CN108152177B - Method for rapidly detecting hematocrit - Google Patents
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- CN108152177B CN108152177B CN201711309844.4A CN201711309844A CN108152177B CN 108152177 B CN108152177 B CN 108152177B CN 201711309844 A CN201711309844 A CN 201711309844A CN 108152177 B CN108152177 B CN 108152177B
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Abstract
The application discloses a method for rapidly detecting the hematocrit, which comprises the steps of settling blood by using an anti-blood cell antibody in a container with a measurable volume, and then calculating the hematocrit. The application also provides the application of the method in the instant examination. The method provided by the application is particularly suitable for being used in point-of-care testing (POCT), the materials are portable, the reaction is rapid, the operation is simple and easy, the result is accurate, the hematocrit can be rapidly obtained, and the rapid implementation of diagnosis and subsequent treatment work is facilitated.
Description
Technical Field
The application relates to the technical field of detection, in particular to a method for rapidly detecting hematocrit.
Background
POCT, point-of-care testing (point-of-care testing), refers to a new method for rapidly obtaining test results by performing clinical tests near patients, which are usually not necessarily performed by clinical testers, and performing analysis immediately at a sampling site, thereby omitting complicated processing procedures of specimens during laboratory tests.
The hematocrit refers to the volume ratio of settled blood cells to whole blood measured after a certain amount of anticoagulated whole blood is centrifugally precipitated. After anticoagulation treatment, blood is separated into two major parts, blood cells and blood plasma under the action of external force. The conventional method for measuring the hematocrit is to place blood in a wenshi tube and centrifuge the blood according to a specified time and speed, after the centrifugation is finished, the red blood cells are completely compacted at the bottom end of the tube, the red blood cells are in close contact with each other, and all plasma among the cells is eliminated as much as possible, at this time, the plasma is completely extruded to the upper layer of the blood cells, and the percentage of the blood cells in the lower layer to the whole blood is the hematocrit to be obtained, namely, the volume fraction (or percentage) of the compacted blood cells.
The hematocrit is one of the important factors affecting the whole blood test result of a partial in vitro diagnostic reagent, in the POCT in vitro diagnostic reagent test with a fixed sample volume, the concentrations of the detection targets in the whole blood sample and the serum and plasma samples are the same, but because the blood cells occupy a certain volume in the whole blood sample, the total amount of the targets is less than that of the serum and plasma samples in the fixed sample volume, and thus the test result of the whole blood sample is often lower than that of the serum and plasma samples in the same sample volume. The hematocrit condition of each individual is different, and the hematocrit condition of different individuals needs to be measured, so that the aim of eliminating the influence of the hematocrit is fulfilled.
The conventional hematocrit measurement method needs specific equipment and centrifugation, and if the method is applied to a POCT in-vitro diagnostic reagent, the defects of additional equipment investment and sample amount increase exist, and the method is not suitable for the characteristics of rapidness, simplicity and convenience of POCT.
Disclosure of Invention
To solve the above technical problems, a first object of the present invention is to provide a method for rapidly detecting hematocrit; the second objective of the present invention is to provide the application of the above method for rapidly detecting hematocrit in point-of-care testing (POCT) to achieve rapid and convenient determination of blood cells in point-of-care testing (POCT).
The technical scheme provided by the invention is as follows:
a method for rapidly detecting the hematocrit is characterized in that in a container with a measurable volume, anti-blood cell antibodies are used for settling blood, and then the hematocrit is calculated.
Preferably, the anti-blood cell antibody is added to a buffer solution to prepare a reaction solution, and the blood is sedimented.
Preferably, the reaction solution is placed in a container with a measurable volume, then the blood is added, the mixture is uniformly mixed, the mixture is stood for sedimentation, and then the hematocrit is calculated.
Preferably, the buffer comprises a pH stabilizer, a surfactant, a preservative, and water.
Preferably, in the buffer solution, the pH stabilizer is any one of a phosphate buffer solution, a boric acid-borax buffer solution, and a MES buffer solution.
Preferably, the surfactant is any one or more of Tween, Poloxamer188 and sodium dodecyl benzene sulfonate.
Preferably, the preservative is any one or more of sodium azide, Proclin300, 5-chloro-2-methyl-4-isothiazolin-3-one, 2-methyl-4-isothiazolin-3-one and 2-methyl-4, 5-trimethylene-4-isothiazolin-3-one.
Preferably, the blood is in particular fresh whole blood ready for use, or anticoagulated whole blood.
Preferably, the volumetric container is embodied as a graduated cuvette.
