WO2021000654A1 - 一种血液中肝素或肝素类物质的检测方法和试剂盒 - Google Patents

一种血液中肝素或肝素类物质的检测方法和试剂盒 Download PDF

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Publication number
WO2021000654A1
WO2021000654A1 PCT/CN2020/090322 CN2020090322W WO2021000654A1 WO 2021000654 A1 WO2021000654 A1 WO 2021000654A1 CN 2020090322 W CN2020090322 W CN 2020090322W WO 2021000654 A1 WO2021000654 A1 WO 2021000654A1
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Prior art keywords
heparin
coagulant
blood sample
mixture
neutralizer
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PCT/CN2020/090322
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English (en)
French (fr)
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李钢
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常熟常江生物技术有限公司
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Publication of WO2021000654A1 publication Critical patent/WO2021000654A1/zh

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483—Physical analysis of biological material
    • G01N33/487—Physical analysis of biological material of liquid biological material
    • G01N33/49—Blood
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483—Physical analysis of biological material
    • G01N33/487—Physical analysis of biological material of liquid biological material
    • G01N33/49—Blood
    • G01N33/4905—Determining clotting time of blood

Definitions

  • the present invention relates to the technical field of heparin, in particular to a detection method and kit for heparin or heparin substances in blood.
  • heparinase must be used to decompose heparin in the blood during the detection.
  • Heparinase is A very expensive substance, and because it is an enzyme in nature, it is easy to inactivate. It must be stored and transported at low temperature in a freeze-dried state, and needs to be reconstituted into a liquid before use. This detection method is expensive and inconvenient to use.
  • the purpose of the present invention is to provide a method for detecting heparin or heparin-like substances in blood.
  • the detection method provided by the present invention has low cost and simple method.
  • the present invention provides a method for detecting heparin or heparin substances in blood, which includes:
  • the first coagulation time is detected after mixing the mixture and the first coagulant to obtain the first coagulation time
  • the second coagulation time is detected to obtain the second coagulation time
  • first coagulant and the second coagulant are the same;
  • the detection method of the first clotting time is the same as the detection method of the second clotting time.
  • the difference between the first clotting time and the second clotting time is greater than 5 seconds, it is determined that the blood sample contains heparin or heparin-like substances.
  • the volume ratio of the first blood sample to the heparin neutralizer is (16-36):1.
  • the heparin neutralizer includes one or two of protamine and toluidine blue.
  • the first coagulant and the second coagulant include one or more of thrombin, ellagic acid, kaolin, tissue factor, phospholipids and calcium chloride.
  • the volume ratio of the mixture and the first coagulant is (16-36):1.
  • the volume ratio of the second blood sample to the second coagulant is (16-36):1.
  • the heparin neutralizer is a heparin neutralizer solution
  • the heparin neutralizer solution includes: a buffer mixture and a heparin neutralizer
  • the buffer mixture includes: Tris-HCl buffer and Proclin300;
  • the mass content of the heparin neutralizer in the buffer mixture is 0.08-0.12%.
  • the first coagulant is a first coagulant solution
  • the first coagulant solution includes: a buffer mixture and a first coagulant
  • the buffer mixture includes: Tris-HCl buffer and Proclin300;
  • the mass content of the first coagulant in the buffer mixture is 0.08-0.12%.
  • the second coagulant is a second coagulant solution
  • the second coagulant solution includes: a buffer mixture and a second coagulant
  • the buffer mixture includes: Tris-HCl buffer and Proclin300;
  • the mass content of the second coagulant in the buffer mixture is 0.08-0.12%.
  • the detection device for the first coagulation time and the second coagulation time is a thromboelastometer.
  • the present invention also provides a kit for detecting heparin or heparin substances in blood, which includes:
  • Heparin neutralizer and coagulant Heparin neutralizer and coagulant.
  • the method provided by the present invention is suitable for detecting heparin or heparin-like substances in a blood sample by detecting blood clotting time by a thromboelastometer.
  • the present invention adds a heparin neutralizer and the clotting time of a blank blood sample to the tested blood sample. Make a comparison to get the test result of heparin or heparin-like substances in the blood sample.
