The
coagulation analyzer is a routine medical device for measuring the content of various components in human blood and the results of quantitative biochemical analysis, providing reliable digital basis for clinical diagnosis of various diseases in patients.
1. Working principle
Different types of coagulation analyzers adopt different principles. The main detection methods currently used are: coagulation method, substrate colorimetric method, immunoassay, latex agglutination method, etc.
1. Coagulation method (biophysical method)
The coagulation method is to detect the changes of a series of physical quantities (light, electricity, mechanical movement, etc.) of plasma under the action of coagulation activators, and then analyze the obtained data by computer and convert it into the final result, so it can also be called biophysical method.
1.1, current method.
The current method uses the characteristics that fibrinogen is non-conductive, while fibrin has conductivity. The sample to be tested is used as part of the circuit, and the formation of fibrin is judged according to the changes in the circuit current during the coagulation process. However, due to the unreliability and singleness of the current method, it was quickly replaced by the more sensitive and more scalable optical method.
1.2, Optical method (turbidimetry).
The optical coagulation instrument measures the coagulation function based on the change of turbidity during the coagulation process. The detection endpoint is determined based on the change of light in the sample to be tested during the coagulation process. When the coagulation activator is added to the sample, the light intensity of the sample gradually increases as the fibrin clot is formed in the sample. The instrument depicts this optical change as a coagulation curve. When the sample is completely coagulated, the light intensity no longer changes. The advantages of the optical coagulation test are high sensitivity, simple instrument structure, and easy automation; the disadvantages are that the optical abnormality of the sample, the finish of the test cup, and the bubbles in the sample will become interference factors in the measurement.
1.3, Magnetic bead method.
In the early magnetic bead method, a magnetic bead was placed in the test cup, which was closely attached to a ferromagnetic metal rod outside the cup in a straight line. After the specimen solidified, the formation of fibrin caused the magnetic bead to shift away from the metal rod, and the instrument detected the coagulation endpoint based on this. This type of instrument can also be called the planar magnetic bead method. The early planar magnetic bead method can effectively overcome the problem of sample background interference in the optical method, but it has disadvantages such as low sensitivity. The modern magnetic bead method appeared in the late 1980s and entered commercialization in the early 1990s. The modern magnetic bead method is called the dual magnetic circuit magnetic bead method. The test principle of the dual magnetic circuit magnetic bead method is as follows: There is a set of driving coils on both sides of the test cup, which generate a constant alternating electromagnetic field to keep the specially made demagnetized small steel balls in the test cup in an oscillating motion with equal amplitude. After the coagulation activator is added, as the production of fibrin increases, the viscosity of the plasma increases, and the movement amplitude of the small steel balls gradually weakens. The instrument senses the changes in the movement of the small steel balls based on another set of measuring coils, and determines the coagulation endpoint when the movement amplitude decays to 50%.
2. Substrate colorimetric method (biochemical method)
The substrate colorimetric method is to infer the content and activity of the measured substance by measuring the absorbance change of the color-producing substrate. This method can also be called the biochemical method. Its principle is to artificially synthesize a small peptide with a similar amino acid sequence to the natural coagulation factor and a specific action site, and connect the chemical gene that can be hydrolyzed and produce color to the amino acid at the action site. During the measurement, since the coagulation factor has the activity of proteolytic enzymes, it can not only act on the natural protein peptide chain, but also on the synthetic peptide chain substrate, thereby releasing the color-producing gene and making the solution color. The depth of the color produced is proportional to the activity of the coagulation factor, so accurate quantification can be performed. At present, there are dozens of artificially synthesized polypeptide substrates, and the most commonly used one is p-nitroaniline (PNA), which is yellow and can be measured at a wavelength of 405mm.
3. Immunological method
In the immunological method, the purified test substance is used as the antigen, the corresponding antibody is prepared, and then the test substance is qualitatively and quantitatively determined by antigen-antibody reaction.
Commonly used methods are:
3.1, immunodiffusion method.
Combine the test object with the corresponding antibody in a certain medium, measure the size of its precipitation ring, compare it with the standard, and calculate the concentration of the test object. This method is simple to operate and does not require special equipment, but it takes too long and has low sensitivity. It is only suitable for the detection of coagulation factors with high content.
3.2, arrow electrophoresis.
