share the same principle, as they assess the ability of serum/plasma from suspected HIT patients to activate fresh platelets from healthy donors in the presence of several concentrations of heparin. Depending on the assay, donors’ platelets are stimulated either in whole blood (WB), platelet-rich plasma (PRP), or in a buffer medium (washed platelets, WP). In addition, the activation endpoint studied varies from one assay to another: platelet aggregation, membrane expression of markers of platelet activation, release of platelet granules. Tests with WP are more sensitive and serotonin release assay (SRA) is considered to be the current gold standard, but functional assays suffer from certain limitations regarding their sensitivity, specificity, complexity, and/or accessibility. However, the strict adherence to adequate preanalytical conditions, the use of selected platelet donors and the inclusion of positive and negative controls in each run are key points that ensure their performances. Full article

(This article belongs to the Special Issue Platelet Counting, Morphology Assessment and Functional Studies: Pitfalls, Uncertainties, Good Practice for Clinical Usefulness)

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27 pages, 5305 KiB

Open AccessReview

Platelet Counting: Ugly Traps and Good Advice. Proposals from the French-Speaking Cellular Hematology Group (GFHC)

by Véronique Baccini, Franck Geneviève, Hugues Jacqmin, Bernard Chatelain, Sandrine Girard, Soraya Wuilleme, Aurélie Vedrenne, Eric Guiheneuf, Marie Toussaint-Hacquard, Fanny Everaere, Michel Soulard, Jean-François Lesesve and Valérie Bardet

J. Clin. Med. 2020, 9(3), 808; https://doi.org/10.3390/jcm9030808 - 16 Mar 2020

Cited by 60 | Viewed by 15427

Abstract

Despite the ongoing development of automated hematology analyzers to optimize complete blood count results, platelet count still suffers from pre-analytical or analytical pitfalls, including EDTA-induced pseudothrombocytopenia. Although most of these interferences are widely known, laboratory practices remain highly heterogeneous. In order to harmonize [...] Read more.

Despite the ongoing development of automated hematology analyzers to optimize complete blood count results, platelet count still suffers from pre-analytical or analytical pitfalls, including EDTA-induced pseudothrombocytopenia. Although most of these interferences are widely known, laboratory practices remain highly heterogeneous. In order to harmonize and standardize cellular hematology practices, the French-speaking Cellular Hematology Group (GFHC) wants to focus on interferences that could affect the platelet count and to detail the verification steps with minimal recommendations, taking into account the different technologies employed nowadays. The conclusions of the GFHC presented here met with a "strong professional agreement" and are explained with their rationale to define the course of actions, in case thrombocytopenia or thrombocytosis is detected. They are proposed as minimum recommendations to be used by each specialist in laboratory medicine who remains free to use more restrictive guidelines based on the patient’s condition. Full article

(This article belongs to the Special Issue Platelet Counting, Morphology Assessment and Functional Studies: Pitfalls, Uncertainties, Good Practice for Clinical Usefulness)

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18 pages, 1741 KiB

Open AccessReview

Strengths and Weaknesses of Light Transmission Aggregometry in Diagnosing Hereditary Platelet Function Disorders

by Marie-Christine Alessi, Pierre Sié and Bernard Payrastre

J. Clin. Med. 2020, 9(3), 763; https://doi.org/10.3390/jcm9030763 - 12 Mar 2020

Cited by 27 | Viewed by 10211

Abstract

Hereditary defects in platelet function are responsible for sometimes severe mucocutaneous hemorrhages. They are a heterogeneous group of abnormalities whose first-line diagnosis typically involves interpreting the results of in vitro light transmission aggregometry (LTA) traces. Interpretation of LTA is challenging. LTA is usually [...] Read more.

Hereditary defects in platelet function are responsible for sometimes severe mucocutaneous hemorrhages. They are a heterogeneous group of abnormalities whose first-line diagnosis typically involves interpreting the results of in vitro light transmission aggregometry (LTA) traces. Interpretation of LTA is challenging. LTA is usually performed in specialized laboratories with expertise in platelet pathophysiology. This review updates knowledge on LTA, describing the various platelet aggregation profiles typical of hereditary platelet disorders to guide the physician in the diagnosis of functional platelet disorders. Full article

(This article belongs to the Special Issue Platelet Counting, Morphology Assessment and Functional Studies: Pitfalls, Uncertainties, Good Practice for Clinical Usefulness)

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15 pages, 477 KiB

Open AccessReview

Platelet Function Test Use for Patients with Coronary Artery Disease in the Early 2020s

by Pierre Fontana, Marco Roffi and Jean-Luc Reny

J. Clin. Med. 2020, 9(1), 194; https://doi.org/10.3390/jcm9010194 - 10 Jan 2020

Cited by 12 | Viewed by 5503

Abstract

In the field of antithrombotics, precision medicine is of particular interest, as it may lower the incidence of potentially life-threatening side effects. Indeed, antiplatelet drugs such as P2Y₁₂ inhibitors are one of the most common causes of emergency admissions for drug-related adverse [...] Read more.

