Blood Res.  2021 Jun;56(2):109-118. 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

• Sharma P
• Sachdeva MUS
• Kumar N
• Bose S
• Bose P
• Uppal V
• Malhotra P
• Bansal D
• Varma N
• Ahluwalia J

• KMID: 2517066
• DOI: http://doi.org/10.5045/br.2021.2020232

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 (κ=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.

Keyword

Platelet function defect; Platelet aggregometry; Flow cytometry; Light transmission aggregometry; Bleeding disorder

Figure

• 

  Fig. 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.

• 

  Fig. 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).

• 

  Fig. 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.

• 

  Fig. 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.

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