  • the present invention does not need to use expensive heparinase, and replaces heparinase with heparin neutralizer. Since heparin neutralizer is a non-enzymatic substance, the biochemical properties are stable, and the neutralization of heparin is rapid and reliable. It can be made into a liquid preparation and used directly.
  • the detection method using heparin neutralizer provided by the present invention can make the detection cost low, and the detection reagent is easy to store and transport, and is suitable for clinical promotion.
  • the present invention provides a method for detecting heparin or heparin substances in blood, which includes:
  • the first coagulation time is detected after mixing the mixture and the first coagulant to obtain the first coagulation time
  • the second coagulation time is detected to obtain the second coagulation time
  • first coagulant and the second coagulant are the same;
  • the detection method of the first clotting time is the same as the detection method of the second clotting time.
  • the difference between the first clotting time and the second clotting time is greater than 5 seconds, it is determined that the blood sample contains heparin or heparin-like substances.
  • the present invention divides the same blood sample, that is, the blood sample to be tested, into two parts of the same quality, which are respectively recorded as the first blood sample and the second blood sample.
  • the first blood sample and the heparin neutralizer are mixed to obtain a mixture.
  • the heparin neutralizer preferably includes one or two of protamine and toluidine blue.
  • the present invention has no special restriction on the source of the heparin neutralizer, which can be purchased in the market.
  • the volume ratio of the first blood sample and the heparin neutralizer is preferably (16-36):1, more preferably (21-31):1, most preferably (24-27):1 , To ensure that if the first blood sample contains heparin or heparin substances, the heparin neutralizer used can completely neutralize the heparin or heparin substances in it.
  • the heparin neutralizer is preferably a heparin neutralizer solution, and the heparin neutralizer solution preferably includes: a heparin neutralizer and a buffer mixture.
  • the buffer mixture preferably includes: Tris-HCl buffer and Proclin 300, and the pH value of the Tris-HCl buffer is preferably 7.0-7.4, more preferably 7.1-7.3, and most preferably 7.2
  • the mass content of the Proclin 300 in the Tris-HCl buffer is preferably 0.1 to 0.3%, more preferably 0.2%; the mass content of the heparin neutralizer in the buffer mixture is preferably 0.08 to 0.12%, More preferably, it is 0.1%.
  • the first coagulation time is detected after mixing the mixture and the first coagulant to obtain the first coagulation time, that is, the coagulation time is detected on the blood sample added with the heparin neutralizer.
  • the first coagulant preferably includes one or more of thrombin, ellagic acid, kaolin, tissue factor, phospholipids and calcium chloride.
  • the present invention has no special restrictions on the source of the first coagulant, which can be purchased in the market.
  • the volume ratio of the mixture and the first coagulant is preferably (16-36):1, more preferably (21-31):1, most preferably (24-27):1.
  • the first coagulant is preferably a first coagulant solution
  • the first coagulant solution preferably includes: a first coagulant and a buffer mixture
  • the buffer mixture The composition is consistent with the buffer mixture described in the above technical solution, and will not be repeated here.
  • the mass content of the first coagulant in the buffer mixture is preferably 0.08-0.12%, more preferably 0.1%.
  • the second blood sample and the second coagulant are mixed and the second coagulation time is detected to obtain the second coagulation time, that is, the coagulation time is detected on the blood sample without heparin.
  • the type and source of the second coagulant are consistent with the type and source of the above-mentioned first coagulant, and will not be repeated here.
  • the second coagulant is preferably a second coagulant solution, and the composition of the second coagulant solution is the same as that of the first coagulant solution, which will not be repeated here.
  • the volume ratio of the second blood sample to the second coagulant is preferably (16-36):1, more preferably (21-31):1, most preferably (24-27): 1.
  • the detection device for the first clotting time and the second clotting time is preferably a thrombological elasticity chart.
  • the invention does not have any special restrictions on the thrombological elasticity chart, and a thromboelastic chart known to those skilled in the art is used. Instrument can be.
  • the present invention has no special restrictions on the detection methods of the first clotting time and the second clotting time.
  • a thromboelastometer is used to detect the blood clotting time according to the instructions or requirements of the thromboelastometer to detect the clotting time.
