Collection and preservation of blood samples

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, as 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. Preparation of individuals before blood collection, such as the length of fasting time, the determination of sampling time, and the posture of patients during blood collection; the application time of tourniquet, infusion, sports, anticoagulant, and stabilizer selection; specimen processing 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 empty tube collection without additives: the inner wall of the blood vessel is uniformly coated with a drug (silicone oil) to prevent 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 testing, 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 centrifuged for use or storage.

2. Collecting 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 at the same time. 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 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 gel can have 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 enzymes, 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 coagulation 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. Red blood cells are needed Fully solidified, the required rest time is longer;

2, the serum is relatively less, and 1 ml of whole blood does not resist coagulation, only 0.2-0.3 ml of serum can be isolated. Second, anti-coagulation collection of plasma: collecting anticoagulated whole blood, gently inverted and fully anticoagulated, can be directly centrifuged to separate plasma for use or preservation. 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, there will be no interference with the relevant indicators, while the anticoagulation ratio is large (anticoagulant: whole blood = 1:30), there is 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, heparin lithium is considered to be the best. This is mainly because people think that sodium heparin can make the sodium value higher, and heparin ammonium can make the blood ammonia value higher (determined by the urease method). Heparin can inhibit some of the tool enzymes commonly used in molecular biologies, 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 a 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 amino polycarboxylic acid, which can effectively bind calcium ions in the blood. Calcium will remove calcium from the reaction point and will prevent and terminate endogenous or external. The source of the blood coagulation process, thereby preventing blood coagulation, compared with other anticoagulants, its effect on blood cell agglutination and blood cell morphology is small, so 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 loss when using this anticoagulant. Specifically, depending on the indicators measured by the customer, it is necessary to use EDTA for anticoagulation.

3. Antimony citrate: Sodium citrate acts as an anticoagulant by acting on calcium ions in blood samples. The National Clinical Laboratory Standardization Committee (NCCLS) recommends a 3.2% or 3.8%, anticoagulant to blood ratio. It is 1:9 and is mainly used for the fibrinolysis 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 attention point of anticoagulation:

1. The amount of anticoagulant added in each sample should be the same, and the amount of whole blood taken should be as consistent as possible;

2. Be sure to gently reverse the anticoagulated whole blood. Full anticoagulation to prevent some blood from contacting the anticoagulant and causing coagulation;

3. The anticoagulated whole blood collects more plasma (1ml anticoagulated whole blood can separate 0.4-0.5ml plasma);

4. Anticoagulation After the collected plasma is cryopreserved, flocculent turbidity 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 any time 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 collection of serum, and plasma used centrifugal speed: 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 rev/min, centrifuge 8-10 minutes;

2 Rats, rabbits: generally 2000-2500 rpm, centrifuge for 8-10 minutes;

3, people: generally 2500-3000 rev/min, centrifuge for 8-10 minutes. Fourth, the impact of high fat and hemolysis factors: the impact of hemolysis: the impact 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 some 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 measured component in the red blood cells is higher than that of the plasma, the measurement result is high;

(2) the measured 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 of 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 the non-hemolytic specimen was 4.8±3.2 mg/dl, and the moderate hemolytic serum Hb was 43.5±13.9 mg/dl. Even slight hemolysis may lead to high results (such as LDH, ACP, K, etc.), and 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 depends on the method used. In general, lipemia affects the accuracy of the test results by sample heterogeneity, water replacement, and absorption of lipophilic components. The visible blood lipid specimens can affect the determination of total protein, electrophoresis and chromatographic analysis. V. Collection and preservation of whole blood or red blood cells To determine the relevant indicators of whole blood or red blood cells, it is necessary to collect anticoagulated whole blood first. 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; 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 anticoagulant, directly centrifuge to remove the plasma, leaving a layer of red blood cells, add 3 times volume 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 in proportion Cold distilled water is used to make lysed blood for testing (some red blood cells will rupture after thawing, so the sample must be quantified 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 under the microscope 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.
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