The coagulation function package includes more than PT, TT, APTT and FIB. Some large hospitals also add an international sensitivity index (ISI) to ensure the standardization of PT. There are many measurement methods: manual method, semi-automatic, fully automatic. The required method must be consistent with the current conditions. There are manufacturers that specialize in reagents, such as Yazhong, Jinyu, and there are many. There are official websites and Baidu. What can be found ~ The following are for reference only: Recommended method of prothrombin time (PT)
[Principle]
Divide tissue thromboplastin (mainly containing tissue factor and lipids) and calcium ions, add it to citrate anticoagulated plasma, incubate it at 37°C, and measure the plasma coagulation time, which is PT. PT is mainly used to screen and detect inhibitors of factors VII, II, V, X and related factors of the exogenous coagulation system.
[Specimen collection and processing]
1. Specimen collection: Use a siliconized or plastic syringe to draw fasting venous blood, and add citron containing 0.109mol/L at a ratio of 9:1 Place the sodium acid anticoagulant in a siliconized or plastic test tube and mix gently.
2. Specimen processing: Centrifuge at 2000~2500g for 15 minutes to separate platelet-poor plasma, and complete the test within 24 hours.
3. Normal control plasma: Choose more than 10 normal healthy men and women, aged 18 to 45 years old. However, it cannot be used by women who are pregnant, menstruating, lactating, or taking oral contraceptives. The collected plasma should be freeze-dried or stored at -80°C.
[Reagent]
1. Anticoagulant: 109mmol/L sodium citrate solution (equivalent to 32g/L sodium citrate containing two molecules of crystal water).
2. Thromboplastin reagent: Commercially available products should be marked with ISI, batch number and expiry date. Lyophilized products should be reconstituted with the specified buffer diluent according to the instructions.
3. Calcium chloride (CaCl2) solution: 25mmol/L. (At present, some commercial reagents have mixed thromboplastin solution and calcium chloride solution, and there is no need to prepare additional CaCl2 solution).
4. Quality control substances: normal and abnormal control plasma
5. The water used to prepare reagents must meet the standards of Grade 1 pure water.
[Instrument]
1. Manual method: stopwatch, constant temperature water bath or electric heating block maintained at 37℃±1℃. The water depth can immerse the test tube for more than 3cm, and the surface has no scratches on the 10×mm swab tube. Standard 0.1mL pipette. Corrected stopwatch.
2. Instrument method: operate various automatic or semi-automatic coagulation meters in strict accordance with the instructions.
[Operating steps]
1. Manual method
(1) The measurement temperature is 36.5~38.5℃. The above reagents and the tested plasma should be pre-heated. Warm to this temperature, but the prewarming of thromboplastin reagent should not exceed 30 minutes, and the prewarming of plasma should generally not exceed 10 minutes.
(2) All test tubes, sample injectors and other equipment that come into contact with plasma are plastic or siliconized glass tubes.
(3) Take 0.1 mL of citrated anticoagulant plasma and add it to a small test tube: add 0.1 mL of thromboplastin reagent, mix well, and place in a 37°C water bath. Then add 0.1 mL of 25 mmol/L CaCl2 solution (you can also mix equal amounts of thromboplastin reagent and CaCl2 solution first, and add 0.2 mL). Mix immediately and start the stopwatch: the test tube is still immersed in the water bath. When it reaches about 10 seconds, take it out of the water bath. Quickly wipe off the water droplets outside the test tube on gauze. Keep tilting the test tube in a bright place and observe whether there are any leaks in the flowing state. Fibrin formation. As soon as fibrin is seen (and the fluid flow will slow down), stop the watch immediately and record the time. Two tubes are measured each time and reported as the average. The pH of the final mixture in this test should be 7.2 to 7.3. Most commercial thromboplastin reagents are prepared with buffer-containing solutions.
(4) Normal and abnormal controls should be done for each batch at the same time, and the method should be exactly the same as the test specimen.
[Reporting method]
1. Report in seconds (S) of PT (nearest 0.5 seconds)
2. Report in patient plasma (S) )/normal control PT(S) ratio (PRT) is reported.
3. When monitoring oral warfarin anticoagulant therapy, the international normalized ratio (INR) should be reported. Most automated instruments can automatically calculate the INR based on the measured PTR and ISI of the thromboplastin reagent. The manual method can be calculated directly with a calculator according to the following formula:
Poller designed a simple nomogram. After obtaining the PTR and ISI values, the INR value can be directly found from the graph. It is very convenient and has been introduced in China.
ICSH regulations no longer use dilution curves or percentage (activity) reporting.
