Evaluation of a New Approach of the Diagnosis of Constitutional Functional Disorders of Platelets

Multicentre Evaluation of a New Laboratory Approach for the Diagnosis of Constitutional Functional Disorders of Platelets

The primary purpose of the study is to evaluate a standardized method of screening for platelet signalling defects in patients with constitutional disorders of platelet function of unknown origin. We hypothesize that such defects are under-diagnosed in patients, due to heavy workup and requirement of relatively large blood sample by conventional biochemical methods. We propose to analyse kinase signalling downstream platelet membrane receptors using multiplex flow cytometry quantification and fluorescent platelet barcoding.

Study Overview

• Status: Completed
• Conditions: Platelet Dysfunction
• Intervention / Treatment: Other: Blood punction

Detailed Description

Little is known of the molecular basis of disorders of signalling pathways potentially responsible of constitutional defects of platelet functions (adhesion, aggregation or secretion) (1-5). Indeed, in routine practice, this investigation is limited by the complexity of the analyse using biochemical methods (western blotting), and the requirement of large amount of platelets.

We have designed a new approach for the quantification of platelet cytoplasmic phosphoproteins by flow cytometry. Fresh platelets in platelet rich plasma are analysed at baseline or after stimulation by major agonists (ADP, TRAP, thromboxane analogue, or collagen-related peptide), with and without relevant inhibitors of each pathway. Multiplex barcoding is used to identify each condition, allowing a high throughput analysis (6, 7). Platelet signalling profiling of Akt, Slp76, P38 MAPK and LIMK can be obtained from a blood sample of less than 10 ml, and within 6h The main objective of the study is to standardize the method between clinical laboratories with a standard expertise in flow cytometry. The study will be performed in 4 academic hospitals members of the French reference network of rare platelet diseases. Three groups of patients referred for mild or severe bleeding disorders will be included: 1) a control group of patients (30 per centre) with a bleeding disorder definitely other than of platelet origin (e.g. "low" von Willebrand); 2) a group of 10 patients per centre with definite constitutional platelet disorder (e.g. Glanzmann thrombasthenia) and 3) a group of 10 patients per centre with a defect of platelet function of unknown origin, potentially defective in signalling pathway.

The control group will serve to standardize the method between centres and to establish the reference values. A quality control will be set up by using frozen platelet preparations. The patients with definite platelet disorder will be useful for detecting potential signalling defects still not described in these pathologies. Platelet signalling defects which could be evidenced in these groups will be further identified by conventional biochemical and molecular methods after confirmation on a new sample.

If this new approach can be proposed to clinical laboratories working on rare platelet diseases, we expect an advance in our knowledge in the field. In addition the method has a potential in pharmaceutical innovation, for identifying (8) or monitoring new antiplatelet agents (9, 10), or identifying platelet defects induced by new "target therapies" designed for other diseases such as cancer or immune pathologies (10).

• Study Type: Interventional
• Enrollment (Actual): 322
• Phase: Not Applicable

Contacts and Locations

Study Locations

• France
  • Toulouse, France, 31000
    • University Hospital Toulouse

Participation Criteria

Eligibility Criteria

• Ages Eligible for Study: 18 years and older (Adult, Older Adult)
• Accepts Healthy Volunteers: No

Description

Inclusion Criteria:

• patient consulting for hemorrhagic symptomatology with a bleeding disorder definitely other than of platelet origin (e.g. "low" von Willebrand) / or constitutional platelet disorder (e.g. Glanzmann thrombasthenia) / or with a defect of platelet function of unknown origin, potentially defective in signalling pathway.
• Informed consent form
• patient with social security insurance or equivalent

Exclusion Criteria:

• treatment interfering with platelets function within 7 days prior to enrollment
• thrombocytopenia <100G/L
• pregnant or lactating females
• subjects under juridical protection guardianship or tutelage measure
• subjects involved in another clinical trial

Study Plan

How is the study designed?

Design Details

• Primary Purpose: Diagnostic
• Allocation: Non-Randomized
• Interventional Model: Parallel Assignment
• Masking: None (Open Label)

