What is the difference between prothrombin time measurement and prothrombin time measurement?

What is the clinical significance of the prothrombin time (PT) assay and the cholinesterase (ChE) viability test?

Prothrombin is a protein synthesized by the liver. The prothrombin test is a test to find out how the blood coagulates and it reflects the liver's clotting function. The prothrombin time (PT) is related to coagulation factors I, V, VII and X, which are also synthesized in the liver. The normal value of prothrombin time is 11-15 seconds (Quiessence method). When the liver is diseased, liver function is poor, and the content of the above coagulation factors decreases due to the synthesis disorder, it can cause the prolongation of the prothrombin time and coagulation disorder occurs. For example, in severe hepatitis, the prolongation of prothrombin time is obvious, and the patient is easy to bleed and has a poor prognosis; in chronic active hepatitis and hepatic cirrhosis, the prolongation of prothrombin time can be mild; in extrahepatic obstruction without obvious hepatocellular damage, the prolongation of prothrombin time can be normal. Long-term extrahepatic obstruction, cholestasis, affecting the absorption of vitamin K, can also lead to prolongation of the prothrombin time; if the patient is injected with vitamin K, the prothrombin time can be normalized.

In addition, due to the short half-life of plasminogen, there is a change of plasminogen time in a short period of time after the onset of acute severe hepatitis, so the determination of plasminogen time has important clinical significance for the diagnosis of severe hepatitis, and the determination of the condition and prognosis.

Serum cholinesterase, also known as "pseudo" or "non-specific" cholinesterase, is a kind of poor specificity synthesized by the liver, which can act on both acetylcholine and other cholinesterase. Its normal value is 0.80 to 1.00 or 40 units or more.

Because of its short half-life, serum cholinesterase is an extremely sensitive test for intrahepatic damage that reflects impaired hepatic enzyme synthesis. The degree of reduction in its activity is often consistent with the severity of liver disease. Such as severe hepatitis patients cholinesterase activity value is generally lower than normal, and the degree of reduction is closely related to the severity of the disease; about 80% of patients, the serum cholinesterase activity can be reduced to 60% of the normal value of the severe risk patients and even down to less than 10%, such patients more quickly die. Therefore, the measurement of serum cholinesterase activity is helpful for the diagnosis of severe hepatitis and the determination of the condition and prognosis. In addition, in patients with severe chronic hepatitis and advanced liver cirrhosis, serum cholinesterase is also reduced to varying degrees.

The clinical importance of thrombosis and hemostasis laboratory tests is increasing day by day, and the content of the tests is expanding, and the workload is increasing; the continuous emergence of method updates and reagents commercialization, operation automation, changing the previous manual operation, self-prepared reagents, and the low efficiency of the work situation. At the same time, method standardization and quality control have become particularly important. However, due to the specificity of thrombosis and hemostasis test ......

Related to (three tests such as prothrombin time (PT), activated partial thromboplastin time (APTT), and fibrinogen (FIB) assay)

Abstract: A review of the international documented tests published by the ICSH, ICTH, or the U.S. National Committee on Clinical Biochemistry (NCCLS) related to the thrombin time (PT), activated partial thromboplastin time (APTT), and fibrinogen (FIB) assay. Activation Time (APTT) and Fibrinogen assay are standardized and their importance is briefly described.

Keywords: prothrombin time, activated partial thromboplastin time, fibrinogen, standardization

Because of the specificity of thrombosis and hemostasis experiments, only prothrombin time (PT), activated partial thromboplastin time (APTT), and fibrinogen assay have been standardized so far. Therefore, so far, only three experiments, such as prothrombin time (PT), activated partial thromboplastin time (APTT) and fibrinogen (Fg), have standardized reagents (e.g., PT), standards (e.g., Fg), quality control products and a unified reporting form, etc.; others, such as platelet function, anticoagulant factors, and fibrinolytic components and other tests, are still lacking in mature standardized protocols. This article only provides a brief introduction to the standardization of the three tests of PT, APTT and Fg, which have been published in the form of documents by international organizations such as the International Committee for Standardization in Hematology (ICSH), the International Committee on Thrombosis and Haemostasis (ICTH), or the U.S. National Committee for Clinical Biochemistry Standards (NCCLS).

