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Method for platelet morphology scan imagery and platelet activation function evaluation

A technology of scanning imaging and platelet-rich plasma, applied in scanning probe technology, scanning probe microscopy, measuring devices, etc., can solve the problem of needle tip or sample damage, which cannot meet the needs of high-resolution observation of platelet microscopic morphology, It will damage the surface structure of platelets and other problems, and achieve the effect of easy operation

Inactive Publication Date: 2014-04-02
GENERAL HOSPITAL OF TIANJIN MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, there are many methods and instruments used to detect the morphology of platelets. Among them, the resolution of ordinary optical microscopes and laser confocal microscopes can only reach about 200 nanometers, which cannot meet the needs of high-resolution observation of platelet microscopic morphology.
Although scanning electron microscopy (SEM) has a sufficiently high resolution, platelets need to be solidified and specially treated to achieve the conductivity of the sample, which will inevitably change or even destroy the microstructure of the platelet surface, so it is not suitable for the observation of living platelets at all.
Atomic force microscopy (AFM), which is a powerful tool for high-resolution bioimaging research in the scanning probe microscope family, has been used to study the three-dimensional topological structure of platelet microtopography, but due to its use of the interaction between the probe tip and the platelet membrane The force is controlled by negative feedback to achieve scanning imaging. The slight contact between the probe and the living platelet is inevitable during scanning, which will not only damage the surface structure of the platelet, but also activate the platelet to cause morphological changes due to mechanical force, so it cannot meet the requirements of platelet activators. The need for microtopography imaging before and after action
[0003] In order to overcome the shortcomings of atomic force microscopy that the scanning probe needs to be in contact with the sample to damage or activate the biological sample, Professor Hansma of the University of California invented the non-contact scanning ion conductance microscopy (SICM) in 1989. The limitations and deficiencies of the negative feedback control method and precise positioning technology, the slender glass micropipette probe often accidentally touches the sample during scanning and causes damage to the needle tip or sample, SICM technology is only applicable for a long time after its invention Scanning and imaging on flat polyester film

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  • Method for platelet morphology scan imagery and platelet activation function evaluation

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Effect test

Embodiment 1

[0024] A method for platelet morphology scanning imaging, comprising the steps of:

[0025] (1) Preparation of platelet samples

[0026] Take 2ml of venous blood from a healthy person and put it into a sodium citrate anticoagulant tube, and centrifuge at 150×g for 15 minutes to obtain platelet-rich plasma; take 200 μL of the platelet-rich plasma and place it in a tube coated with 200 μL of human fibrinogen at a concentration of 2 mg / mL Incubate at room temperature in 35mm cell culture dish for 30 minutes, use PBS buffer to elute the platelets that are not adhered to the bottom of the cell culture dish, add 1.5mL PBS buffer and set aside;

[0027] (2) Place the Ag / AgCl reference electrode connected to the scanning ion conductance microscope on the cell culture dish obtained in step (1); place the detection electrode connected to the scanning ion conductance microscope on the cell culture dish obtained in step (1). In the dish, the detection electrode is an Ag / AgCl electrode ar...

Embodiment 2

[0030] A method for assessing platelet activation function, comprising the steps of:

[0031] (1)-(3) are the same as (1)-(3) of Embodiment 1;

[0032] (4) Then add the activator thrombin to the cell culture dish, so that the final concentration of thrombin is 2U / mL and act for 10 minutes. Use SICM technology to observe the surface morphology of the living platelets after adding thrombin for 10 minutes: SICM control The detector monitors the change of the current flowing into the detection electrode, and keeps a constant distance between the detection electrode and the platelet through negative feedback control. The trajectory of the detection electrode on the cell surface is the three-dimensional topological structure of the surface of the living platelet after adding a certain concentration of activator for 10 minutes, and accurately obtained The number of activated disc-shaped platelets; the three-dimensional topological structure map of the surface morphology of platelets ...

Embodiment 3

[0035] A method for platelet morphology scanning imaging, comprising the steps of:

[0036] (1) Preparation of platelet samples

[0037] Take 2 ml of venous blood and put it into a sodium citrate anticoagulant tube, and centrifuge to obtain platelet-rich plasma; take 200 μL of the platelet-rich plasma and place it in a 35 mm cell culture dish coated with 200 μL of human fibrinogen at a concentration of 2 mg / mL Incubate at room temperature for 20 minutes, elute platelets not adhered to the bottom of the cell culture dish with PBS buffer, add 2 mL of PBS buffer and set aside;

[0038] (2) Same as (2) of Embodiment 1;

[0039](3) Use a scanning ion conductance microscope to monitor the change of the current flowing into the detection electrode, and maintain a set constant distance between the jumping detection electrode and the platelet through negative feedback control (that is, no physical contact between the detection electrode and the platelet), and record the Describe the ...

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Abstract

The invention discloses a method for platelet morphology scan imagery and platelet activation function evaluation. The method for platelet morphology scan imagery comprises the following steps of: (1) preparing a platelet sample; (2) putting a reference electrode and an exploring electrode connected to a scanning ionic conductance microscope into a cell culture dish obtained by the step (1); and (3) monitoring the change of current flowing into the exploring electrode through the scanning ionic conductance microscope, enabling a setting distance to be kept between the jumping exploring electrode and a platelet through degeneration control, recording a position of the exploring element, and drawing to obtain a three-dimensional topology structure chart of platelet surface morphology through a computer. According to the method, the morphology performances of the platelet before and after adding an activating agent can be intuitively observed in real time, and not only can parameters such as the quantity of platelets and mean platelet volume within a scanning range be measured simply, conveniently, easily and accurately, but also the activation function of the platelet can be evaluated. The method is simple and convenient to operate, and is low in cost.

Description

technical field [0001] The invention relates to a method for platelet morphology scanning imaging and platelet activation function evaluation. Background technique [0002] Platelet is one of the formed components with specific morphological structure and biochemical composition in blood, and its main physiological function is to participate in coagulation and hemostasis. The structure of platelets is complex and individual differences are large, and because of their ability to move and deform, they often show polymorphism. Normal platelets in circulating blood are oval or disc-shaped, with an average diameter of about 2 to 4 microns. Platelets are easily activated by mechanical and chemical stimuli and lead to morphological changes. Once the platelets come into contact with the wound surface or the activator, they can expand into disc-shaped platelets to increase the area for coagulation and hemostasis. Thrombin, ADP, epinephrine and collagen are all activators of platel...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01Q60/44
Inventor 张彦军张建宁董京飞
Owner GENERAL HOSPITAL OF TIANJIN MEDICAL UNIV