Number of Arms

3

Arms and Interventions

Participant Group / Arm	Intervention / Treatment
Other: bleeding disorder; Blood punction at patients with bleeding disorder definitely other than of platelet origin (e.g. "low" von Willebrand)	Other: Blood punction; T1 and T2: one blood punction with signalisation test (T1 for each arm, T2 at +6 months only if an anomaly at signalisation test is detected) signalisation test is made by flow cytometry Stage 1: for arm "bleeding disorder" : flow cytometry analysis to establish reference values observed in subjects without thrombopathy for each marker after agonist stimulation and corresponding search of an effect if a center-center effect is observed on the reference values its origin will be sought and corrected. Stage 2: for the two others arms: Several markers will be analyzed, corresponding to routes or levels of different signaling. For each of these markers, the test will evaluate quantitatively the phosphorylation activity of the protein tested.
Other: constitutional platelet disorder; Blood punction at patients with constitutional platelet disorder (e.g. Glanzmann thrombasthenia)	Other: Blood punction; T1 and T2: one blood punction with signalisation test (T1 for each arm, T2 at +6 months only if an anomaly at signalisation test is detected) signalisation test is made by flow cytometry Stage 1: for arm "bleeding disorder" : flow cytometry analysis to establish reference values observed in subjects without thrombopathy for each marker after agonist stimulation and corresponding search of an effect if a center-center effect is observed on the reference values its origin will be sought and corrected. Stage 2: for the two others arms: Several markers will be analyzed, corresponding to routes or levels of different signaling. For each of these markers, the test will evaluate quantitatively the phosphorylation activity of the protein tested.
Other: defect of platelet function; Blood punction at patients with defect of platelet function of unknown origin, potentially defective in signalling pathway.	Other: Blood punction; T1 and T2: one blood punction with signalisation test (T1 for each arm, T2 at +6 months only if an anomaly at signalisation test is detected) signalisation test is made by flow cytometry Stage 1: for arm "bleeding disorder" : flow cytometry analysis to establish reference values observed in subjects without thrombopathy for each marker after agonist stimulation and corresponding search of an effect if a center-center effect is observed on the reference values its origin will be sought and corrected. Stage 2: for the two others arms: Several markers will be analyzed, corresponding to routes or levels of different signaling. For each of these markers, the test will evaluate quantitatively the phosphorylation activity of the protein tested.

What is the study measuring?

Primary Outcome Measures

Outcome Measure	Measure Description	Time Frame
standardise the method between clinical laboratories	This is a pilot study of a new approach of screening platelet signalling pathways disorders by flow cytometry with methodological primary outcomes (12) as follows: 1)to standardise the method between clinical laboratories, 2)to establish reference values of the method and 3)to design a quality control assessment The primary endpoint will focus on the overall variability of reference values observed in subjects without thrombopathy for each marker after agonist stimulation and corresponding search of an effect if a center-center effect is observed on the reference values its origin will be sought (deviation from the protocol, reagent lot .....) and corrected. The "center effect" will also be analyzed by the quarterly quality control, which will compare the results obtained from the same wafer tests analyzed simultaneously in three sites.	3 years

Secondary Outcome Measures

Outcome Measure	Measure Description	Time Frame
To describe the signalling pathways in platelet disorders	To describe the signalling pathways in platelet disorders with definite diagnosis To screen putative defects in bleeding patients with disorders of platelet function of unknown origin To identify more deeply the defects found in these groups. Several markers will be analyzed, corresponding to routes or levels of different signaling. For each of these markers, the test will evaluate quantitatively the phosphorylation activity of the protein tested. We can thus determine for each subject and each marker, the response to the test defined by: The percentage increase in the signal (relative to the rest) after stimulation with a receptor agonist,; The percentage increase in the signal (relative to the rest) in the presence of the agonist to the associated receptor antagonist.	3 years

Collaborators and Investigators

• Sponsor: University Hospital, Toulouse
• Investigators: Principal Investigator: Pierre SIE, MD PhD, University Hospital, Toulouse

Publications and helpful links

General Publications

• Garcia C, Dejean S, Savy N, Bordet JC, Series J, Cadot S, Ribes A, Voisin S, Rugeri L, Payrastre B, Sie P. Multicolor flow cytometry in clinical samples for platelet signaling assessment. Res Pract Thromb Haemost. 2023 May 16;7(4):100180. doi: 10.1016/j.rpth.2023.100180. eCollection 2023 May.

Study record dates

Study Major Dates

• Study Start: February 1, 2013
• Primary Completion (Actual): June 29, 2017
• Study Completion (Actual): June 29, 2017

Study Registration Dates

• First Submitted: June 18, 2013
• First Submitted That Met QC Criteria: September 30, 2013
• First Posted (Estimated): October 8, 2013

Study Record Updates

• Last Update Posted (Estimated): December 10, 2025
• Last Update Submitted That Met QC Criteria: December 2, 2025
• Last Verified: April 1, 2019

More Information

Terms related to this study

• Keywords: constitutional functional disorders of platelets; platelet signalling defects
• Additional Relevant MeSH Terms: Pathologic Processes
• Other Study ID Numbers: 12 068 08; PHRCI 2012 (Other Grant/Funding Number: French Ministry of Health)