In the field of antithrombotics, precision medicine is of particular interest, as it may lower the incidence of potentially life-threatening side effects. Indeed, antiplatelet drugs such as P2Y₁₂ inhibitors are one of the most common causes of emergency admissions for drug-related adverse events. The last ten years have seen a continuous debate on whether platelet function tests (PFTs) should be used to tailor antiplatelet drugs to cardiovascular patients. Large-scale randomized studies investigating the escalation of antiplatelet therapies according to the results of PFTs were mostly negative. Potent P2Y₁₂ inhibitors are recommended as a first-line treatment in acute coronary syndrome patients, bringing the bleeding risk at the forefront. De-escalation from prasugrel or ticagrelor to clopidogrel is now considered, with or without the use of a PFT. This review covers recent advances in escalation and de-escalation strategies based on PFTs in various clinical settings. It also describes the main features of the most popular platelet function tests as well as the potential added value of genetic testing. Finally, we detail practical suggestions on how PFTs could be used in clinical practice. Full article

(This article belongs to the Special Issue Platelet Counting, Morphology Assessment and Functional Studies: Pitfalls, Uncertainties, Good Practice for Clinical Usefulness)

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9 pages, 1226 KiB

Open AccessReview

Sensitivity of Viscoelastic Tests to Platelet Function

by Marco Ranucci and Ekaterina Baryshnikova

J. Clin. Med. 2020, 9(1), 189; https://doi.org/10.3390/jcm9010189 - 10 Jan 2020

Cited by 49 | Viewed by 6742

Abstract

Viscoelastic tests provide a dynamic assessment of coagulation, by exploring the time to clot formation and the clot strength. Using specific activators or inhibitors, additional factors can be explored, like the fibrinogen contribution to clot strength. Since the early days, various attempts have [...] Read more.

Viscoelastic tests provide a dynamic assessment of coagulation, by exploring the time to clot formation and the clot strength. Using specific activators or inhibitors, additional factors can be explored, like the fibrinogen contribution to clot strength. Since the early days, various attempts have been done to measure platelet function with viscoelastic test. In general, the difference between the maximum clot strength and the fibrinogen contribution is considered an index of platelet contribution. However, this parameter does not clearly split platelet count from function; additionally, the extensive thrombin generation of standard activated viscoelastic tests activates platelet through the protease activated receptors, bypassing the other pathways. For this reason, standard viscoelastic tests cannot be used to assess platelet reactivity under the effects of aspirin or P2Y₁₂ inhibitors. To overcome this limitation, a specific test was developed (thromboelastography platelet mapping). This test has been compared with the gold standard of light transmission aggregometry and with other point-of-care tests, with conflicting results. In general, the use of viscoelastic tests to assess the effects of antiplatelet agents is still limited. Conversely, platelet contribution to clot strength in the setting of coagulopathic bleeding is considered an important parameter to trigger platelet transfusion or desmopressin. Full article

(This article belongs to the Special Issue Platelet Counting, Morphology Assessment and Functional Studies: Pitfalls, Uncertainties, Good Practice for Clinical Usefulness)

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Open AccessReview

Clinical Applications, Pitfalls, and Uncertainties of Thrombin Generation in the Presence of Platelets

by Marina Panova-Noeva, Paola E.J. van der Meijden and Hugo ten Cate

J. Clin. Med. 2020, 9(1), 92; https://doi.org/10.3390/jcm9010092 - 30 Dec 2019

Cited by 16 | Viewed by 4165

Abstract

Platelet-dependent thrombin generation is a helpful tool to assess ex vivo the interaction between platelets and plasma coagulation factors in the initiation, amplification, and inhibition of thrombin generation (TG). This review article discusses the most relevant available data on the clinical applications of [...] Read more.

Platelet-dependent thrombin generation is a helpful tool to assess ex vivo the interaction between platelets and plasma coagulation factors in the initiation, amplification, and inhibition of thrombin generation (TG). This review article discusses the most relevant available data on the clinical applications of fluorogenic TG, the most widely used TG assay, performed in the presence of platelets, i.e., in platelet-rich plasma. With respect to prothrombotic states, arterial hypertension and obesity were the most prominent cardiovascular conditions linked to increased platelet-dependent TG. In addition, platelet-associated hypercoagulability, assessed by the TG assay, has been shown in individuals with active cancer. In terms of bleeding, platelet-dependent TG has been applied to assess bleeding risk in individuals with hemophilia, von Willebrand disease, and Glanzmann thrombasthenia as well as in subjects with other congenital or acquired coagulation factor deficiencies. In addition to risk prediction, a role of the TG assay has been suggested in monitoring antiplatelet therapy in prothrombotic conditions and replacement therapy in bleeding diathesis. Finally, for the routine clinical use and as a biomarker of disease development and progression, better standardization and clinical validation of platelet-dependent TG are still needed. Full article

(This article belongs to the Special Issue Platelet Counting, Morphology Assessment and Functional Studies: Pitfalls, Uncertainties, Good Practice for Clinical Usefulness)