The invention also provides the use of the method for detecting hematocrit in any one of the above in a point-of-care assay.
The invention provides a method for rapidly detecting hematocrit, which comprises the steps of mixing blood (such as fresh whole blood ready for use or anticoagulated whole blood) and an anti-blood cell antibody in a container with a measurable volume, reacting, utilizing the specific binding reaction of the antibody and blood cells to agglutinate and precipitate the blood cells, thereby precipitating the blood cells at the bottom of the container with the measurable volume, extruding plasma to the upper layer of the blood cells, measuring the volume of the blood cells and the volume of the whole blood respectively through the container with the measurable volume, and obtaining the hematocrit (namely the percentage of the blood cells at the lower layer to the whole blood) through calculation. The antibody and the blood cells have high reaction speed, so that the blood cells can be precipitated in a short time, and the detection of the hemangiocyte volume can be rapidly carried out. Moreover, the hemangiocrit can be detected only by carrying a container with a measurable volume and the anti-hemocyte antibody during detection by using the anti-hemocyte antibody, equipment such as a centrifuge and the like is not required to be configured, the rapid detection is convenient to be carried out on various occasions, particularly at the bedside of a patient, the time is saved, and the hospitalizing cost is saved.
Drawings
In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings needed to be used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments described in the present application, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a standard curve of different volumes for example 1 of the present invention;
FIG. 2 is a graph showing a comparison between the method of the present invention used in example 2 of the present invention and a conventional test method.
Detailed Description
In order to make those skilled in the art better understand the technical solutions in the present application, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application, and it is obvious that the described embodiments are only a part of the embodiments of the present application, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.
Referring to fig. 1 to 2, an embodiment of the present invention provides a method for rapidly detecting hematocrit, in which an anti-blood cell antibody is used to settle blood in a container with a measurable volume, and then the hematocrit is calculated.
The invention provides a method for rapidly detecting hematocrit, which comprises the steps of mixing blood (such as fresh whole blood ready for use or anticoagulated whole blood) and an anti-blood cell antibody in a container with a measurable volume, reacting, utilizing the specific binding reaction of the antibody and blood cells to agglutinate and precipitate the blood cells, thereby precipitating the blood cells at the bottom of the container with the measurable volume, extruding plasma to the upper layer of the blood cells, measuring the volume of the blood cells and the volume of the whole blood respectively through the container with the measurable volume, and obtaining the hematocrit (namely the percentage of the blood cells at the lower layer to the whole blood) through calculation. The antibody and the blood cells have high reaction speed, so that the blood cells can be precipitated in a short time, and the detection of the hemangiocyte volume can be rapidly carried out. Moreover, the hemangiocrit can be detected only by carrying a container with a measurable volume and the anti-hemocyte antibody during detection by using the anti-hemocyte antibody, equipment such as a centrifuge and the like is not required to be configured, the rapid detection is convenient to be carried out on various occasions, particularly at the bedside of a patient, the time is saved, and the hospitalizing cost is saved.
Preferably, the anti-blood cell antibody is added to a buffer solution to prepare a reaction solution, and the blood is sedimented.
The anti-blood cell antibody is added into the buffer solution to prepare reaction solution, which is more convenient for operators to use. The standard reaction solution can be directly prepared by a factory and carried by an operator, and the standard reaction solution directly reacts with blood when in use, so that the time is further saved. Meanwhile, the anti-blood cell antibody added with the buffer solution can be stably stored in a long storage period, so that the detection result is prevented from being influenced by deterioration.
Preferably, the reaction solution is placed in a container with a measurable volume, then the blood is added, the mixture is uniformly mixed, the mixture is stood for sedimentation, and then the hematocrit is calculated.
When the method of the present invention is used for detecting the hematocrit, the reaction solution is placed in a container with a measurable volume, then the blood is added, the mixture is uniformly mixed (for example, the container is sealed and then is repeatedly turned upside down and uniformly mixed), the blood cells are settled by standing, and then the hematocrit is calculated. Usually, the mixture is allowed to stand for 3 to 10min, preferably 5 to 7 min.
Preferably, the buffer comprises a pH stabilizer, a surfactant, a preservative, and water.
Preferably, in the buffer solution, the pH stabilizer is any one of a phosphate buffer solution, a boric acid-borax buffer solution and a MES buffer solution
The surfactant is one or more of Tween, Poloxamer188 and sodium dodecyl benzene sulfonate.
