A Whole Blood Flow Cytometric Method for Diagnosis of Platelet Function Disorders

R.G. Dave¹, T. Geevar¹, R. V.¹, S. Singh¹, A. Abraham², A. Srivastava², S.C. Nair¹

¹Christian Medical College, Vellore, Department of Transfusion Medicine and Immunohaematology, Vellore, India, ²Christian Medical College, Vellore, Department of Haematology, Vellore, India

Abstract Number: PB1485

Meeting: ISTH 2020 Congress

Theme: Platelet Disorders and von Willebrand Disease » Platelet Function Disorders, Hereditary

Background: Light transmission aggregometry(LTA) is the standard for diagnosis of platelet function disorders(PFDs), but LTA requires a customized aggregometer, large volume of blood and often cannot be used in the setting of thrombocytopenia. Flow cytometry based platelet activation test(PACT) requires very small volume of blood, can be interpreted irrespective of the extent of thrombocytopenia, can be performed on whole blood in a flow cytometer which is increasingly available in diagnostic laboratories for various applications.

Aims: Aims of this study were to develop simplified PACT using whole blood for diagnosis of PFDs and to compare its performance with the current standard LTA.

Methods: Platelet function was assessed after informed consent by LTA(Chrono-log Corporation, Havertown, USA) and PACT on Navios flow cytometer(Beckman Coulter, Miami, USA) in 27 normal controls and cohort of 33 patients with clinical bleeding suspected to be PFD after exclusion of coagulation factor deficiencies. LTA was performed using agonists Ristocetin, ADP, Epinephrine, Collagen, Arachidonic Acid, thrombin receptor-activation peptide-6 (TRAP) and Thromboxane A2 receptor analogue (TRA). ATP release response to collagen and thrombin were assessed. PACT was performed using agonists ADP, convulxin, TRAP and ristocetin. Activation markers like P-selectin (CD62P), antibody against GpIIb-IIIa activation epitope (PAC-1) and anti-Von Willebrand Factor antibody were used. Both tests were compared for agreement using Cohen’s Kappa analysis.

Results: LTA and PACT had almost perfect agreement (kappa = 0.89, standard error 0.13). Using LTA as reference method, sensitivity and positive predictive value of PACT was 96.4%, while specificity and negative predictive value was 96.9%. In 12% of the patients, LTA was inconclusive due to thrombocytopenia while PACT could assess the platelet function. Table 1 shows findings of PACT in study population.

Conclusions: PACT can be alternative to LTA for diagnosis of PFD, especially in the setting of thrombocytopenia, pediatric patients or in the setup where aggregometer is not readily available.

Diagnosis	Number of cases / controls	Glycoprotein expression	Activation Marker Anti-Von Willebrand Factor Antibody:		Activation Marker PAC-1:		Activation Marker CD62P:
			Response to Normal dose Ristocetin	Response to Low dose Ristocetin	Response to ADP	Response to convulxin	Response to ADP	Response to convulxin	Response to TRAP
Bernard Soulier Syndrome	6	Absent expression of CD42b	Absent	Absent	Present	Present	Present	Present	Present
Glanzmann Thrombasthenia	5	Absent expression of CD41	Present	Absent	Absent	Absent	Present	Present	Present
Glanzmann Thrombasthenia Variant	9	Normal / dim expression of CD41	Present	Absent	Absent	Absent	Present	Present	Present
Acquired Platelet Dysfunction with Eosinophilia (APDE)	2	Normal expression of CD41 and CD42b	Present	Absent	Variable	Variable	Variable	Variable	Variable
Mixed Granule Deficiency	4	Normal expression of CD41 and CD42b	Present	Absent	Present	Present	Variable	Variable	Variable
Von Willebrand Disease Type 2B	2	Normal expression of CD41 and CD42b	Present	Present	Present	Present	Present	Present	Present
Normal platelet function with thrombocytopenia	5	Normal expression of CD41 and CD42b	Present	Absent	Present	Present	Present	Present	Present
Normal Controls	27	Normal expression of CD41 and CD42b	Present	Absent	Present	Present	Present	Present	Present

[Diagnosis and Platelet Activation Response to various agonist / activation markers in PACT]

To cite this abstract in AMA style:

Dave RG, Geevar T, RV, Singh S, Abraham A, Srivastava A, Nair SC. A Whole Blood Flow Cytometric Method for Diagnosis of Platelet Function Disorders [abstract]. Res Pract Thromb Haemost. 2020; 4 (Suppl 1). https://abstracts.isth.org/abstract/a-whole-blood-flow-cytometric-method-for-diagnosis-of-platelet-function-disorders/. Accessed January 26, 2022.

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