WO2022061570A1 - 中性粒细胞趋化检测方法 - Google Patents

中性粒细胞趋化检测方法 Download PDF

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WO2022061570A1
WO2022061570A1 PCT/CN2020/117066 CN2020117066W WO2022061570A1 WO 2022061570 A1 WO2022061570 A1 WO 2022061570A1 CN 2020117066 W CN2020117066 W CN 2020117066W WO 2022061570 A1 WO2022061570 A1 WO 2022061570A1
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chemotaxis
neutrophils
calcium
balanced salt
salt solution
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PCT/CN2020/117066
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French (fr)
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孙炳伟
杨云稀
刘璐
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苏州市立医院
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Priority to JP2023532113A priority Critical patent/JP7470276B2/ja
Priority to PCT/CN2020/117066 priority patent/WO2022061570A1/zh
Publication of WO2022061570A1 publication Critical patent/WO2022061570A1/zh
Priority to US18/124,577 priority patent/US11774440B2/en

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    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • G01N33/56972—White blood cells

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  • the invention relates to a neutrophil chemotaxis detection method, which belongs to the technical field of medical detection application.
  • Neutrophils are important innate immune cells in the human body to resist the invasion of pathogens. They are produced by hematopoietic stem cells in the bone marrow, differentiate and develop in the bone marrow and enter the blood or tissues. They are the largest number of white blood cells in the peripheral blood of the body. 40% to 75% of the total. In Wright's stained blood smears, neutrophils have a colorless or very light reddish cytoplasm, and their nuclei are rod-shaped or 2-5 lobulated. Known as polymorphonuclear leukocyte (PMN), it has the functions of chemotaxis, phagocytosis and sterilization.
  • PMN polymorphonuclear leukocyte
  • neutrophils When the human body is invaded by pathogenic bacteria, neutrophils can chemotactic to the infection site, swallow the bacteria into the cell, and kill the bacteria through various ways. It is the first line of defense against bacterial infection in the human body. It plays a very important role in the non-specific immune system.
  • the purpose of the present invention is to provide a method for detecting neutrophil chemotaxis, which can save the time for neutrophil extraction and make the extracted neutrophils have strong chemotactic function.
  • a method for detecting neutrophil chemotaxis comprising:
  • the solution after density gradient centrifugation is divided into three layers, and the supernatant of the upper layer and the PBMC layer of the middle layer are sucked to obtain the cell mass of the bottom layer;
  • Neutrophils were counted, the concentration of neutrophils was adjusted, and the neutrophil suspension was drawn into the chemotaxis model for chemotaxis.
  • the blood samples were allowed to stand at room temperature for 20 minutes, then the supernatant was taken into a centrifuge tube, and centrifuged at 20°C for 10 minutes using a centrifugal force of 400 g.
  • red blood cells in the cell mass need to be lysed twice.
  • the chemotaxis model is a petri dish, and the petri dish is a transparent petri dish to visually observe the migration movement of neutrophils.
  • the beneficial effects of the present invention are: by centrifuging and lysing the blood sample, the neutrophils can be quickly obtained, the extraction speed is improved, and the detection time is shortened, and the method is simple to use, efficient and fast, and improves the performance of the neutrophils. .
  • FIG. 1 is a flow chart of the method for detecting neutrophil chemotaxis of the present application.
  • Figure 2 is a schematic diagram of the chemotaxis of neutrophils extracted by the present application.
  • the terms “installed”, “connected” and “connected” should be understood in a broad sense, unless otherwise expressly specified and limited, for example, it may be a fixed connection or a detachable connection Connection, or integral connection; can be mechanical connection, can also be electrical connection; can be directly connected, can also be indirectly connected through an intermediate medium, can be internal communication between two elements.
  • installed should be understood in a broad sense, unless otherwise expressly specified and limited, for example, it may be a fixed connection or a detachable connection Connection, or integral connection; can be mechanical connection, can also be electrical connection; can be directly connected, can also be indirectly connected through an intermediate medium, can be internal communication between two elements.