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8 pages, 499 KiB

Open AccessBrief Report

Characterization of Thrombin Generation Curve Shape in Presence of Platelets from Acute Venous Thromboembolism Patients

by Jeremy Lagrange, Bianca Wagner, Markus Nagler, Vincent ten Cate, Alejandro Pallares Robles, Thomas Koeck, Steffen Rapp, Jürgen H. Prochaska, Henri M. Spronk, Philip Wenzel, Wolfram Ruf, Hugo ten Cate, Philipp S. Wild and Marina Panova-Noeva

J. Clin. Med. 2020, 9(9), 2892; https://doi.org/10.3390/jcm9092892 - 7 Sep 2020

Cited by 2 | Viewed by 2282

Abstract

Background. Anticoagulant therapy, the cornerstone treatment in acute venous thromboembolism (VTE), strongly impacts thrombin generation (TG). Until now, the appearance of the TG curve in platelet rich plasma (PRP) from patients with acute VTE has not been investigated. Methods. We analyzed the shape [...] Read more.

Background. Anticoagulant therapy, the cornerstone treatment in acute venous thromboembolism (VTE), strongly impacts thrombin generation (TG). Until now, the appearance of the TG curve in platelet rich plasma (PRP) from patients with acute VTE has not been investigated. Methods. We analyzed the shape of TG curves measured in PARP of 180 acute VTE patients. Results. Normal shape of TG curves was observed in 110 patients, 50 patients showed no TG and 20 patients showed biphasic TG curve. The linear regression analysis, adjusted for age, sex, VTE clinical phenotypes and therapy showed that the appearance of biphasic curves is significantly associated with female sex, presence of cancer and therapy with Factor Xa inhibitors. Conclusions. This study demonstrated that despite taking anticoagulants, TG in presence of platelets is still present in the majority of acute VTE patients. Appearance of unusual TG curves is strongly related to the intake of anti-Factor Xa inhibitors. The clinical relevance of biphasic TG curve appearance requires further investigation. Full article

(This article belongs to the Special Issue Platelet Counting, Morphology Assessment and Functional Studies: Pitfalls, Uncertainties, Good Practice for Clinical Usefulness)

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11 pages, 1452 KiB

Open AccessBrief Report

Comparative In Vitro Study of Various α₂-Adrenoreceptor Agonist Drugs for Ticagrelor Reversal

by Guillaume Porta Bonete, Anne Godier, Pascale Gaussem, Tiphaine Belleville-Rolland, Alexandre Leuci, Sonia Poirault-Chassac, Christilla Bachelot-Loza and Anne-Céline Martin

J. Clin. Med. 2020, 9(3), 809; https://doi.org/10.3390/jcm9030809 - 16 Mar 2020

Cited by 1 | Viewed by 2524

Abstract

Ticagrelor, an antiplatelet adenosine diphosphate (ADP)-P2Y₁₂ receptor antagonist, increases the risk of bleeding. Its management is challenging because platelet transfusion is ineffective and no specific antidote is currently available. Epinephrine, a vasopressor catecholamine prescribed during shock, restores platelet functions inhibited by ticagrelor [...] Read more.

Ticagrelor, an antiplatelet adenosine diphosphate (ADP)-P2Y₁₂ receptor antagonist, increases the risk of bleeding. Its management is challenging because platelet transfusion is ineffective and no specific antidote is currently available. Epinephrine, a vasopressor catecholamine prescribed during shock, restores platelet functions inhibited by ticagrelor through stimulation of α_2A-adrenoreceptors. It subsequently inhibits cyclic adenosine monophosphate (cAMP) pathway and PI3K signaling. However, since epinephrine may expose a patient to deleterious hemodynamic effects, we hypothesized that other α₂-adrenoreceptor agonist drugs used in clinical practice with fewer side effects could reverse the antiplatelet effects of ticagrelor. We compared in vitro the efficacy of clonidine, dexmedetomidine, brimonidine, and norepinephrine with epinephrine to restore ADP- and PAR-1-AP-induced washed platelet aggregation inhibited by ticagrelor, as well as resulting platelet cAMP levels. In ticagrelor-free samples, none of the α₂-adrenoreceptor agonists induced aggregation by itself but all of them potentiated ADP-induced aggregation. Compared with epinephrine, norepinephrine, and brimonidine partially restored ADP- and fully restored PAR-1-AP-induced aggregation inhibited by ticagrelor while clonidine and dexmedetomidine were ineffective. Indeed, this lack of effect resulted from a lower decrease in cAMP concentration elicited by these partial α₂-adrenoreceptor agonists, clonidine, and dexmedetomidine, compared with full α₂-agonists. Our results support the development of specific full and systemic α₂-adrenoreceptor agonists for ticagrelor reversal. Full article

(This article belongs to the Special Issue Platelet Counting, Morphology Assessment and Functional Studies: Pitfalls, Uncertainties, Good Practice for Clinical Usefulness)

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