Clinical trial NCT03377439: The Association Between Platelet Reactivity and Bleeding Risk in Adult ITP
Official title: The Association Between Platelet Reactivity and Bleeding Risk in Adult ITP Patients: a Prospective Multicenter Double-blind Study
Study type: OBSERVATIONAL
Overall status: UNKNOWN
Conditions: Immune Thrombocytopenia
Keywords: Platelet function
Bleeding risk
Immune Thrombocytopenia
Lead sponsor: Shandong University
Sponsor class: OTHER
Collaborators: name: Traditional Chinese Medicine Hospital Affiliated to Shandong University
name: Jinan Central Hospital
name: Qianfoshan Hospital
name: Shandong Provincial Hospital
name: Shandong University Second Hospital
First posted: 2017-12-19
Enrollment: 400
Sex: ALL
Minimum age (years): 18
Maximum age (years): 80
Standard ages: ADULT
OLDER_ADULT
Accepts healthy volunteers: yes
Observational model: COHORT
Time perspective: PROSPECTIVE
Sampling method: NON_PROBABILITY_SAMPLE
Study population: Untreated adult ITP patients of both genders between the ages of 18 and 80 years.

Brief summary: It seems reasonable to assume that patients who present significant bleeding symptoms may have different quality of platelets than those without bleeding. This question was addressed in a study that examined platelet function in adult ITP patients, which try to determine whether this correlated with bleeding risk. Previous reports have suggested that measuring platelet function may help define patients at highest risk of bleeding. In addition, Middelburg and colleagues corrected platelet function for quartile of platelet count, using \<32×10\^9/L as the lowest cohort and \>132×10\^9/L as the top quartile. They demonstrated that increased platelet reactivity (as measured by flow cytometry) was associated with decreased risk of bleeding but particularly for those patients with the lowest platelet counts. Further studies in a larger cohort are needed to confirm this correlation. Our study aimed at standardizing a prediction model to evaluate the bleeding risk of adult ITP patients with the use of platelet function tests.

Detailed description: The investigators are undertaking a prospective multicenter double-blind study of 400 adult patients with immune thrombocytopenia from 6 medical centers in China. We adopted three different assays that examined platelet function and reactivity. 1) Flow cytometry: Citrate anticoagulated whole blood was diluted in PBS to result in 20×10\^9/L platelets, and 20 μl was aliquoted into polystyrene test tubes. Ten microliters of anti-CD42b-PE was added and incubated at room temperature for 10 min. Agonists (TRAP-6 12.5 μMol/L, Collagen 20 μg/mL, ADP 2 μM, Epinephrin 20 μM, Arachidonic acid 0.275 mM, Ristocetin 1.5 mg/mL) or PBS were added (10 μl each) and incubated again for 10 min. Then mAb PAC-1-FITC or anti-CD62p-FITC (10 μl each) or the corresponding isotype-matched controls were added. After 15-min incubation in the dark, the reaction was stopped with 500 μl PBS. Samples were analyzed on a flow cytometer (FACScan, Becton-Dickinson) by measuring 10,000 events in the CD42b-positive fraction. 2) Filopodia quantification: Briefly, platelets in Tyrode's buffer were allowed to adhere to VWF (9×10\^6 cells/coverslip) in the presence of botrocetin (1 μg/mL) and Integrilin (40 μg/mL) at 37°C. After 15 min, non-adherent platelets were removed by washing and adherent platelets were fixed with 4% PFA, stained with TRITC-phalloidin (2 μg/mL) and viewed by epifluorescence microscopy for filopodia count. 3) Platelet aggregation: Measured on an automated platelet aggregation analyzer.

Understanding bleeding risk and underlying determinants of bleeding is important in order to help recognize patients that will require pharmacologic therapy even at higher platelet counts. Previous studies have suggested that low platelet counts, increased patient age, use of concurrent medications, and male sex are associated with increased bleeding risk. The present investigation will answer whether platelet function predicts the severity of bleeding in adult ITP patients. Clinical information of recruited participants includes gender, age, platelet count and physical/laboratory examination. Blinding was set between investigators who evaluated bleeding risks and those who performed experiments.

