Prothrombin is a protein synthesized by the liver. Prothrombin test is a test to understand blood coagulation, which can reflect the coagulation function of the liver. 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 1 1 ~ 15 seconds (kjeldahl method). When the liver is diseased, the liver function is poor, and the content of the above coagulation factors is reduced due to synthesis disorder, the prothrombin time can be prolonged, resulting in coagulation disorder. Such as severe hepatitis, prothrombin time is obviously prolonged, patients are prone to bleeding, and the prognosis is poor; Prothrombin time can be slightly prolonged in chronic active hepatitis and cirrhosis; Prothrombin time can be normal when there is extrahepatic obstruction without obvious liver cell damage. Long-term extrahepatic obstruction and cholestasis affect the absorption of vitamin K, and also lead to prolonged prothrombin time; If the patient is injected with vitamin K, the prothrombin time can return to normal.
In addition, due to the short half-life of prothrombin, the prothrombin time changes in a short time after the onset of acute severe hepatitis, so measuring prothrombin time has important clinical significance for the diagnosis, condition and prognosis of severe hepatitis.
Serum cholinesterase, also known as "pseudo" or "nonspecific" cholinesterase, is an enzyme synthesized by the liver with poor specificity and can act on cholinesterase such as acetylcholine. Its normal value is 0.80 ~ 1.00 or more than 40 units.
Because of the short half-life of serum cholinesterase, it is an extremely sensitive test to reflect the disorder of liver enzyme synthesis when liver is damaged. The decrease of its activity is often consistent with the severity of liver disease. For example, the cholinesterase activity of patients with severe hepatitis is generally lower than normal, and the degree of reduction is closely related to the severity of the disease; About 80% patients' serum cholinesterase activity can be reduced to 60% of the normal value, and even to below 10% in critically ill patients. Most of these patients die soon. Therefore, the determination of serum cholinesterase activity is helpful for the diagnosis of severe hepatitis and the judgment of its condition and prognosis. In addition, serum cholinesterase in patients with chronic severe hepatitis and advanced cirrhosis also decreased to varying degrees.
The clinical significance of laboratory examination of thrombosis and hemostasis is increasing day by day, the examination content is expanding and the workload is increasing day by day. The continuous updating of methods, commercialization of reagents and automation of operation have changed the previous situation of manual operation, self-preparation of reagents and low work efficiency. At the same time, method standardization and quality control are particularly important. However, due to the particularity of thrombus and hemostasis test. ......
It is related to (three experiments: determination of prothrombin time (PT), activated partial thromboplastin time (APTT) and fibrinogen (FIB)).
This paper briefly introduces the standardization and importance of three international experiments: ICSH, ICTH or the determination of prothrombin time (PT), activated partial thromboplastin time (APTT) and fibrinogen published by the National Committee of Clinical Biochemistry (NCCLS).
Key words: standardization of time activation of partial thromboplastin and fibrinogen prothrombin
Due to the particularity of thrombosis and hemostasis experiments, so far only three experiments, prothrombin time (PT), activated partial thromboplastin time (APTT) and fibrinogen (Fg), have standardized reagents (such as PT), standards (such as FG), quality control products and unified report forms. Others, such as platelet function, anticoagulant factors and fibrinolytic components, lack mature standardized schemes. This paper briefly introduces the standardization of prothrombin time, APTT and fibrinogen by international organizations such as International Committee for Standardization of Hematology (ICSH), International Committee for Thrombosis and Hemostasis (ICTH) or National Committee for Clinical Biochemical Standards (NCCLS).
I. Importance of standardization and quality control
The standardization and quality control of the detection methods of thrombus and hemostasis refers to the reasonable management, detection and evaluation of the quality level of the technology, operation, instruments, reagents and specimens of thrombus and hemostasis experiments by using statistical principles and standardized physical, chemical and biological methods, so as to plug the errors and improve the precision, accuracy and reliability of the experiments. The importance of standardization and quality control lies in:
1, which provides a reliable basis for clinical diagnosis and treatment: the reliability of experimental results is an important basis and condition for clinical diagnosis and treatment of diseases. If the experimental result is false positive, it will lead to misdiagnosis and mistreatment; If it is false negative, it will lead to missed diagnosis and treatment; The experimental results will affect the diagnosis and differential diagnosis of patients and the doctor's judgment on the condition and curative effect.
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 experiment is reliable, it can reveal the objective law of thrombosis and hemostasis, and even bring great theoretical breakthrough and (or) social and economic benefits; If the reliability of the experiment is poor or there are mistakes, it will cause illusions or theoretical errors.
