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Platelet transmembrane potential detection method

A detection method and technology of transmembrane potential are applied in the field of blood transfusion medicine to achieve the effects of high detection sensitivity, improvement of clinical infusion safety, and reduction of ineffective occurrences

Active Publication Date: 2015-10-07
SHANGHAI BLOOD CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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  • Platelet transmembrane potential detection method

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Embodiment 1

[0039] Example 1 Platelet transmembrane potential detection method of the present invention (1)

[0040] 1 Experimental materials and instruments

[0041] Fresh platelets were collected on the same day, put into platelet storage bags, and stored with shaking at 22°C.

[0042] Bis(1,3-dibutylbarbituric acid)trimethineoxonol and DiBAC4(3) (purchased from Sigma) were dissolved in dimethyl sulfoxide to prepare a working solution of 10 μmol / L, and stored at -20°C.

[0043] Gramicidin (Gramicidin, purchased from Sigma, product number 50845) was prepared into a 2 mg / mL working solution using dimethyl sulfoxide and stored at -20°C.

[0044] Hepes-Tyrodes buffer (calcium-free): 137mmol / L sodium chloride, 2.7mmol / L potassium chloride, 12mmol / L sodium bicarbonate, 1mmol / L magnesium chloride, 0.4mmol / L sodium dihydrogen phosphate, 5.5mmol / L glucose , 10mmol / L Hepes, 1mol / L sodium hydroxide to adjust the pH to 7.4.

[0045] Hepes-Tyrodes buffer (high potassium): 2.7mmol / L sodium chlorid...

Embodiment 2

[0056] Example 2 Platelet transmembrane potential detection method of the present invention (2)

[0057] 1 Experimental materials and instruments

[0058] Fresh platelets were collected on the same day, put into platelet storage bags, and stored with shaking at 22°C.

[0059] Bis(1,3-dibutylbarbituric acid)trimethineoxonol and DiBAC4(3) (purchased from Sigma) were dissolved in dimethyl sulfoxide to prepare a working solution of 10 μmol / L, and stored at -20°C.

[0060] Gramicidin (Gramicidin, purchased from Sigma, product number 50845) was prepared into a 2 mg / mL working solution using dimethyl sulfoxide and stored at -20°C.

[0061] Hepes-Tyrodes buffer (calcium-free): 137mmol / L sodium chloride, 1mmol / L potassium chloride, 12mmol / L sodium bicarbonate, 1mmol / L magnesium chloride, 0.4mmol / L sodium dihydrogen phosphate, 5.5mmol / L glucose, 10mmol / L Hepes, 1mol / L sodium hydroxide to adjust the pH to 7.4.

[0062] Hepes-Tyrodes buffer (high potassium): 2mmol / L sodium chloride, 13...

Embodiment 3

[0073] Example 3 Platelet transmembrane potential detection method of the present invention (3)

[0074] 1 Experimental materials and instruments

[0075] Fresh platelets were collected on the same day, put into platelet storage bags, and stored with shaking at 22°C.

[0076]Bis(1,3-dibutylbarbituric acid)trimethineoxonol and DiBAC4(3) (purchased from Sigma) were dissolved in dimethyl sulfoxide to prepare a working solution of 10 μmol / L, and stored at -20°C.

[0077] Gramicidin (Gramicidin, purchased from Sigma, product number 50845) was prepared into a 2 mg / mL working solution using dimethyl sulfoxide and stored at -20°C.

[0078] Hepes-Tyrodes buffer (calcium-free): 134mmol / L sodium chloride, 5mmol / L potassium chloride, 12mmol / L sodium bicarbonate, 1mmol / L magnesium chloride, 0.4mmol / L sodium dihydrogen phosphate, 5.5mmol / L glucose, 10mmol / L Hepes, 1mol / L sodium hydroxide to adjust the pH to 7.4.

[0079] Hepes-Tyrodes buffer (high potassium): 17mmol / L sodium chloride, 12...

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Abstract

The present invention relates to a platelet transmembrane potential detection method, which comprises: taking a washed platelet sample, carrying out re-suspending dilution with Hepes-Tyrodes buffer containing calcium chloride, adding to a flow tube, and recording as F; taking a washed platelet sample, carrying out re-suspending dilution with Hepes-Tyrodes buffer having 120-150 mmol / L of potassium chloride and 2-20 mmol / L of sodium chloride, adding to a flow tube, and recording as F0; adding gramicidin with a final concentration of 0.1-5 [mu]g / L to the F0 flow tube; adding DiBAC4(3) with a final concentration of 10-200 nmol / L to the F flow tube and the F0 flow tube, and dyeing; loading onto the machine and detecting; and calculating the result according to the corrected Nernst equation. According to the present invention, the method has characteristics of less platelet destruction, high sensitivity, good reproducibility, enrichment of the existing platelet storage quality assessment method, and prediction of the platelet storage damage.

Description

technical field [0001] The invention relates to the field of blood transfusion medicine, in particular to a method for detecting platelet transmembrane potential. Background technique [0002] Platelets are one of the components of blood and play an important role in hemostasis, coagulation and host immunity. Platelets have no nucleus and are about 2-3 μm in diameter. The number of platelets in normal blood is about 150-400×10 9 / L, the longest lifespan in the body is about 8-10 days. [0003] With the development of transfusion medicine, transfusion of platelet products has become an important means of clinical blood transfusion therapy, used to prevent and alleviate thrombocytopenia or dysfunction. However, clinically, platelet transfusion failures often occur due to non-immune factors, such as fever, infection, drugs, and the quality of platelet products, which may affect the effect of platelet transfusion. The morphology, activation, metabolism and apoptosis of plate...

Claims

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Application Information

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IPC IPC(8): G01N15/14
Inventor 陆萍乔振磊朱自严李睿书凌冰
Owner SHANGHAI BLOOD CENT
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