[Reference value]
Due to different instruments/reagents, different results will be obtained, so it is difficult to uniformly specify the reference value. Each laboratory should independently measure a group of healthy people and establish reference values ??based on its own instruments, reagents and other conditions. Thereafter, it shall be re-established at least annually or when conditions change, based on new conditions. All conditions for the reference value PT measurement should be the same as those for the patient plasma PT measurement (including blood collection, containers, anticoagulants, etc.). Healthy blood donors should be selected from at least 20 men aged 18 to 55 and women who are not pregnant or menstruating. They should not take medicine and collect blood in a calm and resting state to reduce individual differences (if possible, a group of elderly people and children can be tested separately. statistics). At the same time, blood collection and measurement should be separated on several days to reduce day-to-day differences. The measurement results are statistically processed and the standard deviation is calculated: two standard deviations (2SD) or 95% confidence limit are used as the reference range. Whether three standard deviations are normal or abnormal needs to be evaluated based on the specific situation. Statistically speaking, some of these people are normal. Using the above criteria, patients (PT abnormalities) will rarely be missed.
3. Standardization of activated partial thromboplastin time (APTT)
(1) Standardization of activated partial thromboplastin time (APTT)
APTT is Screening tests for detecting coagulation factor defects and related inhibitors in the endogenous coagulation system are also currently the main means for detecting coagulation factors, heparin anticoagulant therapy, and lupus anticoagulant substances.
As with the PT assay, different partial thromboplastins, different activators, and different activation times have very different sensitivities to various coagulation factor deficiencies, to heparin, and to lupus anticoagulants. For example, different activators (kaolin, diatomaceous earth, ellagic acid) used in reagents for detecting APTT have different sensitivities for detecting heparin, lupus anticoagulant substances, and factors VIII and IX.
To date, ICSH and ICTH have not yet come up with a feasible plan for standardizing APTT detection methods or reagents. Only NCCLS proposed an interim plan numbered H29-T in 1992.
(2) Recommended method for activated partial thromboplastin time (APTT)
[Principle]
Add a phospholipid and activator to plasma After incubation, add appropriate concentration of calcium ions. The time (in seconds) for the fibrin clot to form is the APTT. This method is mainly used to screen and determine defects in endogenous pathway coagulation factors, such as factors VII, XI, VIII, IX, kallikrein (PK), high molecular weight kininogen (HMWK), and fibrinogen. It is also used for the determination of inhibitors of the above factors, monitoring of heparin treatment and examination of lupus anticoagulant factors.
[Instrument]
The instruments and equipment used in this experiment (including blood collection, blood storage containers, sampling devices and specimen processing, etc.) and their requirements are completely the same as PT. The automated instruments used in PT are also suitable for this experiment.
[Reagent]
1. Partial thromboplastin reagent: supplied by commercial products. Generally, the commonly used activators are prepared together with the activator in proportion (or separately). The commonly used activators are diatomaceous earth (trade name: Celite), white clay, silica particles, tannic acid (ellagic acid) or other available activators. Agent, supplied by the factory.
The APTT reagent/instrument combination should be able to produce abnormally prolonged results in plasma with factor VIII, IX or XI activity lower than 0.3μ/mL (or <30%).
2. Calcium chloride solution (25mmol/L) and other reagents are the same as those used in PT.
[Operation steps]
1. The collection, storage and transportation of samples are the same as those for PT measurement.
Note that clean plastic or siliconized glass blood collection devices should be used to collect and store blood.
2. The preparation of the specimen (platelet-depleted plasma) is the same as that of PT. Note that platelet-depleted plasma should be used for the assay.
3. The temperature of the water bath or electric hot plate is 37℃±1℃. Always check whether it is correct.
4. Contact activation time: The time for adding activator to activate factor XII should be consistent; the regulations of each instrument and reagent manufacturer may be different, so the instructions should be strictly followed. When operating manually, use a stopwatch or similar timing device.
5. Operation: Mix one part of pre-warmed (no more than 30 minutes) APTT reagent with one part of pre-warmed (no more than 10 minutes) test plasma, start the stopwatch immediately; until the specified contact activation At the end of the time, add a portion of CaCl2 solution pre-warmed at 37°C, mix well, and start the stopwatch at the same time. When plasma coagulation occurs, stop the watch and record the plasma coagulation time (in seconds). During manual measurement, two tubes should be measured at the same time and reported as the average. Some automatic or semi-automatic coagulometers whose precision has been greatly improved can only be measured once if appropriate quality control standards are available. Normal and abnormal control plasma should be measured simultaneously. Sorry, TT couldn’t find it, but if I contact the authoritative manufacturer, I think I won’t have to look for it myself~