3. It is helpful to people's health survey and establish the normal range of hematology parameters: In order to understand the health level of a certain population and establish a reference range of hematology with broad significance, a large-scale population health survey must be carried out, and the reliability of experimental results must be guaranteed in the health survey, otherwise the results will be inaccurate or wrong, so quality control is also of great significance to group doctors.
In a word, the quality control of thrombus and hemostasis laboratory reflects the medical level and research level of thrombus and hemostasis in a country, a region and a unit to a certain extent. Therefore, we should pay more attention to it.
Second, the standardization of prothrombin time (PT)
Since 1935 was established by Quick, the determination of prothrombin time (PT) is still an important screening test to check exogenous coagulation system factors and related inhibitors, and PT is also the main means of oral anticoagulant therapy at present. However, the determination of PT is influenced by many factors, so it must be standardized and quality controlled to improve the precision, accuracy and reliability of PT detection.
(A) the standardization of prothrombin time
1. Standardization of tissue thromboplastin: IRP has the following international reference preparations:
(1) international reference of single tissue thromboplastin: it is a tissue extract saline suspension preparation. Based on the unified standard "British comparative thromboplastin (BCT)" used by the British WHO, the WHO first-class reference preparation, such as human tissue thromboplastin, was prepared, with the number of 67/40. At the same time, the secondary reference preparation was standardized according to BCT67/40. Such as bovine brain tissue thromboplastin, the number is 68/434; The rabbit brain is 70/ 178. Later, WHO also published some reference materials of secondary tissue thromboplastin, such as human brain or placenta preparation, such as BCT/253；; ; Rabbit or rabbit monkey tissue mixed preparation, such as RBT/79.
(2) International reference of composite tissue thromboplastin: It is a preparation composed of tissue extract and appropriate amount of fibrinogen, factor V and calcium chloride in physiological saline suspension, such as bovine tissue thromboplastin OBT/79.
At present, these WHO reference materials are widely used in countries all over the world to calibrate the tissue thromboplastin reference materials prepared or produced in their own countries or regions. In this way, there are many kinds of tissue thromboplastin standard substances all over the world.
2. International sensitivity index (ISI) of tissue thromboplastin working preparation.
Because the sensitivity of thromboplastin to coagulation factors is different in different tissues. In order to make tissue thromboplastins with different sensitivities get the same results when detecting PT, it is necessary to develop a sensitivity index with the same * * * *. It is necessary to compare the self-made reagent with the international reference material (IRP), and then get a correction value. The specific method is as follows:
(1) Calibrate the reference preparation (RP) of the country, region or our laboratory with the international reference material (IRP).
(2) Calibrate working preparation (WP) with laboratory reference preparation (RP).
1) samples: 2 normal plasma samples, 6 plasma samples from patients who took anticoagulants orally for 6 weeks, and 60 samples were collected on 10 day.
2) Determination: Determine in a certain order, and repeat the determination twice for each specimen. Point the PT measurement results (seconds) on log-log coordinate paper, with the abscissa representing the PT measurement results of WP and the ordinate representing the PT measurement results of RP, draw the best fitting straight line of each point to get the calibration curve, and then calculate the slope b through WP/RP ratio or regression equation.
3) Calculate the ISI value of WP: The closer the ISI value is to 1.0, the more sensitive the tissue thromboplastin reagent is. Therefore, manufacturers who produce and sell tissue thromboplastin reagents must mark ISI on their products.
3. Reporting method of 3.PT determination results: Theoretically, any tissue thromboplastin can be compared and corrected with international reference materials as long as it is marked with ISI, and can be reported in the same measurement unit. 1985 ICSH and ICTH suggested that the international normalized ratio (INR) should be used as a reporting method for monitoring PT in oral anticoagulants.
The calculation formula of INR is as follows: After using INR, all kinds of tissue thromboplastins with different sensitivities can get the same INR value.
4. The instrument is characterized by ISI: the above PT is suitable for manual method (tube tilt method) according to the reporting method. However, there are still differences between manual method and instrumental method and between instrumental method and instrumental method in determining INR of PT. The research shows that the average values of PT on ACL, Cobas Fibro and Coaga-Pet are 10.7 sec, 12. 1 sec and 1 1 sec, respectively. However, if INR is calculated with the same ISI value, the values obtained are different to varying degrees. Therefore, experts suggest that when the same tissue thromboplastin reagent is used in different instruments, so-called ISI (instrument-specific ISI) or "local ISI" can be used to calculate INR. The thrombin reagent used in each instrument should have a specific ISI value. The method of recalibrating the ISI value is to buy freeze-dried plasma marked with INR, and then recalibrate the ISI value of thrombin reagent used in your own instrument to make the patient's INR comparable.