  • the composition and dosage of the first coagulant and the second coagulant are the same, the detection method of the first coagulation time and the detection method of the second coagulation time are the same, the present invention uses the same coagulant , Coagulant dosage and coagulation time testing methods to ensure that the first coagulation time and the second coagulation time are obtained under the same testing conditions, so that the two are comparable.
  • the heparin or heparin-like substances in the blood sample added with the heparin neutralizer is neutralized by the heparin neutralizer, the anticoagulant effect of heparin disappears, and the clotting time is shortened .
  • the clotting time of the two blood samples is basically the same.
  • the blood sample contains heparin or heparin-like substances That is to say, when the clotting time of the blood sample added with heparin neutralizer is shorter than the clotting time of the blank blood sample and the value is greater than 5 seconds, heparin or heparin-like substances are present in the blood sample.
  • the detection method provided by the present invention can replace the existing heparinase method, and is suitable for detecting heparin in the blood by using a thromboelastometer.
  • the present invention also provides a kit for detecting heparin or heparin substances in blood, which includes:
  • Heparin neutralizer and coagulant Heparin neutralizer and coagulant.
  • the types of the heparin neutralizer and coagulant are the same as the types of the heparin neutralizer and coagulant described in the above technical solution, for example, the heparin neutralizer and coagulant are preferably heparin neutralizers The solution and the coagulant solution will not be repeated here.
  • the kit for detecting heparin or heparin substances in blood provided by the present invention is convenient for clinical detection.
  • the reagents used in the following examples of the present invention are all commercially available products.
  • the Tris, HCl, Proclin 300, calcium chloride, toluidine blue, and protamine used are all purchased from SIGMA, and the automatic thromboelasticity chart used is The TEG5000 model instrument provided for American Blood Technology Corporation.
  • the preparation method of the toluidine blue solution is: adding toluidine blue to the buffer mixture, the buffer mixture includes: The pH is 7.20 Tris-Hcl buffer and Proclin300. The mass content of Proclin300 in the Tris-Hcl buffer is 0.2%; the mass content of toluidine blue in the buffer mixture is 0.1%.
  • the preparation method of the thrombin solution is: adding thrombin to the buffer mixture, and the buffer mixture includes: pH 7.20 Tris-Hcl buffer and Proclin300, the mass content of Proclin300 in the Tris-Hcl buffer is 0.2%; the mass content of thrombin in the buffer mixture is 0.1%.
  • the above-mentioned mixture of thrombin solution and the second blood sample were tested for coagulation time.
  • the result of the test was that the coagulation time of the mixture was 20 seconds, and the coagulation time of the second blood sample was The time is 28 seconds, and the time difference between the clotting time of the mixture and the clotting time of the second blood sample is 8 seconds. Therefore, the blood sample contains heparin or heparin-like substances.
  • the preparation method of the protamine solution is: adding protamine to the buffer mixture
  • the buffer mixture includes: The pH is 7.20 Tris-Hcl buffer and Proclin300. The mass content of Proclin300 in the Tris-Hcl buffer is 0.2%; the mass content of protamine in the buffer mixture is 0.1%.
  • the method for preparing the calcium chloride solution is: adding calcium chloride to a buffer mixture, and the buffer mixture includes: pH It is 7.20 Tris-Hcl buffer and Proclin300, the mass content of Proclin300 in the Tris-Hcl buffer is 0.2%; the concentration of calcium chloride in the buffer mixture is 0.25mol/L.
  • the above-mentioned mixture of calcium chloride solution and the second blood sample were tested for coagulation time respectively.
  • the result of the test was that the coagulation time of the mixture was 30 seconds, and that of the second blood sample The clotting time is 42 seconds, and the time difference between the clotting time of the mixture and the clotting time of the second blood sample is 12 seconds. Therefore, the blood sample contains heparin or heparin-like substances.
  • the heparinase method is used to detect whether the blood sample of Example 1 contains heparin.
  • the detection method is:
  • the above-mentioned mixture with coagulant and the second blood sample were tested for coagulation time respectively.
