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Separation method of hyperpure platelets

A separation method and platelet technology, applied in the fields of medicine and biology, can solve the problems of reducing white blood cell retention, low overall pass rate, and contaminating platelet RNA, etc., to achieve the effect of avoiding background noise

Inactive Publication Date: 2018-11-23
XIAMEN LIFEINT TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, according to the difference in the size of leukocytes and platelets, this method uses a filter membrane with a fixed pore size to retain leukocytes, which is easily affected by individual differences and the size of leukocytes. The fixed pore size cannot be applied to whole blood samples from various sources, resulting in an overall qualified low rate
In addition, the filter will retain liquid and cause loss of platelets. In order to improve the recovery rate of platelets, the applicant tried to use buffer to wash the filter, but this method will reduce the retention of white blood cells, and even cause white blood cells to lyse and release RNase and RNA. Degrades or contaminates platelet RNA

Method used

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  • Separation method of hyperpure platelets

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1: Separation of ultrapure platelets by double centrifugation combined with magnetic bead purification

[0043] Whole blood sample collection: Use BD dipotassium EDTA blood collection tube to collect 2mL of venous blood from the subject, gently invert the blood collection tube several times after collection, so that the anticoagulant and whole blood can be fully mixed, and the whole blood should be processed within 96 hours after collection .

[0044] The first centrifugation: put the blood collection tube into the centrifuge rotor, centrifuge at 60×g for 30 minutes at room temperature, use a pipette to absorb 600 μL of platelet-rich plasma in the upper layer, and transfer it to a new 1.5mL centrifuge tube. The absorption process is as gentle as possible , to avoid stirring the middle buffy coat layer, causing white blood cells to float up and increasing the contamination rate.

[0045] Magnetic beads pre-treatment: CD45 immunomagnetic beads (Invitrogen, 11153D...

Embodiment 2

[0054] 1 mL of venous blood was collected from the subject, and the operation steps were the same as in Example 1. The results are shown in Table 1.

Embodiment 3

[0056] 9 mL of venous blood was collected from the subject, and the operation steps were the same as in Example 1. The results are shown in Table 1.

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Abstract

The invention discloses a separation method of hyperpure platelets. The separation method comprises the following steps of collecting a whole blood sample, and removing cells and other condensation product precipitates with centrifugation for the first time; removing leukocytes and erythrocytes with double-immunomagnetic beads; and performing secondary centrifugation, and collecting platelet precipitates. By adopting the method, the leukocyte contamination rate of the hyperpure platelets acquired from smaller than or equal to 10mL whole blood is lower than 0.0001 percent and is far lower thanthe leukocyte contamination rate of the existing method, and the method is suitable for platelet RNA sequencing, real-time fluorescent quantitative PCR and the like and can be applied to serious disease detection such as tumor screening and diagnosis.

Description

technical field [0001] The invention relates to the fields of medicine and biology, in particular to a method for separating ultrapure platelets. Background technique [0002] In a normal body, platelets mainly play a role in promoting blood coagulation and promoting wound healing through release and aggregation. However, in the microenvironment of major diseases such as acute and chronic inflammation or tumors, splicing of platelet-specific pre-mRNAs can occur, thereby affecting the gene expression profile of platelets. In addition, platelets, the second most abundant cell type in blood, are readily available and easy to isolate, allowing them to be used as new assays. Therefore, RNA sequencing of domesticated platelets, such as tumor conditioned platelets (Tumor Conditioned Platelets), has become a new liquid biopsy method to detect whether a subject suffers from cancer. [0003] The separation of ultra-pure platelets is the key to platelet RNA sequencing. How to minimiz...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N1/34
CPCG01N1/34
Inventor 肖剑萍叶国栋陈欣欣陈茂立许剑雄韩大雄郭奇伟蔡逸民杨燕燕李顺杰董康梅朱莎莎张丽芳宋丹
Owner XIAMEN LIFEINT TECH CO LTD
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