  • the axis direction is consistent with the height direction.
  • a method for detecting neutrophil chemotaxis in a preferred embodiment of the present invention includes:
  • Collect blood samples into EDTA anticoagulation tubes add an equal volume of glucose, mix well, and let stand.
  • the color of the EDTA anticoagulation tube is purple
  • the volume of the blood sample and the glucose is 2ml
  • the concentration of the glucose is 3%.
  • the volume of the blood sample and the glucose can also be other, and the concentration of the glucose can also be other, which is not specifically limited here, and is determined according to the actual situation.
  • the supernatant of the standing blood sample was taken into a centrifuge tube for centrifugation. Specifically, the blood sample needs to stand for 20 minutes at room temperature to precipitate, and then the supernatant is taken into a centrifuge tube, and centrifuged at 20° C. for 10 minutes using a centrifugal force of 400 g.
  • the conditions of the centrifugation treatment can also be other, which are not specifically limited here, and are determined according to the actual situation.
  • 1X calcium-magnesium-free Hanks Balanced Salt Solution to the centrifuged blood samples to resuspend the bottom cell pellet.
  • the 1 ⁇ calcium-magnesium ion-free Hanks balanced salt solution is a pre-configured stock solution. At this time, there is no need to make any dilution of the stock solution, and the stock solution can be directly added to the centrifuged blood sample.
  • the volume of 1 ⁇ calcium-magnesium-free Hanks balanced salt solution is 3 ml.
  • the "density gradient centrifugation treatment” specifically includes: centrifuging at 20° C. for 35 minutes with a centrifugal force of 400 g.
  • the solution after density gradient centrifugation was divided into three layers, and the supernatant of the upper layer and the PBMC layer of the middle layer were aspirated to obtain the cell mass of the bottom layer.
  • the PBMC layer is the (Peripheral Blood Mononuclear Cell, peripheral blood mononuclear cell) layer, which contains a large number of monocytes and lymphocytes, and the bottom is red blood cells and mature neutrophils.
  • the erythrocytes in the cell mass are lysed by using sterile water.
  • the red blood cells in the cell mass need to be lysed twice, and the volume of the sterile water is 3 ml.
  • After gently blowing and sucking add 2 ⁇ Hanks balanced salt solution without calcium and magnesium ions and 1 ⁇ Hanks balanced salt solution without calcium and magnesium ions to mix well, and centrifuge again to obtain neutrophils; "Specifically: use a centrifugal force of 400g and centrifuge at 20°C for 7 minutes.
  • the chemotaxis model is a petri dish, and the petri dish is a transparent petri dish to visually observe the migration of neutrophils.
  • the culture dish is the device for the cell transmembrane migration test disclosed in Chinese patent CN105062865B and the model for making it, which will not be repeated here.
  • the time required for conventional extraction of neutrophils is about 2 hours, and the time required to extract neutrophils according to the above method is doubled, and the steps are simplified, so as to improve the extraction speed and shorten the detection time.
  • the chemotaxis results of neutrophils in the chemotaxis model obtained by this method are the first in the world to establish accurate analysis indicators.
  • the indicators are as follows :
  • Chemotaxis Distance the farthest distance that neutrophils can reach by chemotaxis in the agarose chemotaxis model for two hours, and the farthest distance is greater than or equal to 1755.85 ⁇ m.
  • Chemo Cell Ratio the percentage of the total number of chemotactic cells to the total number of chemotactic cells (10 5 ), and the percentage is greater than or equal to 3.34%.
  • the formula for calculating this percentage is as follows:
  • CCR (total number of chemotactic cells/total number of chemotactic cells) ⁇ 100%.
  • Chemotactic index (Chemo Index, CI): the ratio of the number of chemotactic cells in zone I and zone II to the total number of chemotactic cells, the ratio is greater than or equal to 39.63, and the formula for the ratio is as follows:
  • CI [(number of cells in area I + number of cells in area II)/number of all chemotactic cells] ⁇ 100.
  • Vmax the ratio of the longest distance that neutrophils can achieve chemotaxis for two hours to the chemotaxis time (120min), the ratio is greater than or equal to 14.63um/min, calculated The formula is as follows:
  • neutrophils can be quickly obtained, the extraction speed can be increased, and the detection time can be shortened.
  • the method is simple, efficient and fast, and improves the performance of neutrophils.

