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PL-chip

Summary

The PL-chip was specifically designed for the quantitative analysis of platelet thrombus formation process involving platelet adhesion & aggregation, granule secretion, and thrombus growth.

The platelet thrombus formation process can be quantified and visualized by measuring flow-pressure waveform and video microscopy, respectively.


Microscope video

Microscope video

Platelet thrombus formation inside PL-chip at shear rates of 1000 (left) and 2000 s-1 (right)

Characteristics of the PTF-assay using PL-chip

  • Single-use microchip (PL-chip) produced by a high-precision injection molding
  • Small amount of whole blood sample (350 μl)
  • Simple setting of the optional wall shear rate (500-2500 s-1)
  • Dual-monitoring system (by flow pressure waveform and real-time video imaging)
  • Easy operation controlled by a computer interlocking system

Assay principle

A blood sample is placed in the reservoir that is connected to a high-precision micro pump. The micro pump infuses mineral oil into the reservoir, which impels the sample blood through the inlet port and into the flow path that leads to a collagen-coated analytical path consisting of 25 capillary channels.

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The process of platelet thrombus formation in the flow chamber is continuously monitored by a video-microscope (10x) positioned under the flow chamber and by a pressure sensor that tracks pressure changes in the flow path. The flow rate of the micro pump can be easily adjusted as needed based on the desired experimental conditions (range: 4-40 μl/min).
When blood flows through the analytical path of the microchip, platelets adhere and aggregate on the surface of the collagen-coated capillary channels. The platelet aggregates gradually increase in size, and in the process, occlude the capillary, resulting in an increase of flow pressure.

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Parameters

Flow pressure analysis

When blood flows through the analytical path of the PL-chip, platelets adhere and aggregate on the surface of the collagen-coated capillary channels. The platelet aggregates gradually increase in size and eventually occlude the capillary, resulting in an increase of flow pressure. Thus, flow pressure patterns reflect the platelet thrombus formation process.

 

Definitions of the parameters used for quantification of the platelet thrombus formation process

Two specific terms, T10 and AUC10, are used to quantify platelet thrombus formation inside the PL-chip.

T10 (= time to reach 10 kPa) is defined as the onset of platelet thrombus formation and represents the duration (sec) for the flow pressure to increase to 10 kPa from baseline.

AUC10 (= area under the flow pressure curve for 10 min) represents total thrombogenicity and reflects onset time, as well as growth and stability, of formed thrombi.

 

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Antiplatelet agents

In general, direct inhibition of platelet adhesion and aggregation by GPIbα- or GPIIbIIIa-inhibitors causes a shift in the flow pressure waveform (i.e., delay of onset). In contrast, inhibitions of auto- and para-crine activations mediated by granule secretion by aspirin and clopidogrel suppress a rise of the pressure curve, and often show parabolic flow pressure patterns due to the reduced stability and sustainability of thrombi.

Thus, flow-pressure waveforms, as well as parameters of T10 and AUC10, provide useful information to analyze the effects of antiplatelet agents and to diagnose congenital and acquired platelet dysfunctions.

Assay protocol

1.Place a PL-chip on the assay stage and let stand for 1-3 min to allow stabilization of the temperature.
2.Connect the reservoir to the nozzle, and pipette 350 μl of hirudin-treated whole blood into the reservoir.
3.Close the lid, and then remove its cap.
4.Connect the reservoir to the PL-chip.
  5.Push the start button.

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