Coagulation (biophysical method)
Coagulation can also be called biophysical method, by detecting the changes of a series of physical quantities (light, electricity, mechanical movement, etc.). ) The plasma is treated under the action of coagulation activator, and then the data obtained are analyzed by computer and converted into the final result.
A. Current methodology
At present, the method is to use the characteristic that fibrinogen is not conductive but fibrin is conductive, take the sample to be tested as a part of the loop, and judge the formation of fibrin according to the change of loop current during coagulation. However, due to the unreliability and singleness of the current method, it was quickly eliminated by more sensitive and extensible optical methods.
B. Optical method (turbidimetry)
Optical hemagglutination instrument is used to measure coagulation function according to the change of turbidity during coagulation.
According to the change of light in the solidification process of the sample to be measured, the solidification state of the sample is judged. When coagulant is added to the sample, the light intensity of the sample gradually increases with the formation of fibrin clots in the sample. The instrument describes this optical change as a solidification curve, and the light intensity of the sample remains unchanged after complete solidification. Generally, the starting point of solidification is 0%, the end point of solidification is 100%, and 50% is the solidification time. The light detector receives this light change, converts it into an electrical signal, amplifies it and then transmits it to the monitor for processing, and draws a solidification curve.
The advantages of optical coagulation test are high sensitivity, simple instrument structure and easy automation. The disadvantage is that the optical abnormality of the sample, the smoothness of the test cup and the bubbles in the sample will all become interference factors in the measurement.
C. magnetic bead method
The early magnetic bead method was to put a magnetic bead in the detection cup and stick it in a straight line with a ferromagnetic metal rod outside the cup. After the specimen is solidified, due to the formation of fibrin, the magnetic beads move away from the metal rod, and the instrument detects the solidification end point accordingly. This instrument can also be called planar magnetic bead method. The early planar magnetic bead method can effectively overcome the problem of sample background interference in optical method, but it has the disadvantage of low sensitivity.
Modern magnetic bead method appeared in the late 1980s and commercialized in the early 1990s. Modern magnetic bead method is called double magnetic bead method. The testing principle of double magnetic circuit magnetic bead method is that there are a set of driving coils on both sides of the test cup to generate a constant alternating electromagnetic field, so that the specially-made degaussing steel ball in the test cup can keep constant amplitude oscillation. After adding blood coagulation activator, with the increase of fibrin production, the plasma viscosity increases, and the movement range of small steel balls gradually weakens. According to the change of small steel ball motion induced by another set of measuring coils, the instrument determines the solidification end point when the motion amplitude decays to 50%.
Substrate chromogenic method (biochemical method)
Substrate chromogenic method is to estimate the content and activity of the tested substance by measuring the absorbance change of chromogenic substrate, which can also be called biochemical method. The detection channel uses halogen lamp as the detection light source, and the wavelength is generally 405nm. The detector is in a straight line with the light source, similar to a colorimeter.
The principle of using chromogenic substrate to detect thrombus and hemostasis index by hemagglutination instrument is to artificially synthesize a small peptide with a specific action site similar to the amino acid sequence of natural coagulation factor, and connect the hydrolyzable chromogenic chemical gene with the amino acid of the action site. Because coagulation factor has proteolytic activity, it can not only act on natural protein peptide chain, but also on synthetic peptide chain substrate, thus releasing chromogenic genes and making the solution chromogenic. The color produced is directly proportional to the activity of coagulation factors, so it can be accurately quantified. At present, there are dozens of synthetic polypeptide substrates, and the most commonly used one is p-nitroaniline (PNA), which is yellow and can be determined at a wavelength of 405 mm.
Immunological method
In the immunological method, the purified substance is used as antigen to prepare the corresponding antibody, and then the substance is qualitatively and quantitatively determined by antigen-antibody reaction. Commonly used methods are:
A. immunodiffusion method. Combine the tested substance with the corresponding antibody in a certain medium, measure the size of its precipitation ring, and compare with the standard to calculate the concentration of the tested substance. This method is simple to operate and does not need special equipment, but it takes too long and has low sensitivity, and is only suitable for the detection of coagulation factors with high content.
B. arrow electrophoresis. Under a certain electric field, the tested substance in the gel support combines with its corresponding antibody to form a "rocket peak", and the height of the rocket peak is directly proportional to its content. Measure the peak height and compare it with the standard of quantitative determination. This method is complicated in operation and less in clinical application.
C. two-dimensional immunoelectrophoresis. Some coagulation factors with abnormal molecular structure can be separated by horizontal and vertical electrophoresis.
D. enzyme-linked immunosorbent assay (ELISA). The enzyme-labeled antigen or antibody reacts with the detected object, and the unbound antigen or antibody and interfering substances in the sample are removed by washing, leaving the antigen-antibody complex fixed on the tube wall, and then the substrate of the enzyme is added to react with the chromogenic substance to generate colored substances, and the color depth is directly proportional to the concentration of the detected object. This method has high sensitivity and specificity, and has been used to detect many hemostatic and thrombotic components.
E. immunoturbidimetry. The test object is mixed with its corresponding antibody to form a complex, so as to generate sufficiently large precipitated particles, which are determined by transmission turbidity or scattering turbidity. This method is simple, accurate and convenient for automation.