RP% Measurement by FCM as a Diagnostic Test for ITP

A Multi-Center Prospective Blind Study of RP% Measurement by Flow Cytometry in the Diagnosis of Primary Immune Thrombocytopenia

Immature platelets-also termed reticulated platelets (RP)-are platelets newly released into the circulation, and have been associated with a variety of pathological bleeding events including primary immune thrombocytopenia (ITP). They can be assessed by flow cytometry (FCM) after staining with thiazole orange (TO) at low concentration and expressed as a fraction of the total platelet count (RP%). The diagnosis of primary ITP is based on differential diagnosis and the measurement of RP% can serve as an alternative diagnostic test that are useful in daily practice. Our study aimed at distinguishing primary ITP from other thrombocytopenic disorders, especially aplstic (hypoplastic) or chemotherapy-induced thrombocytopenia by FCM. The sensitivity and specificity of the assay as well as agreement between RP% measurement and monoclonal antibody-specific immobilization of platelet antigen (MAIPA) were analyzed accordingly.

Study Overview

• Status: Unknown
• Conditions: Immune Thrombocytopenia
• Intervention / Treatment: Other: RP% Measurement by FCM as a Diagnostic Test for ITP

Detailed Description

The investigators are undertaking a multi-center, prospective blind trial of 500 adults with thrombocytopenic disorders with a platelet count less than 60*10^9/L from 4 medical centers in China. In brief, 15 μl aliquots of anti-coagulated whole blood were incubated for 70 min with 5 μl of phycoerythrin-conjugated anti-CD42b monoclonal antibody (BD Pharmingen, Tokyo, Japan) and 1 ml of thiazole orange (Retic-COUNT; Becton-Dickinson, San Jose, CA, USA) diluted 10 times by phosphate-buffered saline. RP% was analyzed on a flow cytometer (FACScan, Becton-Dickinson) by measuring 10,000 events in the CD42b-positive fraction.

Clinical information of all participants including gender, age, platelet count and definitive diagnosis were recorded by an exclusive investigator. RP% results were revealed at the end of recruitment and after all FCM measurements were completed. The agreement between clinical diagnosis and RP% results were analyzed to identify primary ITP.

• Study Type: Observational
• Enrollment (Anticipated): 500

Contacts and Locations

Study Locations

• China
  • Shandong
    • Jinan, Shandong, China
      • Recruiting
      • Qilu Hospital, Shandong University
      • Contact: Ming Hou, Dr.; Phone Number: 9879 +86-531-82169114; Email: houming@medmail.com.cn
      • Principal Investigator: Ming Hou, Dr.

Participation Criteria

Eligibility Criteria

• Ages Eligible for Study: 18 years to 75 years (ADULT, OLDER_ADULT)
• Accepts Healthy Volunteers: No
• Genders Eligible for Study: All

• Sampling Method: Probability Sample

Study Population

500 adults with thrombocytopenic disorders with a platelet count less than 60000/uL from 4 medical centers in China.

Description

Inclusion Criteria:

1. Untreated adult patients of both gender between the ages of 18 and 75 years
2. Each participant showed a platelet count 60*10^9/L, with or without bleeding manifestations
3. Thrombocytopenic disorders including autoimmune-mediated, aplastic (hypoplastic) or chemotherapy-induced thrombocytopenia

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). Unstable cardiovascular disease or uncontrolled hypertension.
5. Patients who are deemed unsuitable for the study by the investigator

Study Plan

How is the study designed?

Number of groups / cohorts

1

Cohorts and Interventions

Group / Cohort

Intervention / Treatment

RP% Measurement by FCM as a Diagnostic Test for ITP; The investigators are undertaking a multi-center, prospective blind trial of 500 adults with thrombocytopenic disorders with a platelet count less than 60000/uL from 4 medical centers in China. In brief, 15 ul aliquots of anti-coagulated whole blood were incubated for 70 min with 5 ul of phycoerythrin-conjugated anti-CD42b monoclonal antibody (BD Pharmingen, Tokyo, Japan) and 1 ml of thiazole orange (Retic-COUNT; Becton-Dickinson, San Jose, CA, USA) diluted 10 times by phosphate-buffered saline. RP% was analyzed on a flow cytometer (FACScan, Becton-Dickinson) by measuring 10,000 events in the CD42b-positive fraction.

Other: RP% Measurement by FCM as a Diagnostic Test for ITP

What is the study measuring?

Primary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Total platelet count (RP%)	We defined an upper limit for healthy control subjects as mean + 3SD in this study.	The day upon enrollment

Collaborators and Investigators

• Sponsor: Shandong University
• Collaborators: Qianfoshan Hospital; Jinan Central Hospital

Publications and helpful links

General Publications

• Sakuragi M, Hayashi S, Maruyama M, Kabutomori O, Kiyokawa T, Nagamine K, Kato H, Kashiwagi H, Kanakura Y, Tomiyama Y. Clinical significance of IPF% or RP% measurement in distinguishing primary immune thrombocytopenia from aplastic thrombocytopenic disorders. Int J Hematol. 2015 Apr;101(4):369-75. doi: 10.1007/s12185-015-1741-0. Epub 2015 Jan 25.
• Ibrahim H, Nadipalli S, Usmani S, DeLao T, Green L, Kleiman NS. Detection and quantification of circulating immature platelets: agreement between flow cytometric and automated detection. J Thromb Thrombolysis. 2016 Jul;42(1):77-83. doi: 10.1007/s11239-016-1338-3.

Study record dates

Study Major Dates

• Study Start: November 1, 2016
• Primary Completion (ANTICIPATED): December 1, 2017
• Study Completion (ANTICIPATED): December 1, 2017

Study Registration Dates

• First Submitted: November 16, 2016
• First Submitted That Met QC Criteria: November 17, 2016
• First Posted (ESTIMATE): November 18, 2016

Study Record Updates

• Last Update Posted (ESTIMATE): November 18, 2016
• Last Update Submitted That Met QC Criteria: November 17, 2016
• Last Verified: November 1, 2016

More Information

Terms related to this study

• Keywords: Immune Thrombocytopenia; Reticulated Platelets
• Additional Relevant MeSH Terms: Pathologic Processes; Immune System Diseases; Autoimmune Diseases; Hematologic Diseases; Hemorrhage; Hemorrhagic Disorders; Blood Coagulation Disorders; Skin Manifestations; Blood Platelet Disorders; Thrombotic Microangiopathies; Purpura, Thrombocytopenic; Purpura; Purpura, Thrombocytopenic, Idiopathic; Thrombocytopenia
• Other Study ID Numbers: ITP-Reticulated Platelets