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Abstract

一种中性粒细胞趋化检测方法，包括：采集血液样本至抗凝管中，加入葡萄糖混匀后并静置；取上清液至离心管中，进行离心；在离心处理后的血液样本中加入1×无钙镁离子Hanks平衡盐溶液以重悬底部细胞团块；均匀吹散，并从离心管的底部缓慢加入聚蔗糖溶液，进行密度梯度离心处理；密度梯度离心后的溶液分三层，将上层的清液及中间层的PBMC层吸去，以得到底层的细胞团；采用灭菌用水对细胞团中的红细胞进行裂解处理，轻柔吹吸后依次加入2×无钙镁离子Hanks平衡盐溶液、1×无钙镁离子Hanks平衡盐溶液混匀，并再次进行离心处理，得到中性粒细胞；对中性粒细胞进行计数，吸取中性粒细胞悬液至趋化模型中进行趋化。该方法能够提高中性粒细胞的提取速度，缩短检测时间。

Description

中性粒细胞趋化检测方法 技术领域
本发明涉及一种中性粒细胞趋化检测方法，属于医疗检测应用技术领域。
背景技术
中性粒细胞是人体重要的抵御病原体入侵的先天性免疫细胞，由骨髓造血干细胞产生，在骨髓中分化发育，进入血液或组织，是机体外周血中数量最多的白细胞，正常生理状态下占白细胞总数的40％～75％。中性粒细胞在瑞氏染色血涂片中，胞质呈无色或极浅的淡红色，细胞核呈杆状或2～5分叶状，叶与叶间有细丝相连，所以也常被称为多形核白细胞(polymorphonuclear leukocyte，PMN)，具有趋化、吞噬、杀菌的功能。当人体受到致病菌的侵袭时，中性粒细胞能趋化至感染部位，将细菌吞入胞内，并通过多种途径杀灭细菌，是人体抵抗细菌感染的第一道防线，在人体非特异性免疫系统中发挥着非常重要的作用。
近年来大量国内外的研究表明，感染、重症、肿瘤及糖尿病等各类疾病均与中性粒细胞息息相关：患者的中性粒细胞趋化异常，使得中性粒细胞不能到达感染部位清除病原微生物,同时过多的中性粒细胞募集到非炎症部位而引起器官损伤。因此，对中性粒细胞趋化功能的检测与分析对病人的免疫状况的精准诊断和治疗提供了新的方向，具有非常重要的临床意义。
发明内容
本发明的目的在于提供一种中性粒细胞趋化检测方法，其可以节省中性粒细胞提取的时间，且使得提取的中性粒细胞具有较强的趋化功能。
为达到上述目的，本发明提供如下技术方案：一种中性粒细胞趋化检测方法，所述方法包括：
采集血液样本至EDTA抗凝管中，加入等体积的葡萄糖混匀后并静置；
取静置后血液样本的上清液至离心管中，进行离心处理；
在离心处理后的血液样本中加入1×无钙镁离子Hanks平衡盐溶液以重悬底部细胞团块；
将重悬后的底部细胞团块均匀吹散，并从离心管的底部缓慢加入聚蔗糖溶液，进行密度梯度离心处理；
密度梯度离心后的溶液分三层，将上层的清液及中间层的PBMC层吸去，以得到底层的细胞团；
采用灭菌用水对细胞团中的红细胞进行裂解处理，轻柔吹吸后依次加入2×无钙镁离子Hanks平衡盐溶液、1×无钙镁离子Hanks平衡盐溶液混匀，并再次进行离心处理，得到中性粒细胞；
对中性粒细胞进行计数，并调整中性粒细胞的浓度，吸取中性粒细胞悬液至趋化模型中进行趋化。
进一步地，所述“取静置后血液样本的上清液至离心管中，进行离心处理”具体为：
血液样本在室温下静置20分钟，然后取上清液至离心管中，采用400g的离心力，并在20℃的条件下离心10分钟。
进一步地，所述“密度梯度离心处理”具体为：
采用400g的离心力，在20℃的条件下离心35分钟。
进一步地，所述细胞团中的红细胞需要裂解两次。
进一步地，所述“并再次进行离心处理”具体为：
采用400g的离心力，在20℃的条件下离心7分钟。
进一步地，所述趋化模型为培养皿，所述培养皿为透明培养皿以直观的观察中性粒细胞的迁移运动。
本发明的有益效果在于：通过对血液样本离心、裂解等处理，以快速得到中性粒细胞，提高提取速度，缩短检测时间，且该方法使用简单，高效快速，提高了中性粒细胞的性能。
上述说明仅是本发明技术方案的概述，为了能够更清楚了解本发明的技术手段，并可依照说明书的内容予以实施，以下以本发明的较佳实施例并配合附图详细说明如后。
附图说明
图1为本申请的中性粒细胞趋化检测方法的流程图。
图2为本申请所提取的中性粒细胞的趋化示意图。
具体实施方式
下面将结合附图对本发明的技术方案进行清楚、完整地描述，显然，所描述的实施例是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
在本发明的描述中，需要说明的是，术语“中心”、“上”、“下”、“左”、“右”、“竖直”、“水平”、“内”、“外”等指示的方位或位置关系为基于附图所示的方位或位置关系，仅是为了便于描述本发明和简化描述，而不是指示或暗示所指的装置或元件必须具有特定的方位、以特定的方位构造和操作，因此不能理解为对本发明的限制。此外，术语“第一”、“第二”、“第三”仅用于描述目的，而不能理解为指示或暗示相对重要性。