Eligibility criteria: Inclusion Criteria:

1. Untreated adult ITP patients of both genders between the ages of 18 and 80 years.
2. Participants of either acute or chronic phase; with or without thrombocytopenia; with or without bleeding manifestation.

Exclusion Criteria:

1. Received high-dose steroids or IVIG within 3 weeks prior to the test.
2. Received second-line ITP-specific treatments (eg, cyclophosphamide, 6-mercaptopurine, vincristine, vinblastine, etc) within 3 months prior to the test.
3. Current HIV infection, hepatitis B virus or hepatitis C virus infections.
4. Severe medical condition (liver and kidney function impairment).

Primary outcomes:
measure: The severity of bleeding manifestations at onset was assessed using a previously described clinical scoring system with minor modifications. The total bleeding score was calculated by adding the scores for each item.
timeFrame: No more than three months after platelet function assessment.
Secondary outcomes:
Other outcomes:
Locations:
city: Jinan
country: China
facility: Qilu Hospital, Shandong University
state: Shandong
Study officials:
affiliation: Qilu Hospital, Shandong University
name: Jun Peng, MD, PhD
role: STUDY_DIRECTOR
References:
citation: Khellaf M, Michel M, Schaeffer A, Bierling P, Godeau B. Assessment of a therapeutic strategy for adults with severe autoimmune thrombocytopenic purpura based on a bleeding score rather than platelet count. Haematologica. 2005 Jun;90(6):829-32.
citation: Middelburg RA, Roest M, Ham J, Coccoris M, Zwaginga JJ, van der Meer PF. Flow cytometric assessment of agonist-induced P-selectin expression as a measure of platelet quality in stored platelet concentrates. Transfusion. 2013 Aug;53(8):1780-7. doi: 10.1111/trf.12001. Epub 2012 Dec 7.
citation: Middelburg RA, Carbaat-Ham JC, Hesam H, Ragusi MA, Zwaginga JJ. Platelet function in adult ITP patients can be either increased or decreased, compared to healthy controls, and is associated with bleeding risk. Hematology. 2016 Oct;21(9):549-51. doi: 10.1080/10245332.2016.1180097. Epub 2016 May 9.
citation: Panzer S, Hocker L, Koren D. Agonists-induced platelet activation varies considerably in healthy male individuals: studies by flow cytometry. Ann Hematol. 2006 Feb;85(2):121-5. doi: 10.1007/s00277-005-0029-5. Epub 2005 Nov 10.
citation: Panzer S, Rieger M, Vormittag R, Eichelberger B, Dunkler D, Pabinger I. Platelet function to estimate the bleeding risk in autoimmune thrombocytopenia. Eur J Clin Invest. 2007 Oct;37(10):814-9. doi: 10.1111/j.1365-2362.2007.01855.x. Epub 2007 Aug 28.
citation: Mangin P, Yuan Y, Goncalves I, Eckly A, Freund M, Cazenave JP, Gachet C, Jackson SP, Lanza F. Signaling role for phospholipase C gamma 2 in platelet glycoprotein Ib alpha calcium flux and cytoskeletal reorganization. Involvement of a pathway distinct from FcR gamma chain and Fc gamma RIIA. J Biol Chem. 2003 Aug 29;278(35):32880-91. doi: 10.1074/jbc.M302333200. Epub 2003 Jun 17.
citation: Maurer E, Tang C, Schaff M, Bourdon C, Receveur N, Ravanat C, Eckly A, Hechler B, Gachet C, Lanza F, Mangin PH. Targeting platelet GPIbbeta reduces platelet adhesion, GPIb signaling and thrombin generation and prevents arterial thrombosis. Arterioscler Thromb Vasc Biol. 2013 Jun;33(6):1221-9. doi: 10.1161/ATVBAHA.112.301013. Epub 2013 Apr 4.