Whole blood is divided into non-coagulation and anticoagulation according to the conditions of collection. At the same time, the upper yellow liquid that is not separated by anticoagulation is called serum; the upper yellow liquid separated by anticoagulation is called plasma.
The storage temperature and time of the specimen, the method steps of serum, plasma and cell separation are also important factors influencing the test results before analysis. After blood is drawn, blood cells need to consume some nutrients due to metabolism, so when these components are measured, they need to be centrifuged in time to separate the cellular components.
The preparation of the individual before blood collection, such as the length of the fasting time, the determination of the sampling time and the posture of the patient during blood collection; the application time of the tourniquet, the infusion, the selection of sports, anticoagulants and stabilizers; the treatment of specimens, etc. Affects the level of certain test indicators. Therefore, the specimen collection process should be standardized to minimize pre-analytical errors.
First, do not anti-coagulation to collect serum:
1. Dry air tube collection without additives:
The inner wall of the blood vessel is evenly coated with a medicament (silicone oil) for preventing wall hanging. It uses the principle of natural coagulation of blood to coagulate blood, and after the serum is naturally precipitated, it is centrifuged. Mainly used for serum biochemistry (liver function, kidney function, myocardial enzymes, amylase, etc.), electrolytes (serum potassium, sodium, chlorine, calcium, phosphorus, etc.), thyroid function, drug detection, HIV testing, tumor markers, serum immunity Learn to test.
The whole blood can also be collected directly by using a dry EP tube, and the sample is fully allowed to stand so that the red blood cells are fully naturally coagulated, and the serum is separated by centrifugation for use or storage.
2. Collect with a coagulation tube:
The inner wall of the blood collection tube is evenly coated with silicone oil to prevent wall hanging, and a coagulant is added. The coagulant activates fibrin to turn soluble fibrin into insoluble fibrin aggregates, which in turn form a stable fibrin clot. If you want to get results quickly, you can use a coagulation tube. Generally, it is allowed to stand for half an hour to 1 hour, and the serum is directly centrifuged for use or preservation, and is often used for emergency biochemistry.
3. Collection of blood collection tubes containing separation gel and coagulant:
The wall of the tube is silicified and coated with a coagulant to accelerate the coagulation of the blood and shorten the inspection time. The separation gel is added to the tube, and the separation gel has a good affinity with the PET tube, and indeed acts as a barrier. Generally, even in a common centrifuge, the separation glue can liquid components (serum) and solid components (blood cells) in the blood. Thoroughly separate and accumulate in the test tube to form a barrier. After centrifugation, no oil droplets are produced in the serum, mainly used for serum biochemistry (liver function, kidney function, myocardial enzyme, amylase, etc.), electrolytes (serum potassium, sodium, chlorine, calcium, phosphorus, etc.), thyroid function, drug detection, AIDS testing, tumor markers, serum immunology.
Advantages of using a coagulation tube:
1. Accelerate the solidification of red blood cells without standing for a long time;
2, with separation gel, effectively separate the serum to better avoid hemolysis;
Disadvantages of collecting serum:
1, the need for full solidification of red blood cells, the required rest time is longer;
2, the serum is relatively small, 1ml of whole blood does not resist anticoagulation can only separate 0.2-0.3ml serum.
Second, anticoagulation collects plasma:
After collecting anticoagulated whole blood and gently inverting it for full anticoagulation, the plasma can be directly centrifuged for use or stored. According to the performance characteristics of different anticoagulants, choose the appropriate anticoagulant. Common anticoagulants used in laboratories are various salts of heparin, EDTA and sodium citrate.
1. Heparin anticoagulation:
The most commonly used anticoagulant, under normal circumstances, will not interfere with the relevant indicators, and the anticoagulation ratio is large (anticoagulant: whole blood = 1:30), there is basically no dilution effect on the blood.
Heparin is a mucopolysaccharide containing a sulfate group with a strong negative charge, which enhances the anti-thrombin III inactivation of serine proteases, thereby preventing the formation of thrombin and preventing various anticoagulation such as platelet aggregation. effect. Heparin tubes are generally used for the detection of biochemical and hemorheological changes, and are the best choice for electrolyte detection. When testing sodium ions in blood samples, heparin sodium cannot be used to avoid affecting the test results. It can also not be used for white blood cell counting and classification, because heparin causes leukocyte aggregation.
Although there is no significant difference in the electrolyte concentration obtained by anticoagulation with the three salts of heparin, lithium heparin is considered to be the best. This is mainly because people think that sodium heparin can make the measured value of sodium higher, and heparin ammonium can make the blood ammonia value higher (measured by urease method).
Heparin can inhibit some of the tool enzymes commonly used in molecular biology, such as restriction enzymes, Taq enzymes, etc., which can affect the experimental results of PCR. Some methods can be used to eliminate the inhibitory effect of heparin, such as using heparinase, or washing leukocytes twice or more with buffer after separating leukocytes. However, many experimenters are still reluctant to use heparin as an anticoagulant in molecular biology experiments.
2, EDTA anticoagulation:
Ethylenediaminetetraacetic acid is an aminopolycarboxylic acid that effectively binds calcium ions in the blood. Calcium removal of calcium removes calcium from the reaction site and prevents and terminates endogenous or exogenous coagulation. It prevents blood coagulation. Compared with other anticoagulants, it has little effect on blood cell agglutination and blood cell morphology. Therefore, EDTA salt (2K, 3K, 2Na) is usually used as an anticoagulant. For general hematology examination, not for blood coagulation, trace elements and PCR.