在本发明的描述中，需要说明的是，除非另有明确的规定和限定，术语“安装”、“相连”、“连接”应做广义理解，例如，可以是固定连接，也可以是可拆卸连接，或一体地连接；可以是机械连接，也可以是电连接；可以是直接相连，也可以通过中间媒介间接相连，可以是两个元件内部的连通。对于本领域的普通技术人员而言，可以具体情况理解上述术语在本发明中的具体含义。
此外，下面所描述的本发明不同实施方式中所涉及的技术特征只要彼此之间未构成冲突就可以相互结合。在本发明描述中，轴线方向与高度方向一致。
请参见图1，本发明的一较佳实施例中的一种中性粒细胞趋化检测方法，所述方法包括：
采集血液样本至EDTA抗凝管中，加入等体积的葡萄糖混匀后并静置。在本实施例中，EDTA抗凝管的颜色为紫色，且血液样本与葡萄糖的体积为2ml，葡萄糖的浓度为3％。诚然，在其他实施例中，血液样本与葡萄糖的体积也可为其他，葡萄糖的浓度也可为其他，在此不做具体限定，根据实际情况而定。
取静置后血液样本的上清液至离心管中，进行离心处理。具体的，血液样本在室温下需静置20分钟以沉淀，然后取上清液至离心管中，采用400g的离心力，并在20℃的条件下离心10分钟。诚然，在其他实施例中，离心处理的条件也可为其他，在此不做具体限定，根据实际情况而定。
在离心处理后的血液样本中加入1×无钙镁离子Hanks平衡盐溶液以重悬底部细胞团块。其中，1×无钙镁离子Hanks平衡盐溶液为预先配置好的原液，此时无需对该原液做任何稀释，直接在离心处理后的血液样本中加入该原液即可。呈上述，1×无钙镁离子Hanks平衡盐溶液的体积为3ml。
将重悬后的底部细胞团块均匀吹散，并从离心管的底部缓慢加入聚蔗糖溶液，进行密度梯度离心处理。所述“密度梯度离心处理”具体为：采用400g的离心力，在20℃的条件下离心35分钟。
密度梯度离心后的溶液分三层，将上层的清液及中间层的PBMC层吸去，以得到底层的细胞团。其中，PBMC层为(Peripheral Blood Mononuclear Cell，外周血单核细胞)层，其包含大量单核细胞和淋巴细胞等，底部的则为红细胞及成熟的中性粒细胞。
采用灭菌用水对细胞团中的红细胞进行裂解处理，在本实施例中，所述细胞团中的红细胞需要裂解两次，且灭菌用水的体积为3ml。轻柔吹吸后依次加入2×无钙镁离子Hanks平衡盐溶液、1×无钙镁离子Hanks平衡盐溶液混匀，并再次进行离心处理，得到中性粒细胞；所述“并再次进行离心处理”具体为：采用400g的离心力，在20℃的条件下离心7分钟。
请结合图2，对中性粒细胞进行计数，并调整中性粒细胞的浓度，吸取中性粒细胞悬液至趋化模型中进行趋化。所述趋化模型为培养皿，所述培养皿为透明培养皿以直观的观察中性粒细胞的迁移运动。具体的，该培养皿为中国专利CN105062865B所公开的细胞透膜迁移试验用装置及制作用型模，在此不做赘述。
常规提取中性粒细胞所需时间大概要2h，按照上述方法提取中性粒细胞所需的时间提高了快一倍时间，且简化了步骤，达到提高提取速度，缩短检测时间的效果。
请结合图2，并且，经最后趋化试验结果所示，利用本方法所采取的中性粒细胞在趋化模型中的趋化结果在国际上首次建立了精准分析的指标，各项指标如下：
1、趋化距离(Chemotaxis Distance，CD)：中性粒细胞于琼脂糖趋化模型中趋化两小时所能达到的最远距离，该最远距离大于等于1755.85μm。
2、趋化细胞百分比(Chemo Cell Ratio，CCR)：全部趋化细胞数量占趋化细胞总数(10 5个)的百分比，该百分比大于等于3.34％。该百分比的计算公式如下：
CCR＝(全部趋化细胞数/趋化细胞总数)×100％。
3、趋化指数(Chemo Index，CI)：Ⅰ区与Ⅱ区的趋化细胞数量，占全部趋化细胞数量的比值，该比值大于等于39.63，且该比值的公式如下：
CI＝【(Ⅰ区细胞数+Ⅱ区细胞数)/全部趋化细胞数】×100。
4、最大趋化速度(Maximum Speed of Chemotaxis，Vmax)：中性粒细胞趋化两小时所能达到的最远距离与趋化时间(120min)的比值，该比值大于等于14.63um/min，计算公式如下：
Vmax＝趋化距离/趋化时间＝CD/120。
综上所述：通过对血液样本离心、裂解等处理，以快速得到中性粒细胞，提高提取速度，缩短检测时间，且该方法使用简单，高效快速，提高了中性粒细胞的性能。
以上所述实施例的各技术特征可以进行任意的组合，为使描述简洁，未对上述实施例中的各个技术特征所有可能的组合都进行描述，然而，只要这些技术特征的组合不存在矛盾，都应当认为是本说明书记载的范围。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。