I. Importance of standardization and quality control

The so-called standardization and quality control of thrombosis and hemostasis testing methods refers to the adoption of statistical principles, the use of standardized physical, chemical, and biological methods, and the quality level of thrombosis and hemostasis experiments in terms of technology, operation, instrumentation, reagents, and specimens to carry out reasonable management, testing, and evaluation, and to standardize and quality control to plug errors and improve the quality of the experiments. Standardization and quality control are used to prevent errors and improve the precision, accuracy and reliability of the experiments. The importance of standardization and quality control is:

1, for clinical diagnosis and treatment to provide a reliable basis: the reliability of the experimental results is an important basis for clinical diagnosis and treatment of diseases and conditions. If the experimental results are false-positive, it will cause misdiagnosis and mistreatment; if it is false-negative, it will cause omission of diagnosis and treatment; the experimental results of high or low will affect the diagnosis of the patient, differential diagnosis, and affect the physician's judgment on the condition and efficacy of the treatment.

2, improve the efficiency and value of basic research on thrombosis and hemostasis: the form of basic research on thrombosis and hemostasis is to conduct various experiments. If the reliability of experiment is good, it can reveal the objective law of thrombosis and hemostasis, and even bring about major theoretical breakthroughs and/or social and economic benefits; if the reliability of experiment is poor or there are mistakes, it will cause false or wrong theories.

3. It helps to investigate the health of the population and establish the normal range of hematological parameters: in order to understand the health level of a certain group of people and to establish the range of hematological reference values of wide significance, it is necessary to carry out a large-scale health investigation of the population, and the health investigation has to be guaranteed by the reliability of the experimental results; otherwise, it will lead to the inaccurate or incorrect results, so the quality control is also of great significance to group medicine.

In short, the quality control of thrombosis and hemostasis laboratory, to a certain extent, reflects a country, a region, a unit of thrombosis and hemostasis medical level and research level. Therefore, it should be emphasized.

The standardization of prothrombin time (PT)

Since Quick created the prothrombin time (PT) measurement method in 1935, it is still an important screening test to check the factors of exogenous coagulation system and the related inhibitors, and PT is also the main means of oral anticoagulant treatment at present. However, PT assay is affected by many factors, and it must be standardized and quality controlled to improve the precision, accuracy and reliability of PT assay.

(I) Standardization of prothrombin time (PT)

1, standardization of tissue thromboplastin: the application of the international reference preparation IRP, there are the following kinds:

(1) a single tissue thromboplastin international reference preparation: it is a kind of tissue extracts Based on the unified standard "British comparative thromboplastin" (BCT) used by WHO in the United Kingdom, the primary reference preparation of WHO is prepared, such as human brain tissue thromboplastin, No. 67/40, and at the same time, it is also used for the preparation of the international reference preparation IRP. At the same time, BCT67/40 was used as the basis for the preparation of secondary reference preparation, such as bovine brain tissue thromboplastin (BCT). For example, bovine brain tissue thromboplastin, No. 68/434, and rabbit brain, No. 70/178. Later, WHO also published some secondary reference preparations for thromboplastin, such as human brain or placenta preparations, e.g., BCT/253, and rabbit or rabbit-monkey tissue mixed preparations, e.g., RBT/79.

(2) Compound tissue thromboplastin international reference products: it is the saline suspension of tissue extracts added to the appropriate amount of fibrinogen, factor V and calcium chloride in the composition of the preparation, such as bovine tissue thromboplastin OBT/79 and so on.

These WHO references are now widely used around the world to calibrate tissue thromboplastin references prepared or produced in their own countries or regions. In this way, there are a variety of tissue thromboplastin reference products in the world.

2, tissue thromboplastin working preparation (working preparation, WP) of the international sensitivity index (international sensitivity index, ISI)

Because of the different tissue thromboplastin sensitivity to coagulation factors. In order for tissue thromboplastin with different sensitivities to give the same results in the assay PT, it is necessary to develop a ****similar sensitivity index. This involves comparing a homemade reagent with an international reference product (IRP) and then deriving a calibration value. The specific methods are as follows:

(1) Calibrate the reference preparation (RP) of the country, region or our laboratory with the international reference product (IRP).

(2) Calibration of the working preparation (WP) with the laboratory reference preparation (RP).

1) Specimens: 2 normal human plasma and 6 plasma from patients who have been on oral anticoagulants for up to 6 weeks, collected for 10 days, ***60 specimens.