  • the result of the test was that the coagulation time of the mixture was 35 seconds, and the coagulation time of the second blood sample The time is 43 seconds, and the time difference between the clotting time of the mixture and the clotting time of the second blood sample is 8 seconds. Therefore, the blood sample contains heparin or heparin-like substances.
  • Example 2 of the present invention contained heparin, and the test result was that the blood sample contained heparin or heparin-like substances.
  • the method provided by the examples of the present invention is consistent with the detection results obtained by the methods provided by the comparative examples in the prior art.
  • the method provided by the present invention can more accurately detect whether heparin or heparin is contained in the blood. Heparin substances.
  • the present invention provides a method for detecting heparin or heparin substances in blood, including: dividing the same blood sample into two parts of the same quality, and recording them as the first blood sample and the second blood sample; The first blood sample and the heparin neutralizer are mixed to obtain a mixture; the mixture and the first coagulant are mixed and then the first clotting time is detected to obtain the first clotting time; and the second blood sample is combined with After the second coagulant is mixed, the second coagulation time is detected to obtain the second coagulation time; the composition and dosage of the first coagulant and the second coagulant are the same; the detection method of the first coagulation time and The detection method of the second clotting time is the same; if the time difference between the first clotting time and the second clotting time is greater than 5 seconds, the blood sample contains heparin or heparin-like substances.