Claims (6)

  1. 一种中性粒细胞趋化检测方法，其特征在于，所述方法包括：
    采集血液样本至EDTA抗凝管中，加入等体积的葡萄糖混匀后并静置；
    取静置后血液样本的上清液至离心管中，进行离心处理；
    在离心处理后的血液样本中加入1×无钙镁离子Hanks平衡盐溶液以重悬底部细胞团块；
    将重悬后的底部细胞团块均匀吹散，并从离心管的底部缓慢加入聚蔗糖溶液，进行密度梯度离心处理；
    密度梯度离心后的溶液分三层，将上层的清液及中间层的PBMC层吸去，以得到底层的细胞团；
    采用灭菌用水对细胞团中的红细胞进行裂解处理，轻柔吹吸后依次加入2×无钙镁离子Hanks平衡盐溶液、1×无钙镁离子Hanks平衡盐溶液混匀，并再次进行离心处理，得到中性粒细胞；
    对中性粒细胞进行计数，并调整中性粒细胞的浓度，吸取中性粒细胞悬液至趋化模型中进行趋化。
  2. 如权利要求1所述的中性粒细胞趋化检测方法，其特征在于，所述“取静置后血液样本的上清液至离心管中，进行离心处理”具体为：
    血液样本在室温下静置20分钟，然后取上清液至离心管中，采用400g的离心力，并在20℃的条件下离心10分钟。
  3. 如权利要求1所述的中性粒细胞趋化检测方法，其特征在于，所述“密度梯度离心处理”具体为：
    采用400g的离心力，在20℃的条件下离心35分钟。
  4. 如权利要求1所述的中性粒细胞趋化检测方法，其特征在于，所述细胞团中的红细胞需要裂解两次。
  5. 如权利要求1所述的中性粒细胞趋化检测方法，其特征在于，所述“并再次进行离心处理”具体为：
    采用400g的离心力，在20℃的条件下离心7分钟。
  6. 如权利要求1所述的中性粒细胞趋化检测方法，其特征在于，所述趋化 模型为培养皿，所述培养皿为透明培养皿以直观的观察中性粒细胞的迁移运动。
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