2) Measurement: the measurement was performed in a certain order and repeated 2 times for each specimen. The results of PT measurement (seconds) point in double logarithmic coordinate paper, the horizontal coordinate represents the PT measurement results of WP, the vertical coordinate represents the PT measurement results of RP, the best point fitting straight line is drawn, and the calibration curve is obtained, and the slope b is found through the ratio of WP/RP, or the regression equation.

3) Calculation of ISI value of WP: The closer ISI value is 1.0, the more sensitive the reagent is, therefore, the more sensitive the production of WP is, the more sensitive the reagent is. Monitoring of oral anticoagulants is reported as the international normalizde ratio (INR).

The formula for INR is as follows: The same INR value is obtained for all tissue thromboplastin with different sensitivities after the use of INR.

4, instrument-specific has ISI: the above PT according to the reporting method applies to the manual method (test-tube tilt method) to determine the PT. but between the manual method and the instrumental method and the instrumental method and the instrumental method to determine the PT of the INR, there is still a difference. It was shown that the mean values for the determination of PT on three automated instruments, ACL, Cobas Fibro and Coaga-Pet, using the same Thromborel S reagent with an ISI of 1.12, were 10.7, 12.1 and 11.1 seconds, respectively. However, if the same ISI value was used to calculate the INR, the resulting values varied to varying degrees. For this reason, experts have suggested that when the same tissue thromboplastin reagent is used in different instruments, the INR can be calculated using so-called "instrument specific ISIs" (instrument specific ISIs) or so-called local ISIs, and that each thromboplastin reagent used in each instrument should have a specific ISI, and that recalibration of ISIs should be done by purchasing frozen reagents labeled with the INR. To recalibrate the ISI, one should purchase lyophilized plasma labeled with the INR and then recalibrate the ISI of the prothrombin reagent used on one's own instrument so that the patient's INR is comparable.

(II) Recommended method of prothrombin time (PT)

[Principle]

Tissue thromboplastin (mainly containing tissue factor and lipid) and calcium ions are added to citrate anticoagulated plasma, which is kept warm at 37°C, and the plasma clotting time, which is PT, is determined.PT is mainly used for screening detection of factor VII, II, V, X and related factors of exogenous coagulation system of the inhibitors of the test.

[Specimen collection and processing]

1. Specimen collection: Fasting venous blood was drawn with a silicone or plastic syringe, and added to a silicone or plastic test tube containing 0.109 mol/L sodium citrate anticoagulant at a ratio of 9:1, and mixed gently.

2. Specimen processing: centrifuge the blood at 2000-2500g for 15 minutes to separate the spent platelet plasma and complete the test within 24 hours.

3, normal control plasma: choose normal healthy men and women each lO or more, age 18 ~ 45 years old. But can not be pregnancy, menstruation, breastfeeding and oral contraceptives of women, taken plasma freeze-dried preservation or -80 ℃ preservation.

[Reagents]

1, anticoagulant: 109 mmol/L sodium citrate solution (equivalent to 32 g/L of sodium citrate containing two molecules of crystalline water).

2, thrombin reagent: commercially available goods, should be labeled with ISI, batch number and expiration date. The lyophilized ones should be reconstituted with specified buffer diluent according to the instructions.

3, calcium chloride (CaCl2) solution: 25mmol / L. (Some commercial reagents have been mixed with calcium chloride thromboplastin solution, CaCl2 solution can be dispensed with).

4, quality control substances: normal and abnormal control plasma

5, the preparation of reagents with water must meet the standards of class 1 pure water.

[Apparatus]

1, manual method: stopwatch, maintain 37 ℃ ± 1 ℃ constant temperature water bath or electric heating block. Water depth can be immersed test tube more than 3cm, the surface without scratches 10 × mm swab tube. Standardized 0.1mL pipette. Calibrated stopwatch.

2, instrumental method: a variety of automatic or semi-automatic hemagglutination instrument, strictly in accordance with the instructions.

[Steps]

1, manual method

(1) Measurement temperature 36.5 ~ 38.5 ℃, the above reagents and the plasma to be measured, should be pre-warmed to this temperature, but the pre-warming of thrombin reagents should not exceed 30 minutes, plasma pre-warming should not exceed 10 minutes.

(2) The test tubes and sampling apparatus used to contact the plasma are plastic or siliconized glass tubes.