  • the method provided by the present invention uses a heparin neutralizer instead of the traditional use of

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Abstract

一种血液中肝素或肝素类物质的检测方法，包括：将同一血液样本分为质量相同的两份，记为第一血液样本和第二血液样本；将第一血液样本和肝素中和剂混合，得到混合物；将混合物和第一致凝剂混合后进行第一凝固时间的检测，得到第一凝固时间；以及将第二血液样本和第二致凝剂混合后进行第二凝固时间的检测，得到第二凝固时间；第一致凝剂和第二致凝剂的成分和用量相同；第一凝固时间的检测方法和第二凝固时间的检测方法相同；如果第一凝固时间和第二凝固时间的差异大于5秒，则血液样本中含有肝素或肝素类物质。检测方法的检测成本降低，易于操作，适于临床推广。

Description

一种血液中肝素或肝素类物质的检测方法和试剂盒 技术领域
本发明涉及肝素技术领域，尤其涉及一种血液中肝素或肝素类物质的检测方法和试剂盒。
背景技术
目前，检测血液中的肝素通常使用血栓弹力图仪，血栓弹力图仪具有灵敏度高，速度快，准确的优点，但是在进行检测时必须使用肝素酶来分解血液中的肝素，肝素酶是一种价格很昂贵的物质，而且因为其本质是酶，易于失活，存储运输时必须采用冻干状态低温保存，用前需要复溶成为液体，这种检测方法检测费用高，使用不便。
发明内容
有鉴于此，本发明的目的在于提供一种血液中肝素或肝素类物质的检测方法，本发明提供的检测方法成本低、方法简便。
在一个方面，本发明提供了一种血液中肝素或肝素类物质的检测方法，其包括：
将同一血液样本分为质量相同的两份，分别为第一血液样本和第二血液样本；
将所述第一血液样本和肝素中和剂混合，得到混合物；
将所述混合物和第一致凝剂混合后进行第一凝固时间的检测，得到第一凝固时间；以及
将所述第二血液样本和第二致凝剂混合后进行第二凝固时间的检测，得到第二凝固时间；
其中，所述第一致凝剂和所述第二致凝剂相同；
其中，所述第一凝固时间的检测方法和所述第二凝固时间的检测方法相同；并且
其中，如果所述第一凝固时间和所述第二凝固时间的差值大于5秒，则确定所述血液样本中含有肝素或肝素类物质。
在一个优选的实施方式中，所述第一血液样本和肝素中和剂的体积比为(16～36)：1。
在一个优选的实施方式中，所述肝素中和剂包括鱼精蛋白和甲苯胺蓝中的一种或两种。
在一个优选的实施方式中，所述第一致凝剂和第二致凝剂包括凝血酶、鞣花酸、高岭土、组织因子、磷脂和氯化钙中的一种或多种。
在一个优选的实施方式中，所述混合物和第一致凝剂的体积比为(16～36)：1。
在一个优选的实施方式中，所述第二血液样本和第二致凝剂的体积比为(16～36)：1。
在一个优选的实施方式中，所述肝素中和剂为肝素中和剂溶液，所述肝素中和剂溶液包括：缓冲液混合液和肝素中和剂；
所述缓冲液混合液包括：Tris-HCl缓冲液和Proclin300；
所述肝素中和剂在缓冲液混合液中的质量含量为0.08～0.12％。
在一个优选的实施方式中，所述第一致凝剂为第一致凝剂溶液，所述第一致凝剂溶液包括：缓冲液混合液和第一致凝剂；
所述缓冲液混合液包括：Tris-HCl缓冲液和Proclin300；
所述第一致凝剂在缓冲液混合液中的质量含量为0.08～0.12％。
在一个优选的实施方式中，所述第二致凝剂为第二致凝剂溶液，所述第二致凝剂溶液包括：缓冲液混合液和第二致凝剂；
所述缓冲液混合液包括：Tris-HCl缓冲液和Proclin300；
所述第二致凝剂在缓冲液混合液中的质量含量为0.08～0.12％。
在一个优选的实施方式中，所述第一凝固时间和第二凝固时间的检测设备为血栓弹力图仪。
在另一方面，本发明还提供了一种检测血液中肝素或肝素类物质的试剂盒，其包括：
肝素中和剂和致凝剂。
本发明提供的方法适用于通过血栓弹力图仪检测血液凝固时间对血液样本中肝素或肝素类物质进行检测，本发明通过在被检测的血液样本中加入肝素中和剂与空白血液样本的凝固时间进行对比，得到血液样本中是否有肝素或肝素类物质的检测结果。与现有的肝素检测方法相比，本发明不需要使用昂贵的肝素酶，采用肝素中和剂取代肝素酶，由于肝素中和剂是非酶类物质，生化性质稳定，中和肝素迅速可靠，可以制成液体制剂直接使用，本发明提供的使用肝素中和剂的检测方法能够使检测成本低，而且检测试剂易于保存运输，适于临床推广。
