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AR-chip

Summary

The AR-chip was specifically designed for the quantitative analysis of the white thrombus formation process mediated by the activation of both platelets and coagulation system under flow conditions. Development of white thrombi consisting of activated platelets and fibrin can be analyzed and visualized by measuring flow pressure waveforms and video-microscopic inspection, respectively.


Microscope video

Microscope video

White thrombus formation inside an AR-chip at shear rates of 240 (left) and 600 s-1 (right)

Image analysis of white thrombi formation

img-ar_08

Characteristics of the AR-chip assay

  • Uses a tissue thromboplastin- and collagen-coated microchip
  • Requires only a small amount of whole blood (450 μl)
  • Simple setting of optional wall shear rate (200-800 s-1
  • Dual-monitoring system (by flow pressure waveform and real-time video imaging)
  • Easy operation controlled by the computer interlocking system

Assay principle

A recalcified blood sample containing CTI (corn trypsin inhibitor) is placed in a 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 the tissue thromboplastin- and collagen-coated analytical path.
The process of white thrombus formation (WTF) in the flow chamber is continuously monitored by a video-microscope (10x) positioned under the flow chamber and by a pressure sensor that records 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 AR-chip, the extrinsic coagulation pathway and platelets are simultaneously activated on the tissue thromboplastin- and collagen-coated surfaces. The activated coagulation system and platelets then amplify their respsonses resulting in thrombus growth, which occludes the capillary and increases the flow pressure. 

Parameters

Flow pressure analysis

When blood flows through the analytical path of AR-chip, the platelets and coagulation system are simultaneously activated on the collagen- and tissue thromboplastin-coated surface. Small white thrombi that consist of activated platelets and fibrin fibers gradually increase in size and eventually occlude the capillary, resulting in an increase of flow pressure. Thus, flow pressure patterns reflect the white thrombus formation process.

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

Three specific parameters, T10, T80, and AUC30, are used to quantify white thrombus formation inside the AR-chip.

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

T80 (= time to reach 80 kPa) is defined as the complete occlusion of capillary due to thrombus formation and represents the duration (min) for the flow pressure to increase to 80 kPa from baseline.

AUC30 (= area under the flow pressure curve for 30 min) is used to quantify thrombogenicity when T80 cannot be obtained due to reduced thrombus formation or fragile thrombi.

As white thrombi that develop in AR-chips consist of activated platelets and fibrin fibers containing abundant thrombin, the AR-chip assay is capable of analyzing single and integrated efficacies, based on common parameters, of various antithrombotic agents, including anticoagulant, antiplatelet, and thrombolytic agents, under variable flow conditions.

Antithrombotic agents


White thrombi formed in an AR-chip
(red, fibrin; green, activated platelets)

Image analysis of white thrombus formation

White thrombus formation in the AR-chip can be automatically monitored and recorded using a built-in video microscope at three-minute intervals.

Images of white thrombi formed in the capillary can be automatically processed from the recorded video, and the area covered with white thrombi can be identified and calculated using the ZIA program. Thus, in addition to flow pressure analysis, the white thrombus formation process can be optically analyzed and quantified in AR-chip measurements.

Assay protocol

1.Place an AL-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 AL-chip.
  5.Push the start button.

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