(3) Aspirate 0.1mL of citrate anticoagulated plasma and add to a small test tube: add 0.1mL of thrombin reagent and mix well, then place in a 37℃ water bath. Then add 0.1mL of 25mmol/L CaCl2 solution, (you can also mix equal amounts of thrombin reagent and CaCl2 solution first and add 0.2mL). Immediately mix and turn on the stopwatch: the test tube remains immersed in the water bath until about 10 seconds, remove it from the water bath, quickly wipe off the droplets of water outside the test tube on gauze, tilt the test tube continuously in a bright place, and observe the formation of fibrin in the flowing state. Once fibrin is seen (at the same time there will be a slowing down of the liquid flow), stop the meter immediately and record the time. Two tubes were measured at a time and reported as an average. The final mixture for this test should have a pH of 7.2 to 7.3, and most commercial thromboplastin reagents are prepared in buffer-containing solutions.

(4) Normal and abnormal controls are made at the same time for each batch, and the method should be identical to that of the assay specimen.

[Reporting method]

1. Reported as seconds (S) of PT (to the nearest 0.5 sec)

2. Reported as the ratio of patient plasma (S)/normal control PT (S) (PRT).

3. The international normalized ratio (INR) should be reported when monitoring treatment with oral warfarin-based anticoagulants. Most of the automated instruments can be based on the measured PTR and ISI of thrombin reagents, automatically calculate the INR. manual method can be based on the following formula, using a calculator to calculate directly:

Poller designed a simple column line graph, in the acquisition of the PTR and ISI values, you can find out from the graph of the value of the INR, it is very convenient, the domestic has been introduced.

ICSH stipulates that dilution curves or percentage (activity) reports are no longer used.

[Reference values]

It is difficult to standardize the reference values because different instruments/reagents will produce different results. Each laboratory should determine a batch of healthy individuals on its own and establish reference values according to its own conditions of instruments and reagents. Thereafter, at least annually or when conditions change, re-establish them according to the new conditions. All conditions for reference value PT determination should be the same as for patient plasma PT determination (including blood collection, containers, anticoagulants, etc.). Healthy blood donors should be selected from at least 20 men and non-pregnant, non-menstruating women aged 18-55 years, not taking medication, and blood should be collected in a calm, resting state in order to minimize individual differences (when possible, a batch of elderly people and pediatrics can be measured and counted separately). Blood should be collected and measured on several separate days to minimize differences between days. The results were statistically processed and standard deviations were calculated: two standard deviations (2SD) or 95% confidence limits were used as the reference range. Whether three standard deviations is normal or abnormal needs to be assessed on a case-by-case basis. Statistically, some of them are normal. Using the above criteria, patients (with abnormal PT) are rarely missed.

III. Standardization of activated partial thromboplastin time (APTT)

(I) Standardization of activated partial thromboplastin time (APTT)

APTT is a screening test for detecting defects in endogenous coagulation systems of coagulation factors and related inhibitors, and is the main tool currently used for the detection of coagulation factors, heparin anticoagulation therapy, and anticoagulant substances in lupus.

As with the PT assay, the sensitivity of different partial thromboplastin enzymes, different activators, and different activation times to various coagulation factor defects, to heparin, and to lupus anticoagulant substances varies widely. For example, different activators (white clay, diatomaceous earth, ellagic acid) are used in the reagents used to detect APTT, and they have different sensitivities to detecting heparin, lupus anticoagulant substances, and factors VIII and IX.

To date, ICSH and ICTH have not standardized the APTT method or reagents standardization of the feasible program has been published, only the NCCLS in 1992, proposed a number of H29-T tentative program.

(ii) Recommended method for activated partial thromboplastin time (APTT)

[Principle]

A phospholipid and activator are added to plasma, and after incubation, calcium ions are added at appropriate concentrations. The time (in seconds) for the formation of its fibrin clot is known as APTT.This method is mainly used for the screening determination of defects in endogenous pathway coagulation factors, such as factors VII, Ⅺ, VIII, IX, kinin-releasing enzyme proteins (PK), high-molecular-weight kininogen (HMWK), and fibrinogen, etc. It is also used for the determination of inhibitors of these factors. It is also used for the determination of inhibitors of the above factors, monitoring of heparin therapy, and screening for lupus anticoagulant factors.

[Instrumentation]

The instruments and equipment used in this study (including blood collection, blood storage containers, spiking devices, and specimen handling) and their requirements are identical to those of the PT, and the automated instrumentation used in the PT is also suitable for this study.