具体实施方式
下面将对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅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。
在一方面，本发明提供了一种血液中肝素或肝素类物质的检测方法，其包括：
将同一血液样本分为质量相同的两份，分别为第一血液样本和第二血液样本；
将所述第一血液样本和肝素中和剂混合，得到混合物；
将所述混合物和第一致凝剂混合后进行第一凝固时间的检测，得到第一凝固时间；以及
将所述第二血液样本和第二致凝剂混合后进行第二凝固时间的检测，得到第二凝固时间；
其中，所述第一致凝剂和所述第二致凝剂相同；
其中，所述第一凝固时间的检测方法和所述第二凝固时间的检测方法相同；并且
其中，如果所述第一凝固时间和所述第二凝固时间的差值大于5秒，则确定所述血液样本中含有肝素或肝素类物质。
本发明将同一血液样本即待检测的血液样本分为质量相同的两份，分别记为第一血液样本和第二血液样本。
本发明将第一血液样本和肝素中和剂混合，得到混合物。在本发明中，所述肝素中和剂优选包括鱼精蛋白和甲苯胺蓝中的一种或两种。本发明对所述肝素中和剂的来源没有特殊的限制，可由市场购买获得。在本发明中，所述第一血液样本和肝素中和剂的体积比优选为(16～36)：1，更优选为(21～31)：1，最优选为(24～27)：1，以保证如果第一血液样本中含有肝素或肝素类物质，所使用的肝素中和剂能够将其中的肝素或肝素类物质完全中和。
在本发明中，所述肝素中和剂优选为肝素中和剂溶液，所述肝素中和剂溶液优选包括：肝素中和剂和缓冲液混合液。在本发明中，所述缓冲液混合液优选包括：Tris-HCl缓冲液和Proclin 300，所述Tris-HCl缓冲液的pH值优选为7.0～7.4，更优选为7.1～7.3，最优选为7.2；所述Proclin 300在Tris-HCl缓冲液中的质量含量优选为0.1～0.3％，更优选为0.2％；所述肝素中和剂在缓冲液混合液中的质量含量优选为0.08～0.12％，更优选为0.1％。
本发明将所述混合物和第一致凝剂混合后进行第一凝固时间的检测，得到第一凝固时间，即对加入肝素中和剂的血液样本进行凝固时间的检测。在本发明中，所述第一致凝剂优选包括凝血酶、鞣花酸、高岭土、组织因子、磷脂和氯化钙中的一种或多种。本发明对所述第一致凝剂的来源没有特殊的限制，可由市场购买获得。在本发明中，所述混合物和第一致凝剂的体积比优选为(16～36)：1，更优选为(21～31)：1，最优选为(24～27)：1。
在本发明中，所述第一致凝剂优选为第一致凝剂溶液，所述第一致凝剂溶液优选包括：第一致凝剂和缓冲液混合液，所述缓冲液混合液的成分与上述技术方案所述缓冲液混合液一致，在此不再赘述。在本发明中，所述第一致凝剂在缓冲液混合液中的质量含量优选为0.08～0.12％，更优选为0.1％。
本发明将第二血液样本和第二致凝剂混合后进行第二凝固时间的检测，得到第二凝固时间，即对不含肝素的血液样本进行凝固时间的检测。在本发明中，所述第二致凝剂的种类和来源与上述第一致凝剂的种类和来源一致，在此不再赘述。在本发明中，所述第二致凝剂优选为第二致凝剂溶液，所述第二致凝剂溶液的成分与第一致凝剂溶液的成分一致，在此不再赘述。
在本发明中，所述第二血液样本和第二致凝剂的体积比优选为(16～36)：1，更优选为(21～31)：1，最优选为(24～27)：1。
在本发明中，所述第一凝固时间和第二凝固时间的检测设备优选为血栓弹力图仪，本发明对所述血栓弹力图仪没有特殊的限制，采用本领域技术人员熟知的血栓弹力图仪即可。本发明对所述第一凝固时间和第二凝固时间的检测方法没有特殊的限制，优选采用血栓弹力图仪按照血栓弹力图仪检测血液凝固时间的使用说明或者要求进行凝固时间的检测即可。
在本发明中，所述第一致凝剂和第二致凝剂的成分和用量相同，第一凝固时间的检测方法和第二凝固时间的检测方法相同，本发明在使用相同的致凝剂、致凝剂用量以及凝固时间的检测方法下以保证第一凝固时间和第二凝固时间是在相同的检测条件下获得的，从而使二者具有可比性。
在本发明中，如果血液样本中含有肝素或肝素类物质，加入肝素中和剂的血液样本中肝素或肝素类物质被肝素中和剂中和，肝素的抗凝作用消失，造成其凝固时间缩短，将其与未加入肝素中和剂的同一血液样本的凝固时间对比，二者凝固时间会出现时间差；如果血液样本中无肝素存在，加入肝素中和剂的血液样本与未加入肝素中和剂的血液样本，二者凝固时间基本相同。