The preservative is any one or more of sodium azide, Proclin300, 5-chloro-2-methyl-4-isothiazolin-3-one, 2-methyl-4-isothiazolin-3-one and 2-methyl-4, 5-trimethylene-4-isothiazolin-3-one.
The buffer solution can keep the stability of the anti-blood cell antibody in a long storage period, does not deteriorate, and avoids the influence on the sedimentation reaction, thereby ensuring the accuracy of the detection result. The buffer typically includes a pH stabilizer, a surfactant, a preservative, and the balance water.
In the buffer solution, the pH stabilizer is any one of phosphate buffer solution, boric acid-borax buffer solution and MES buffer solution, and the use concentration is usually 0.01M-0.2M, preferably 0.1M; phosphate buffer is preferably used, and phosphate buffer is usually used in a concentration of 0.1mol/L while controlling pH to 7.2 to 7.6, preferably pH 7.4. The surfactant is one or more of Tween, Poloxamer188 and sodium dodecyl benzene sulfonate, and is used at a concentration of 0.05-1%, preferably 0.5%. The surfactant is preferably a Tween type, more preferably Tween 20. The surfactant can enhance the effect of antibody reaction, simultaneously reduce the residual plasma on the surface of blood cells, and ensure that the separation of the blood cells and the plasma is more thorough, thus obtaining more accurate hematocrit data. The preservative is usually any one or more of sodium azide, Proclin300, 5-chloro-2-methyl-4-isothiazolin-3-one, 2-methyl-4, 5-trimethylene-4-isothiazolin-3-one, and is used usually at a concentration of 0.05% to 0.5%, preferably 0.1%. The commercially available Proclin300 is preferably used. The preservative may also be a commercially available finished product such as Proclin 300.
Preferably, the blood is in particular fresh whole blood ready for use, or anticoagulated whole blood.
The ready-to-use fresh whole blood is taken as it is without the adventitial coagulation reaction. The fresh whole blood (ready to use) has not yet completed the blood coagulation reaction, and the anti-erythrocyte antibody binds to the blood coagulation reaction to inhibit the blood coagulation reaction and also to cause the same reactions such as sedimentation, so that the hematocrit can be calculated. The method of the invention can be used for detecting the hematocrit of fresh whole blood which is taken and used immediately, and can be made into a detection device or other devices, which can be convenient for clinical detection beside a patient or used by the patient and family members for self detection.
Anticoagulated whole blood is obtained by adding an anticoagulant to collected blood to inhibit the action of thrombin and maintain the liquid state of the blood, thereby allowing a blood cell precipitation reaction to occur. It is preferred to use anticoagulants that do not interfere with blood sedimentation or other biochemical tests. The anticoagulant is any one of heparin, alkali metal salt of heparin, ethylene diamine tetraacetic acid salt, citrate and oxalate. Such as heparin, heparin sodium, sodium ethylene diamine tetracetate, potassium ethylene diamine tetracetate, sodium citrate, potassium citrate, sodium oxalate and the like.
Preferably, the volumetric container is embodied as a graduated cuvette.
The container for measuring the volume used in the present invention may be any container as long as it can contain blood and a reaction solution and measure the volume of blood cells and whole blood. For example, graduated test tubes or centrifuge tubes may be used.
The invention also provides the use of the method for detecting hematocrit in any one of the above in a point-of-care assay.
The method is particularly suitable for use in point-of-care testing (POCT), has the advantages of portable materials, quick response, simplicity, easy operation and accurate result, can quickly obtain the hematocrit, and is convenient for quick diagnosis and subsequent treatment.
EXAMPLE 1 measurement of hematocrit Standard Curve
Preparing an anti-blood cell antibody buffer solution:
preparing 0.1M PB buffer solution with the pH value of 7.4, and adding 10ml of the PB buffer solution into a 100ml volumetric flask after the preparation is finished; 0.9g of sodium chloride is weighed and added into the same 100ml volumetric flask; weighing 0.5g of Tween20 into a beaker, adding a small amount of ultrapure water to dissolve the Tween20, adding the dissolved Tween into the same 100ml volumetric flask, rinsing the beaker by using a small amount of ultrapure water, and adding rinsing liquid into the same 100ml volumetric flask; measuring 100ul of Proclin300, and adding into the same 100ml volumetric flask; adding 5mg of anti-blood cell antibody into the same 100ml volumetric flask; adding ultrapure water into the volumetric flask to reach 100 ml.
Pre-loading anti-blood cell antibody buffer solution:
1000ul of anti-blood cell antibody buffer was pre-filled into a graduated cylindrical tube (centrifuge tube) for use.