[Reagents]

1. Partial thromboplastin reagent: supplied by commercial products. Generally has been with diabolic activator proportionally together (or separate preparation) commonly used activator by diatomaceous earth (trade name Celite), white clay, silica particles, ellagic acid (ellagic acid) or other available activator, from the factory supporting the supply.

The APTT reagent/instrument combination should result in an abnormally prolonged result for plasma with factor VIII, IX, or Ⅺ activity less than 0.3 μ/mL (or <30%).

2. Calcium chloride solution (25 mmol/L) and other reagents are the same as those used for PT.

[Procedure]

1. Sample taking, storage, and delivery are the same as for PT assay. Note that a clean plastic or siliconized glass blood collector should be used for blood collection and storage.

2. Preparation of specimens (platelet-removed plasma) was the same as for PT. Attention, application of platelet-removed plasma assay.

3, the temperature of the water bath or hot plate is 37 ℃ ± 1 ℃, and should be frequently checked for correctness.

4, contact activation time: add activator activation factor Ⅻ time should be consistent; each instrument and reagent manufacturers may not be the same provisions, should be strictly in accordance with the requirements of the instructions. For manual operation, a stopwatch or similar timing device should be used.

5, operation: pre-warm (not more than 30 minutes) APTT reagent and pre-warm (not more than 10 minutes) of the service test plasma a mixture of a stopwatch, immediately start the stopwatch timing; to the end of the specified contact activation time, add a pre-warmed 37 ℃ CaCl2 liquid a copy of the mixing, and at the same time, start the stopwatch. When plasma coagulation occurs, stop the watch and record the plasma coagulation time (in seconds). Two tubes should be measured at the same time for manual determination and reported as an average. Some automated or semiautomated coagulometers with greatly improved precision may be used for only one measurement if appropriate quality control standards are in place. Normal and abnormal control plasma should be measured simultaneously.

[Reference value] Refer to PT

[Special interpretation]

1. Sensitivity to heparin: APTT is commonly used in the monitoring of heparin therapy, and the ratio of the patient's APTT to the normal plasma APTT in the range of 1.5-2.5 is usually used as the therapeutic control range: however, different reagents/instrumentation systems have different sensitivities to heparin. The sensitivity of their reagents/instrumentation systems to heparin can be determined. In vitro sensitlvity refers to the clinical use of the same type of fishing heparin according to its therapeutic concentration, added to the normal plasma, the determination of APTT. in vitro sensitivity and in vivo sensitivity (in vivo sensitivity) is not equivalent, but can be used as a reference.

2, lupus anticoagulant factor: lupus anticoagulant factor is a kind of anti-phospholipid autoantibody, because it is anti-clotting important component of phospholipids, so it can interfere with blood clotting. APTT will be prolonged if lupus anticoagulant factor exists in the blood. However, the sensitivity of APTT reagents to lupus anticoagulant factor varies considerably, and adequate instructions should be provided by the reagent manufacturer, and information on lupus anticoagulant factor is available from the appropriate national agencies. However, it is important to realize that there is also a great deal of variation among individual patients and that no single reagent can measure all lupus anticoagulant factors.

Four, fibrinogen assay (Fg) standardization

(A) fibrinogen assay (Fg) standardization

fibrinogen (dead) synthesized by the liver, exists in the plasma and body fluids, the structure and function of the basically clarified, but so far there is still no ideal clinical testing methods; the literature has reported that the assay more than 10 kinds of methods, some of the precision, There are more than 10 methods reported in the literature, some of which have better precision and accuracy, but are too complicated and cumbersome; some of which are simple and rapid, but have poorer precision and accuracy.

In 1992, the British National Institute for Biological Standards and Control (NIBSC), research completed a fibrinogen standard, No. 89/644, recommended to the WHO Expert Committee on Biological Standards (ECBS), [15] and was approved by the ECBC as an international reference product (IRP). Since then, all countries have introduced this standard to standardize their own or manufacturers' subpolar standards (the Clinical Laboratory Center of the Ministry of Health of China has also been introduced). The standardization of fibrinogen determination has taken a key step. Therefore, according to the survey, each standard product fibrinogen content varies greatly.

(B) Recommended methods for the determination of fibrinogen (Fg)

Recommended method of Jacobsson's improved method when each family uses IRP to standardize their own standards, which is described as follows.