在本发明中，如果第一凝固时间和第二凝固时间之间时间差大于5秒，即如果第一凝固时间小于第二凝固时间的数值大于5秒时，则血液样本中含有肝素或肝素类物质，也就是说加入肝素中和剂的血液样本的凝固时间短于空白血液样本的凝固时间的数值大于5秒时，血液样本中存在肝素或肝素类物质。
本发明提供的检测方法能够取代现有的肝素酶方法，适用于采用血栓弹力图仪进行血液中肝素的检测。
在另一方面，本发明还提供了一种检测血液中肝素或肝素类物质的试剂盒，其包括：
肝素中和剂和致凝剂。
在本发明中，所述肝素中和剂和致凝剂的种类与上述技术方案所述肝素中和剂和致凝剂的种类一致，如肝素中和剂和致凝剂优选为肝素中和剂溶液和致凝剂溶液，在此不再赘述。
本发明提供的检测血液中肝素或肝素类物质的试剂盒便于临床检测使用。
下面结合实施例，对本发明的具体实施方式作进一步描述。以下实施例仅用于更加清楚地说明本发明的技术方案，而不能以此来限制本发明的保护范围。
本发明以下实施例所采用的试剂均为市售商品，所采用的Tris、HCl、Proclin 300、氯化钙、甲苯胺兰、鱼精蛋白均购买于SIGMA公司，所采用的自动血栓弹力图仪为美国血液技术公司提供的TEG5000型号的仪器。
实施例1
将同一血液样本分成质量(或体积)相同的两份加入到两个测试杯中，分别记为第一血液样本和第二血液样本，第一血液样本和第二血液样本的体积均为0.34mL，然后向第一血液样本中加入0.01mL的甲苯胺兰溶液，得到混合物；所述甲苯胺兰溶液的制备方法为：向缓冲液混合液中加入甲苯胺兰，所述缓冲液混合液包括：pH为7.20的Tris-Hcl缓冲液和Proclin300，Proclin300在Tris-Hcl缓冲液中的质量含量为0.2％；甲苯胺兰在缓冲液混合液中的质量含量为0.1％。
向上述混合物和第二血液样本中分别加入0.01mL的凝血酶溶液，所述凝血酶溶液的制备方法为：向缓冲液混合液中加入凝血酶，所述缓冲液混合液包括：pH为7.20的Tris-Hcl缓冲液和Proclin300，Proclin300在Tris-Hcl缓冲液中的质量含量为0.2％；凝血酶在缓冲液混合液中的质量含量为0.1％。
按照自动血栓弹力图仪的操作手册，将上述加入凝血酶溶液的混合物和第二血液样本分别进行凝固时间的检测，得到的检测结果为，混合物的凝固时间为20秒，第二血液样本的凝固时间为28秒，混合物的凝固时间与第二血液样本的凝固时间的时间差为8秒，因此，血液样本中含有肝素或肝素类物质。
实施例2
将同一血液样本分成质量(或体积)相同的两份加入到两个测试杯中，分别记为第一血液样本和第二血液样本，第一血液样本和第二血液样本的体积均为0.34mL，然后向第一血液样本中加入0.01mL的鱼精蛋白溶液，得到混合物；所述鱼精蛋白溶液的制备方法为：向缓冲液混合液中加入鱼精蛋白， 所述缓冲液混合液包括：pH为7.20的Tris-Hcl缓冲液和Proclin300，Proclin300在Tris-Hcl缓冲液中的质量含量为0.2％；鱼精蛋白在缓冲液混合液中的质量含量为0.1％。
向上述混合物和第二血液样本中分别加入0.02mL的氯化钙溶液，所述氯化钙溶液的制备方法为：向缓冲液混合液中加入氯化钙，所述缓冲液混合液包括：pH为7.20的Tris-Hcl缓冲液和Proclin300，Proclin300在Tris-Hcl缓冲液中的质量含量为0.2％；氯化钙在缓冲液混合液中的浓度为0.25mol/L。
按照自动血栓弹力图仪的操作手册，将上述加入氯化钙溶液的混合物和第二血液样本分别进行凝固时间的检测，得到的检测结果为，混合物的凝固时间为30秒，第二血液样本的凝固时间为42秒，混合物的凝固时间和第二血液样本的凝固时间的时间差为12秒，因此，血液样本中含有肝素或肝素类物质。
比较例1
使用肝素酶方法检测实施例1的血液样本中是否含有肝素，检测方法为：
将同一血液样本分成质量(或体积)相同的两份加入到两个测试杯中，分别记为第一血液样本和第二血液样本，第一血液样本和第二血液样本的体积均为0.34mL，然后向第一血液样本中加入0.2mL肝素酶溶液(美国血液技术公司提供的普通杯试剂盒)得到混合物，向上述混合物和第二血液样本中分别加入0.2mL的氯化钙溶液和0.2mL的致凝剂(氯化钙溶液和致凝剂均为上述试剂盒中提供的试剂)。按照自动血栓弹力图仪的操作手册，将上述加入致凝剂的混合物和第二血液样本分别进行凝固时间的检测，得到的检测结果为，混合物的凝固时间为35秒，第二血液样本的凝固时间为43秒，混合物的凝固时间与第二血液样本的凝固时间的时间差为8秒，因此，血液样本中含有肝素或肝素类物质。