(2) The recommended method of prothrombin time (PT)
[principle]
Tissue thromboplastin (mainly containing tissue factor and lipid) and calcium ion were added to citric acid anticoagulant plasma, and the coagulation time of plasma was measured at 37℃, which was PT. PT is mainly used to screen and detect inhibitors of coagulation factors ⅶ, Ⅱ, ⅴ, ⅹ and exogenous coagulation system related factors.
[Specimen Collection and Processing]
1. Specimen collection: Take fasting venous blood with a silicified or plastic syringe, add it into a silicified or plastic test tube containing 0. 109mol/L sodium citrate anticoagulant according to the ratio of 9: 1, and mix it gently.
2. Specimen processing: centrifuge at 2000 ~ 2500 g 15 minutes, separate platelet-poor plasma, and complete the experiment within 24 hours.
3. Normal control plasma: Choose 10 or more normal healthy men and women, aged 18 ~ 45 years old. However, women who are not pregnant, menstruating, breastfeeding or taking oral contraceptives should freeze-dry their plasma or store it at -80℃.
[reagent]
1. Anticoagulant: 109mmol/L sodium citrate solution (equivalent to 32g/L sodium citrate containing bimolecular crystal water).
2. Thromboplastin reagents: commercially available products, which should be marked with ISI, batch number and expiration date. Freeze-dried people should add the specified buffer diluent according to the instructions for re-dissolution.
3. Calcium chloride (CaCl2) solution: 25 mmol/L (at present, some commercially available reagents have mixed thromboplastin solution with CaCl2 solution, so it is unnecessary to prepare CaCl2 solution separately).
4. Quality control substances: normal and abnormal control plasma.
5. The preparation of reagent water must meet the standard of 1 grade pure water.
[instrument]
1, manual method: stopwatch, 37℃ constant temperature water bath box or electric heating block 1℃. 10×mm swab, with water depth greater than 3cm and no scratches on the surface. Standard 0. 1 ml pipette. A calibrated stopwatch.
2. Instrument method: Operate all kinds of automatic or semi-automatic hemagglutination instruments in strict accordance with the instructions.
[Operation Steps]
1, manual mode
(1) The measuring temperature is 36.5 ~ 38.5℃. The above reagents and plasma to be tested should be preheated to this temperature, but the temperature of thromboplastin reagent should not exceed 30 minutes, and the temperature of plasma should generally not exceed 10 minutes.
(2) Instruments used for contacting plasma, such as test tubes and samplers, are all plastic or silicified glass tubes.
(3) Suck 0. 1mL citric acid anticoagulant plasma and put it in a small test tube: add 0. 1mL thromboplastin reagent, mix well and put it in a water bath at 37℃. Add 0. 1 ml of 25 mmol/l calcium chloride solution (or mix the thromboplastin reagent with the same amount of calcium chloride solution first, then add 0.2 ml). Immediately mix well and start the stopwatch: when the test tube is still immersed in the water bath for about 10 second, take it out of the water bath, quickly wipe off the water drops outside the test tube on the gauze, and continuously tilt the test tube in the bright place to observe whether cellulose forms in the flowing state. Once you see fibrin (at the same time, the liquid flow will slow down), stop the watch immediately and record the time. Measure two tubes at a time, and report the average value. The final pH value of the mixed solution in this experiment should be 7.2 ~ 7.3, and most of the commercially available thromboplastin reagents are prepared with solutions containing buffer.
(4) Each batch should be compared with normal and abnormal at the same time, and the method should be exactly the same as that of specimen determination.
[Reporting method]
1, reported in seconds of PT (nearest 0.5 seconds)
2. Report the ratio (PRT) of patient plasma to normal control PT.
3. When monitoring the treatment of oral warfarin anticoagulants, the international normalized ratio (INR) should be reported. Most automatic instruments can automatically calculate INR according to PTR and ISI measured by thromboplastin reagents. Manual method can be directly calculated by calculator according to the following formula:
Poller designed a simple nomogram. After getting PTR and ISI values, INR values can be found directly from the diagram, which is very convenient and has been introduced in China.
ICSH stipulates that the dilution curve or percentage (activity) will no longer be reported.
