A comparative study between light transmission aggregometry and flow cytometric platelet aggregation test for the identification of platelet function defects in patients with bleeding

Praveen Sharma; Man Updesh Singh Sachdeva; Narender Kumar; Sunil Bose; Parveen Bose; Varun Uppal; Pankaj Malhotra; Deepak Bansal; Neelam Varma; Jasmina Ahluwalia

doi:10.5045/br.2021.2020232

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Original Article

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Blood Res 2021; 56(2):

Published online June 30, 2021

https://doi.org/10.5045/br.2021.2020232

A comparative study between light transmission aggregometry and flow cytometric platelet aggregation test for the identification of platelet function defects in patients with bleeding

Praveen Sharma¹, Man Updesh Singh Sachdeva¹, Narender Kumar¹, Sunil Bose¹, Parveen Bose¹, Varun Uppal¹, Pankaj Malhotra², Deepak Bansal³, Neelam Varma¹, Jasmina Ahluwalia¹

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¹Department of Hematology, ²Clinical Hematology Division, Department of Internal Medicine, ³Pediatric Hematology-Oncology Division, Department of Pediatrics, Advanced Pediatric Centre, Postgraduate Institute of Medical Education and Research, Chandigarh, India

Correspondence to : Jasmina Ahluwalia, M.D.
Department of Hematology, Postgraduate Institute of Medical Education and Research, 5th floor, Research block A, Chandigarh 160012, India
E-mail: jasminapgi@gmail.com

Received: September 8, 2020; Revised: June 10, 2021; Accepted: June 18, 2021

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

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Abstract

Background
Platelet aggregation studies using conventional light transmission aggregometry (LTA) have several disadvantages and require strict pre-analytical measures for reliable results. We aimed to examine the utility of flow cytometric platelet aggregation (FCA) assay in detecting platelet function defects (PFDs) in patients with a history of bleeding symptoms.
Methods
Sixty-four participants (24 patients and 40 healthy controls) were included in this study. LTA and FCA assay were performed simultaneously in patients and healthy controls. In the FCA assay, two portions of platelets from the same individual were labeled separately with CD31-FITC and CD31-PE. After mixing and stimulation with agonists, the double- colored platelet aggregates were visualized using a flow cytometer. The results generated using the two techniques were compared and correlated.
Results
The patients' median age was 17 years (range, 3‒72 yr) with a male-to-female ratio of 1:1.7. There was substantial agreement between LTA and FCA assay in detecting a PFD (=0.792). Four patients showing a Glanzmann thrombasthenia-like pattern on LTA exhibited an abnormal FCA. A functional defect in collagen binding was detected on the FCA assay conducted in two immune thrombocytopenic patients with severe bleeding.
Conclusion
FCA assay can be used to identify functional defects in platelets, with potential applications in thrombocytopenic individuals. It also facilitates the diagnosis of inherited bleeding disorders with platelet defects.

Keywords Platelet function defect, Platelet aggregometry, Flow cytometry, Light transmission aggregometry, Bleeding disorder

Article

Original Article

Blood Res 2021; 56(2): 109-118

Published online June 30, 2021 https://doi.org/10.5045/br.2021.2020232

A comparative study between light transmission aggregometry and flow cytometric platelet aggregation test for the identification of platelet function defects in patients with bleeding

Praveen Sharma¹, Man Updesh Singh Sachdeva¹, Narender Kumar¹, Sunil Bose¹, Parveen Bose¹, Varun Uppal¹, Pankaj Malhotra², Deepak Bansal³, Neelam Varma¹, Jasmina Ahluwalia¹

Correspondence to:Jasmina Ahluwalia, M.D.
Department of Hematology, Postgraduate Institute of Medical Education and Research, 5th floor, Research block A, Chandigarh 160012, India
E-mail: jasminapgi@gmail.com

Received: September 8, 2020; Revised: June 10, 2021; Accepted: June 18, 2021

Abstract

Keywords: Platelet function defect, Platelet aggregometry, Flow cytometry, Light transmission aggregometry, Bleeding disorder

Abstract

Fig 1.

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Figure 1.Flow cytometric platelet aggregation of a healthy control. Dot plots showing platelets in unstimulated (t=0 min) and ADP-stimulated state (t=10 min) in a healthy control (left and right, respectively). The platelets are gated on a log forward (FSC) and side scatter (SSC) (upper panel) and examined on CD31-FITC and CD31-PE combination (lower panel). The double-colored events (Q2) represent the platelet aggregates, Q1 represents the CD31-PE-labeled platelets, Q4 represents the CD31-FITC-labeled platelets, and Q3 represents the unstained events (plasma, debris, etc.). The % double-colored events in this control in unstimulated t=0 min and ADP-stimulated platelet mix at t=10 min are 1.8% and 41.4% respectively.

Blood Research 2021; 56: 109-118https://doi.org/10.5045/br.2021.2020232

Fig 2.