比较例2
按照比较例1的方法测试本发明实施例2中的血液样本是否含有肝素，检测结果为，血液样本中含有肝素或肝素类物质。
通过实施例和比较例可以看出，本发明实施例提供的方法与现有技术中比较例提供的方法得到的检测结果一致，本发明提供的方法能够较为准确的检测出血液中是否含有肝素或肝素类物质。
由以上实施例可知，本发明提供了一种血液中肝素或肝素类物质的检测方法，包括：将同一血液样本分为质量相同的两份，记为第一血液样本和第二血液样本；将所述第一血液样本和肝素中和剂混合，得到混合物；将所述混合物和第一致凝剂混合后进行第一凝固时间的检测，得到第一凝固时间；将所述第二血液样本和第二致凝剂混合后进行第二凝固时间的检测，得到第二凝固时间；所述第一致凝剂和第二致凝剂的成分和用量相同；所述第一凝固时间的检测方法和第二凝固时间的检测方法相同；如果第一凝固时间和第二凝固时间之间的时间差大于5秒，则血液样本中含有肝素或肝素类物质。本发明提供的方法通过使用肝素中和剂替代传统使用肝素酶分解血液样本中的肝素进行检测，使检测成本降低，易于操作，适于临床推广。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明技术原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (10)

  1. 一种血液中肝素或肝素类物质的检测方法，其包括：
    将同一血液样本分为质量相同的两份，分别为第一血液样本和第二血液样本；
    将所述第一血液样本和肝素中和剂混合，得到混合物；
    将所述混合物和第一致凝剂混合后进行第一凝固时间的检测，得到第一凝固时间；以及
    将所述第二血液样本和第二致凝剂混合后进行第二凝固时间的检测，得到第二凝固时间；
    其中，所述第一致凝剂和所述第二致凝剂相同；
    其中，所述第一凝固时间的检测方法和所述第二凝固时间的检测方法相同；并且
    其中，如果所述第一凝固时间和所述第二凝固时间的差值大于5秒，则确定所述血液样本中含有肝素或肝素类物质。
  2. 根据权利要求1所述的方法，其中，所述第一血液样本和肝素中和剂的体积比为(16～36)：1。
  3. 根据权利要求1所述的方法，其中，所述肝素中和剂包括鱼精蛋白和甲苯胺蓝中的一种或两种。
  4. 根据权利要求1所述的方法，其中，所述第一致凝剂和第二致凝剂包括凝血酶、鞣花酸、高岭土、组织因子、磷脂和氯化钙中的一种或多种。
  5. 根据权利要求1所述的方法，其中，所述混合物和第一致凝剂的体积比为(16～36)：1。
  6. 根据权利要求1所述的方法，其中，所述第二血液样本和第二致凝剂的体积比为(16～36)：1。
  7. 根据权利要求1所述的方法，其中，所述肝素中和剂为肝素中和剂溶液，所述肝素中和剂溶液包括：缓冲液混合液和肝素中和剂；
    所述缓冲液混合液包括：Tris-HCl缓冲液和Proclin300；
    所述肝素中和剂在缓冲液混合液中的质量含量为0.08～0.12％。
  8. 根据权利要求1所述的方法，其中，所述第一致凝剂为第一致凝剂溶液，所述第一致凝剂溶液包括：缓冲液混合液和第一致凝剂；
    所述缓冲液混合液包括：Tris-HCl缓冲液和Proclin300；
    所述第一致凝剂在缓冲液混合液中的质量含量为0.08～0.12％；并且
    其中，所述第二致凝剂为第二致凝剂溶液，所述第二致凝剂溶液包括：缓冲液混合液和第二致凝剂；
    所述缓冲液混合液包括：Tris-HCl缓冲液和Proclin300；
    所述第二致凝剂在缓冲液混合液中的质量含量为0.08～0.12％。
  9. 根据权利要求1所述的方法，其中，所述第一凝固时间和第二凝固时间的检测设备为血栓弹力图仪。
  10. 一种检测血液中肝素或肝素类物质的试剂盒，其包括：
    根据权利要求1至9中任一项所述的肝素中和剂和致凝剂。
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