In a certain electric field, the test object in the gel support combines with its corresponding antibody to form a "rocket peak". The height of the rocket peak is proportional to its content. The peak height is measured and compared with the standard for quantitative determination. This method is complicated to operate and is less clinically used.
3.3, two-way immunoelectrophoresis.
Certain coagulation factors with abnormal molecular structures can be separated by electrophoresis in both horizontal and vertical directions.
3.4, enzyme-linked immunosorbent assay (ELISA method).
The enzyme-labeled antigen or antibody is used to react with the test object to carry out antigen binding reaction. After washing, the unbound antigen or antibody and the interfering substances in the specimen are removed, leaving the antigen-antibody complex fixed on the tube wall. Then, the substrate of the enzyme and the chromogenic substance are added to react to produce colored substances, which are measured by an enzyme marker. The color depth is proportional to the concentration of the test object. This method has high sensitivity and strong specificity. It has been used in many hemostasis and thrombosis component detection.
3.5, immunoturbidimetry.
The test object is mixed with its corresponding antibody to form a complex, thereby producing sufficiently large precipitation particles, which are measured by transmission turbidimetry or scattering turbidimetry. This method is simple to operate, accurate, and easy to automate.
2. Development history
In 1910, Kottman invented the world's first coagulation instrument, which reflects the plasma coagulation time by measuring the change in viscosity during blood coagulation.
In 1922, Kugelmass used a turbidimeter to measure the change in transmitted light to reflect the plasma coagulation time.
In 1950, Schnitger and Gross invented a coagulation instrument based on the current method.
In the 1960s, mechanical coagulation analyzers were developed, and early planar magnetic bead methods appeared.
After the 1970s, due to the development of machinery and electronics industries, various types of fully automatic coagulation analyzers came out one after another.
In the 1980s, due to the emergence of chromogenic substrates and their application in the detection of blood coagulation, fully automatic coagulation analyzers can not only perform general screening tests, but also detect single factors of coagulation, anticoagulation, and fibrinolysis systems, making the detection of anticoagulation and fibrinolysis possible.
In the late 1980s, the invention of the dual magnetic circuit magnetic bead method brought new concepts to the detection of thrombosis and hemostasis. Due to its unique design principle, some influencing factors of optical detection no longer exist on this type of detection instrument.
In the 1990s, the development of the immune channel of the fully automatic coagulation analyzer integrated various detection methods, and the detection items were more comprehensive, providing a new means for the detection of thrombosis and hemostasis.
3. Classification
According to the degree of automation, coagulation analyzers can be divided into fully automatic and semi-automatic instruments
1. Fully automatic:
The characteristics of fully automatic instruments are fast detection speed, many measurement items, complex detection principle and intelligent instrument design. When using a fully automatic coagulation analyzer, as long as the separated plasma sample is placed in the designated position, the instrument can complete the whole process of sample addition, preheating, detection and report printing. Most fully automatic coagulation analyzers can arbitrarily select different combinations of items for detection. The detection of samples is random, and the data processing and storage functions of the instrument are also strong.
2. Semi-automatic:
Semi-automatic coagulation analyzers require manual sample addition, slow detection speed, simple principle, few detection items, and limited software functions equipped with the instrument.
4. Detection indicators
The basic detection indicators of thrombosis and hemostasis are: prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen (FIB), thrombin time (TT), endogenous coagulation factors, exogenous coagulation factors, high molecular weight heparin, low molecular weight heparin, protein C, protein S, etc.
V. Precautions
1. After purchasing the instrument, the instrument should be evaluated according to the performance parameters that the instrument should be able to achieve as indicated in the manual. If any problems are found, contact the manufacturer in time.
2. The sampler used in the test process should be calibrated to ensure the accuracy of reagent dilution and sample addition.
3. For test items with calibrated plasma, the standard curve must be established with calibrated plasma. When the reagent batch number or type is changed, the calibrated plasma should be used to re-establish the standard curve.
4. In-house quality control must be performed when testing specimens, and the detection of semi-automatic instruments should be double-measured.
5. It is very important to do a good job of quality control before analysis. The collection and storage of specimens should be carried out strictly in accordance with relevant requirements.
6. The placement time of reagents in the preheating tank should be strictly limited according to the requirements of the reagent manual. Extended placement time will affect the accuracy of the test results.