The test method comprises the following steps:
1. whole blood samples of 30%, 35%, 40%, 45%, 50%, 55%, 60% hematocrit were prepared.
2. 1000ul of whole blood samples are taken and added into a graduated cylindrical tube (a centrifugal tube) pre-filled with an anti-hemocyte antibody buffer solution, the cylindrical tube is placed on a tube frame after being inverted and mixed uniformly, the timing is simultaneously carried out for 5 minutes, after the timing for 5 minutes is finished, the graduation occupied by the hemocyte on the cylindrical tube is read, and each packed sample is tested for 5 times.
3. And fitting a curve equation by taking the number of the packed products of the samples as a Y value and the read scale average value as an X value.
And (3) testing results:
according to the test result, preparing standard curves with different volume ratios, and obtaining a standard equation as follows: y is 0.0003x2+0.0028x+0.1719，R20.9997. The standard curve is shown in figure 1.
The activities of different batches of anti-blood cell antibodies are different, so that the standard equation for measuring the hematocrit by using different batches of anti-blood cell antibodies is different, and different equations are used among different batches. The standard equation is provided for each batch of the commercial product, and the standard equation is the same as the code modulation of the existing diagnostic product kit in this embodiment, and each batch has one code modulation, and the equation corresponding to each batch is provided in the form of an electronic tag or an IC chip.
EXAMPLE 2 actual sample testing for hematocrit
Sample preparation:
blood samples from 20 volunteers were collected and the hematocrit value of each sample was determined using a conventional method (winy tube).
The test and calculation method comprises the following steps:
the calculation equation of the volume value was determined using the method in example 1, then the volume test was performed on each sample, and the measured value of the sample was substituted into the calculation equation to calculate the volume value, and compared with the measured value of the conventional method.
And (3) testing results:
| sample number | Value of volume of pressure | Value of conventional test | Deviation of |
| 1 | 46.6％ | 45％ | 3.60％ |
| 2 | 42.9％ | 43％ | -0.14％ |
| 3 | 48.6％ | 48％ | 1.15％ |
| 4 | 39.5％ | 40％ | -1.25％ |
| 5 | 44.8％ | 43％ | 4.07％ |
| 6 | 37.9％ | 39％ | -2.90％ |
| 7 | 41.2％ | 42％ | -1.93％ |
| 8 | 42.9％ | 43％ | -0.14％ |
| 9 | 44.8％ | 44％ | 1.70％ |
| 10 | 36.3％ | 37％ | -1.89％ |
| 11 | 41.2％ | 41％ | 0.46％ |
| 12 | 46.6％ | 47％ | -0.81％ |
| 13 | 44.8％ | 43％ | 4.07％ |
| 14 | 39.5％ | 40％ | -1.25％ |
| 15 | 44.8％ | 45％ | -0.56％ |
| 16 | 50.5％ | 49％ | 3.14％ |
| 17 | 41.2％ | 42％ | -1.93％ |
| 18 | 41.2％ | 42％ | -1.93％ |
| 19 | 46.6％ | 46％ | 1.35％ |
| 20 | 44.8％ | 45％ | -0.56％ |
The test result detected by the method provided by the invention is compared with the result of the conventional test method, and the standard equation of y is 0.8185x +0.0774, R20.9521. See FIG. 2 for an alignment chart.
Therefore, the relative deviation of the hematocrit value calculated by the method provided by the invention and the conventional method is not more than 5%, and the correlation of the test result and the conventional method test result is better.
Example 3 comparison of the results of a test to calculate the hematocrit with the results of a test to calculate the plasma
Sample preparation:
a certain amount of blood samples of volunteers were collected, the content of plasma C-reactive protein was determined using a Meyer's C-reactive protein assay kit, and 20 samples were selected.
The test and calculation method comprises the following steps:
the hematocrit value is tested by using the method, the content of the C-reactive protein is tested by using a fluorescence lateral chromatography test strip and an instrument (in a plasma sample test mode), the measured hematocrit value is used for correction (the correction value is equal to a plasma mode test value/(1-hematocrit value)), and the result is compared with the result of the C-reactive protein detection kit of Merrill on the basis of the plasma test result and the result of the fluorescence lateral chromatography test strip and the instrument (in a whole blood sample test strip mode).
The detection results are as follows:
from the above results, the result of performing the hematocrit correction is less deviated from the result of the plasma test, and the accuracy is more excellent compared to the whole blood mode. (Whole blood model tests generally use the median of normal physiologic volume values as the number of volumes, and then increase sample loading to match plasma serum results.)