[Reagents]

1, buffer: Na2O-2H2O 0.882g; KH2PO4 2.77gl liters, this is the storage buffer; l part of the storage buffer, add 2 parts of saline that is, the application buffer, pH 6.35.

2, saline: 0.15mol/L

3, human or bovine Thrombin: 500IU/mL, saline solution.

4, clot dissolver: 400g of urea dissolved in a small amount of evaporated water, 200mL 1.Omol/L NaOH, and then add water to l liter.

[Procedure]

Fibrinogen lyophilized (standard) product (derived from 89/644) was redissolved in lmL of distilled water, added to a plexiglass shallow dish or other container containing 2mL of application buffer, and then added to the thrombin solution of 50 μL, quickly mixed, and left to stand for 2 hours at room temperature. Place the container upside down on an absorbent cloth (absorbent paper may be placed underneath), and then absorb the clot with an appropriate substance (plus filter paper). Remove the clot and place it in 5OmL of saline for 2 washes. After each wash, the clot should be blotted dry (a glass can be used to squeeze the clot), and the liquid contained in the clot should be removed as much as possible to avoid other plasma proteins remaining in the liquid. If necessary, it can be squeezed on a clean cotton cloth.

Carefully add the clot to a 7.5-mL container of clot dissolver and shake well until the clot is completely dissolved. Pour into a colorimetric cup with an optical diameter of 1cm, and read the absorbance (A) at 280nm and 315nm using the clot dissolver as a blank.

(C) Applicable fibrinogen (Fg) assay (Clauss method)

[Principle]

Plasma coagulation occurs when thrombin is added to the plasma to turn fibrinogen into fibrin. In the presence of sufficient amounts of thrombin to interact with varying levels of fibrinogen, the time to develop plasma coagulation is negatively correlated with the fibrinogen content.

[Reagents]

1, bovine thrombin l00NIHU/mL

2, fibrinogen standard (IRP substandard)

3, buffer (either one of the following two):

(1) barbiturate buffer (PH7.5); barbiturates 2.5g, barbiturates sodium 2.75g, sodium lu chloride 7.3g dissolved in 750mL deionized water, corrected to PH7.5, add water to 1L.

(2) Imidazole (Imidazole, or Glyoxaline) buffer: Imidazole 3.4g (0.05mol/L), sodium chloride 5.85g, added to about 500mL of water; add O.1mol/L hydrochloric acid 186mL, adjust PH to 7.3-7.4, and finally add distilled water to 1 liter.

[Procedure]

1. Manual method

(1) Dilute the standard into 0.8, 1.6, 2.4, and 4.0g/L fibrinogen concentration with the above buffer, and then dilute the concentration with buffer for 1:10.

(2) Patient plasma and quality control material were diluted 1:10 with buffer.

(3) Plasma containing 100 NIHU/mL, bovine thrombin, was mixed and stored at room temperature. If the instrumental method is used, 100 NIHU/mL, bovine thrombin, need not be diluted.

(4) In a test tube, add 0.2 mL of diluted plasma and place in a 37°C water bath for 4 minutes.

(5) Add O.2 mL of lOONIHU/mL thrombin solution prewarmed at 37°C, shake well and immediately turn on the stopwatch, constantly observing the coagulation time. Stop the stopwatch until coagulation occurs.

(6) Each specimen was measured twice and averaged. Simultaneous determination of the standard tube and the control (quality control) tube, the same method, and accurately record the time.

(7) Calculation: Use double logarithmic paper to make a graph, with the coagulation time as the longitudinal coordinate, the fibrinogen concentration as the horizontal coordinate, the standard tube of each corresponding concentration of the coagulation time, the corresponding points on the graph and connected to a straight line, to make a standard curve. Then according to the patient and quality control plasma measured coagulation time. The fibrinogen content was checked on the graph.

If the plasma fibrinogen content is greater than 4 g/L, the plasma should be diluted and retested, and the result multiplied by the dilution factor. If the plasma fibrinogen content is lower than O.8g/L, the original plasma should be diluted 1:2 or 1:5, and the result should be found on the standard curve and divided by 5 or 2.

2. Instrumental method: This method can be measured by automatic or semi-automatic coagulometer according to the prothrombin time (TT) assay. The results can be printed out automatically. The instrumental method is generally more precise than the manual method.