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Figure 2.Scatter diagrams showing the distribution of test results. The % maximum amplitude of response to ADP, collagen, and ristocetin in light transmission aggregometry (A) and the % double-colored events of ADP-, collagen-, and ristocetin-stimulated platelet mix on flow cytometric platelet aggregation assay of healthy controls and patients are shown (B).

Blood Research 2021; 56: 109-118https://doi.org/10.5045/br.2021.2020232

Fig 3.

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Figure 3.Scatter plots showing the test results of patients 1, 2, 3, and 4 with a GT-like pattern on light transmission aggregometry (LTA). (A) % aggregation of LTA and (B) % aggregation of flow cytometric platelet aggregation to agonists ADP, collagen, and ristocetin are plotted. Note that patient 1 in the Fig. 3A has superimposed ADP and collagen values.

Blood Research 2021; 56: 109-118https://doi.org/10.5045/br.2021.2020232

Fig 4.

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Figure 4.Flow cytometric platelet aggregation assay of a patient with Glanzmann thrombasthenia (GT). Dot plots showing platelets in unstimulated (t=0 min) and agonist-stimulated state (t=10 min) in a GT patient. The % double-colored events in this patient in unstimulated t=0 min and ADP-, collagen-, and ristocetin-stimulated platelet mix at t=10 min are 0.6% and 0.7%, and 14.3% and 29.9% respectively.

Blood Research 2021; 56: 109-118https://doi.org/10.5045/br.2021.2020232

Table 1 . Demographic and baseline laboratory data of the study patients..

Parameter	Results
N	22^a)
Median age in years (range)	17 (3–72)
Median BAT score (range)	4.5 (3–11)
PT in seconds: mean (±2SD)	13 (±2.83)
aPTT in seconds: mean (±2SD)	30.9 (±6.85)
Fibrinogen level in g/dL: mean (±2SD)	3.17 (±1.43)
Hemoglobin in g/L: median (range)	113 (57–148)
Total leucocyte count ×10⁹/L: median (range)	7.2 (2.7–113.5)
Platelet count ×10⁹/L: median (range)	217 (123–425)
VWF antigen assay: % median (range)	123.05 (68.8–223.6)
VWF GPIbR (RiCoF activity) assay: % median (range)	109.3 (58.7–221.3)

^a)Includes 22 non-thrombocytopenic (parallel LTA and FCA data available). Two thrombocytopenic patients (only FCA data available) were discussed separately..

Abbreviations: aPTT, activated partial thromboplastin time; BAT, bleeding assessment tool; PT, prothrombin time; RiCoF, ristocetin cofactor activity assay; VWF, von Willebrand factor..

Table 2 . Agreement between the results of light transmission aggregometry and flow cytometric platelet aggregation assay performed simultaneously in 22 patients..

Technique	Flow cytometric platelet aggregation
Light transmissi on aggregometry		Normal	Abnormal
	Normal	14	2
	Abnormal	0	6

Table 3 . Studies utilizing the flow cytometric platelet aggregation technique..

Research study	Sample size	Patients	Platelet labelling	N of platelet agonists tested	Platelet agonists used	Remarks
De Cuyper et al. (2013) [5]	NA	Human and mouse platelets	1.CD31 for labelling of platelets in whole blood 2.CFSE and PKH26 dyes for washed platelets	3–4	PMA, type I collagen, Aggretin A, or ristocetin	Platelet aggregation using flow cytometry can be performed with small starting volume or low platelet count.
van Bladel et al. (2014) [6]	33	Pediatric chronic ITP patients	CFSE and PKH26 dyes for washed platelets	2	PMA or ristocetin	Decreased platelet function is seen in patients with severe bleeding phenotype.
Vinholt et al. (2017) [7]	20	TCP patients diagnosed with acute myeloid leukemia or myelodysplastic syndrome	CAMU and CV450 dyes for washed platelets	3	Collagen-related peptide TRAP and ADP	Platelet aggregation assay is applicable in TCP patients to identify a bleeding risk.
Present study (2020)	24	Bleeding patients suspected to have a platelet function defect including two TCP patients	CD31 for washed platelets	3	ADP, type-I collagen, Ristocetin	FCA is a potential technique for identification of PFD comparable to LTA, with applicability in TCP patients.

Abbreviations: ADP, adenosine diphosphate; AM, acetoxymethyl ester; CAMU, calcein acetoxymethyl ester ultrapure grade; CFSE, carboxyfluorescein diacetate succinimidyl ester; CV450, calcein-AM Violet 450; FCA, flow cytometric platelet aggregation; ITP, immune thrombocytopenic purpura; LTA, light transmission aggregometry; NA, not available; PFD, platelet function defect; PKH26, PKH26 Red Fluorescent Cell Linker Kit for General Cell Membrane Labeling; PMA, phorbol myristate acetate; TCP, thrombocytopenia; TRAP, thrombin receptor-activating peptide..