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (9)
1. A method for rapidly detecting the hematocrit is characterized in that an anti-blood cell antibody is added into a buffer solution in a container with a measurable volume to prepare a reaction solution, blood is settled, and the hematocrit is calculated according to a standard curve;
the standard curve for hematocrit was determined as follows:
(1) preparing a whole blood sample with the hematocrit of 30%, 35%, 40%, 45%, 50%, 55% and 60%;
(2) taking 1000ul of a whole blood sample, adding the whole blood sample into a graduated cylindrical tube preloaded with an anti-hemocyte antibody buffer solution, turning the whole blood sample upside down, uniformly mixing the whole blood sample and the anti-hemocyte antibody buffer solution, placing the cylindrical tube on a tube frame, timing for 5 minutes simultaneously, reading the graduation occupied by hemocytes on the cylindrical tube after the 5-minute timing is finished, and testing each packed sample for 5 times;
(3) and fitting a curve equation by taking the number of the packed products of the samples as a Y value and the read scale average value as an X value to prepare a standard curve.
2. The method of claim 1, wherein the reaction solution is placed in a container with a measurable volume, the blood is added, the mixture is mixed uniformly, the mixture is left to settle, and then the hematocrit is calculated.
3. The method for rapidly detecting the hematocrit according to claim 2, wherein the buffer comprises a pH stabilizer, a surfactant, a preservative, and water.
4. The method for rapidly detecting the hematocrit according to claim 3, wherein the pH stabilizer in the buffer solution is any one of a phosphate buffer solution, a boric acid-borax buffer solution and a MES buffer solution.
5. The method for rapidly detecting the hematocrit as claimed in claim 3, wherein the surfactant is any one or more of Tween type, Poloxamer188 and sodium dodecyl benzene sulfonate.
6. The method for rapidly detecting the hematocrit according to claim 3, wherein the preservative is any one or more of sodium azide, Proclin300, 5-chloro-2-methyl-4-isothiazolin-3-one, 2-methyl-4, 5-trimethylene-4-isothiazolin-3-one.
7. The method for rapid detection of hematocrit according to any one of claims 1 to 6, wherein the blood is in particular fresh whole blood ready for use, or anticoagulated whole blood.
8. Method for the rapid detection of hematocrit according to any one of claims 1-6, wherein the container of quantifiable volume is embodied as a graduated cuvette.
9. Use of the method for rapid detection of hematocrit according to any one of claims 1 to 8 in a point-of-care assay.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1196486A (en) * | 1996-11-22 | 1998-10-21 | S·C·沃德罗 | Process for enhancing aggregation and/or agglutination of erythrocytes prior to centrifugation |
| US6017764A (en) * | 1997-02-04 | 2000-01-25 | Streck Laboratories, Inc. | Erythrocyte sedimentation rate control |
| US6521460B1 (en) * | 1996-05-21 | 2003-02-18 | Rhein Biotech Gesellschaft Fur Neue Biotechnologische Prozesse Und Produkte Mbh | Method of carrying out blood tests |
| CN102016578A (en) * | 2008-03-21 | 2011-04-13 | 艾博特健康公司 | Method and apparatus for determining the hematocrit of a blood sample utilizing the intrinsic pigmentation of hemoglobin contained within the red blood cells |
| CN106840828A (en) * | 2017-03-29 | 2017-06-13 | 天津中新科炬生物制药股份有限公司 | The method and separator of quick separating blood plasma in a kind of micro whole blood |
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2017
- 2017-12-11 CN CN201711309844.4A patent/CN108152177B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6521460B1 (en) * | 1996-05-21 | 2003-02-18 | Rhein Biotech Gesellschaft Fur Neue Biotechnologische Prozesse Und Produkte Mbh | Method of carrying out blood tests |
| CN1196486A (en) * | 1996-11-22 | 1998-10-21 | S·C·沃德罗 | Process for enhancing aggregation and/or agglutination of erythrocytes prior to centrifugation |
| US6017764A (en) * | 1997-02-04 | 2000-01-25 | Streck Laboratories, Inc. | Erythrocyte sedimentation rate control |
| CN102016578A (en) * | 2008-03-21 | 2011-04-13 | 艾博特健康公司 | Method and apparatus for determining the hematocrit of a blood sample utilizing the intrinsic pigmentation of hemoglobin contained within the red blood cells |
| CN106840828A (en) * | 2017-03-29 | 2017-06-13 | 天津中新科炬生物制药股份有限公司 | The method and separator of quick separating blood plasma in a kind of micro whole blood |
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