Since EDTA will chelate heavy metal ions, some macromolecular enzymes with metal ions will have a large amount of loss when using this anticoagulant. Specifically, depending on the indicators measured by the customer, it is necessary to use EDTA for anticoagulation.
3, citrate anticoagulation:
Sodium citrate acts as an anticoagulant by acting on calcium ions in blood samples. The National Committee for Clinical Laboratory Standards (NCCLS) recommends 3.2% or 3.8%, and the ratio of anticoagulant to blood is 1:9. Lysis system (prothrombin time, thrombin time, activated partial thrombin time, fibrinogen). Blood collection should pay attention to the amount of blood collected to ensure the accuracy of the test results, immediately after the blood collection should be gently inverted and mixed 5-8 times. Because the anticoagulation ratio is small (anticoagulant: whole blood = 1:9), there is a certain dilution of the blood, which is generally not recommended.
Select the point of attention for anticoagulation:
1. The amount of anticoagulant added to each sample should be the same, and the amount of whole blood taken should be as consistent as possible;
2, after collecting anticoagulated whole blood must be gently inverted, fully anti-coagulation, to prevent part of the blood from contacting the anticoagulant and causing coagulation;
3. The plasma collected by anticoagulated whole blood is relatively more (1ml anticoagulated whole blood can separate 0.4-0.5ml plasma);
4. After cryopreservation of the anticoagulated plasma, flocculation may occur during thawing, and if necessary, it may be centrifuged to remove turbidity for measurement.
After the serum and plasma are collected, they are not measured at the moment, and can be cryopreserved immediately. The lower the temperature, the better. If the temperature is not repeated in the middle, it can be stored for one month below -20 °C, and can be stored for three months below -70 °C.
Third, the centrifugal speed used to collect serum and plasma:
Separation of serum or plasma, depending on the species, the centrifugal speed is also different, the recommended centrifugal speed is:
1. Mice: generally 1000-1500 rpm, centrifuged for 8-10 minutes;
2. Rats and rabbits: generally 2000-2500 rpm, centrifuged for 8-10 minutes;
3, person: generally 2500-3000 rev / min, centrifuge for 8-10 minutes.
Fourth, the impact of high fat and hemolysis factors:
The effect of hemolysis:
The effect of hemolysis on certain test indicators depends not only on the method used, but also on the analytical instrument used. The dual-wavelength measurement can eliminate the color interference of Hb to a certain extent, but the interference of Hb can not be completely eliminated, especially when Hb can react with the reagents used. In general, mild hemolysis did not significantly interfere with the determination of most clinical chemistry indicators. Severe hemolysis may have two effects: (1) when the concentration of the component in the red blood cells is higher than that of the plasma, the measurement result is high; (2) the determination component is slightly diluted when the concentration in the RBC is lower than that of the plasma. effect. Observing whether a specimen is hemolyzed with the naked eye can only be a rough judgment. Hemolysis can only be seen by the naked eye when the serum hemoglobin concentration exceeds 20 mg/dl. It has been reported that 0.1% of red blood cells are hemolyzed, and their serum appearance is consistent with non-hemolytic specimens; after hemolysis occurs in 1% red blood cells, the serum is clear and is cherry red, and such specimens represent moderate hemolysis. It has been suggested to use serum Hb concentration to judge the degree of hemolysis. The serum free Hb of non-hemolytic specimens was 4.8±3.2 mg/dl, and the moderate hemolytic serum Hb43.5±13.9 mg/dl. Even slight hemolysis may result in higher determination of the corresponding indicators (such as LDH, ACP, K, etc.), such specimens should be avoided as much as possible.
The effect of high fat:
The effect of turbidity produced by hyperlipidemia on the determination of certain indicators is also dependent on the assay used. In general, lipemia affects the accuracy of the test results by sample heterogeneity, water replacement, and absorption of lipophilic components. Macroscopic blood lipid specimens can affect total protein determination, electrophoresis, and chromatographic analysis.
fifth. Collection and preservation of whole blood or red blood cells
To measure whole blood or related indicators in red blood cells, it is first necessary to collect anticoagulated whole blood.
Whole blood: Collect anticoagulated whole blood, gently invert and fully anticoagulation, directly absorb the whole blood, use cold distilled water to prepare dissolved blood and use it for different indicators. It can also quantitatively absorb whole blood and transfer to EP tube. Cryopreservation (the lower the temperature, the better), thawed before the measurement and proportionally added cold distilled water to make lysed blood for testing.
Red blood cells: Collect anticoagulated whole blood, gently invert and fully anticoagulate, directly centrifuge to remove the plasma, leave layer of red blood cells, add 3 times of normal saline, gently invert and mix, 500~1000 rpm, centrifuge for 5 minutes. Discard the precipitated red blood cells and repeat 2 to 3 times until the supernatant is colorless (washing red blood cells).
1. Directly absorb red blood cells, add cold distilled water in proportion to make dissolved blood to be tested;
2. Quantitatively absorb red blood cells, transfer to EP tube, immediately freeze at low temperature (the lower the temperature, the better), thaw before measurement, and add cold distilled water to make dissolved blood for detection (some red blood cells will rupture after thawing, so the sample Quantification must be performed before cryopreservation).
Preparation of dissolved blood: quantitatively absorb red blood cells or whole blood, add cold distilled water in proportion, fully vortex and mix to prepare lysed blood (observed by light, the solution is clear and translucent, can be observed microscopically whether the red blood cells are broken, if not broken, the mixing time can be extended) , diluted to different concentrations for the detection of different indicators.