[Reference value]
Different instruments/reagents will give different results, and it is difficult to uniformly specify reference values. Each laboratory should determine a group of healthy people and establish reference values according to its own instruments, reagents and other conditions. After that, it will be re-established at least once a year according to new conditions or when conditions change. All conditions for determining the reference value PT should be the same as those for determining the patient's plasma PT (including blood collection, containers, anticoagulants, etc.). ). Healthy blood donors should choose at least 20 men and non-pregnant and menstruating women aged 18 ~ 55 to collect blood in a quiet rest state to reduce individual differences (when conditions permit, a group of old people and children can be measured and counted separately). At the same time, blood collection and determination should be separated for several days to reduce the day difference. Statistically process the measurement results and calculate the standard deviation: take two standard deviations (2SD) or 95% confidence limit as the reference range. Whether the three standard deviations are normal or abnormal needs to be evaluated in combination with specific circumstances. Statistically, some of them are normal. According to the above criteria, patients are rarely missed (abnormal PT).
Thirdly, standardization of activated partial thromboplastin time (APTT)
Standardization of activated partial thromboplastin time (APTT)
APTT is a screening test to detect various coagulation factor defects and related inhibitors in endogenous coagulation system, and it is also the main means of coagulation factor detection, heparin anticoagulant therapy and lupus anticoagulant therapy.
Like PT determination, different partial thromboplastins, different activators and different activation times have different sensitivities to various coagulation factor defects, heparin and lupus anticoagulants. For example, in the reagents for detecting APTT, the activators (clay, diatomite and ellagic acid) used are different, and the sensitivity for detecting heparin, lupus anticoagulant, factors VIII and VIII is also different.
So far, ICSH and ICTH have not come up with a feasible scheme to standardize the detection methods or reagents of APTT. Only NCCLS has put forward a temporary scheme numbered H29-T in 1992.
(2) Recommended method for activating partial thromboplastin time (APTT)
[principle]
Phospholipids and activators are added to the plasma, and after incubation, calcium ions with appropriate concentration are added. The time (in seconds) for fibrin clot formation is APTT. This method is mainly used to screen and determine the defects of endogenous coagulation factors, such as coagulation factors ⅶ, ⅷ, ⅸ, kallikrein (PK), high molecular weight kininogen (HMWK) and fibrinogen. At the same time, it is also used for the determination of inhibitors of the above factors, the monitoring of heparin therapy and the examination of lupus anticoagulant factors.
[instrument]
The instruments and equipment used in this experiment (including blood collection, blood storage container, sample adding device and sample processing, etc.). ) and their requirements are exactly the same as PT. The automatic instrument used in PT is also suitable for this experiment.
[reagent]
1, partial thromboplastin reagent: provided by commodities. Usually, the activator mixed with the activator in proportion (or prepared separately) is diatomite (trade name Celite), white clay, silica particles, ellagic acid or other available activators, which are provided by the manufacturer.
APTT reagent combined with instrument should prolong the plasma with coagulation factor Ⅷ, Ⅷ or Ⅷ activity less 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 of PT determination. Pay attention to the use of clean plastic or silicified glass blood collectors to collect and store blood.
2. The preparation of the specimen (platelet-removed plasma) is the same as that of PT. Attach importance to the application of platelet-removed plasma determination.
3. The temperature of the water bath box or electric heating plate is 37℃+ 1℃, so it should be checked frequently for correctness.
4. Contact activation time: the time of adding activator and activating factor should be the same; The regulations of each instrument and reagent manufacturer may be different, so it should be carried out in strict accordance with the requirements of the manual. For manual operation, a stopwatch or similar timing device shall be used for timing.
5. Operation: Mix a preheated APTT reagent (no more than 30 minutes) with a preheated plasma (no more than 10 minutes), and immediately start the stopwatch to time; At the end of the specified contact activation time, add CaCl2 solution preheated to 37℃, mix evenly, and start the stopwatch at the same time. When plasma coagulation occurs, stop the watch and record the plasma coagulation time (in seconds). When manually measuring, two pipes should be measured at the same time and the average value should be reported. Some automatic or semi-automatic coagulators whose accuracy has been greatly improved can only be measured once if there are appropriate quality control standards. Normal and abnormal control plasma should be determined simultaneously.
[reference value] refers to PT.
[Special instructions]
1. Sensitivity to heparin: APTT is often used to monitor the treatment of heparin. Usually, the ratio of patients' APTT to normal plasma APTT is 1.5 ~ 2.5 as the treatment control range. However, different reagent/instrument systems have different sensitivities to heparin. The sensitivity of its reagent/instrument system to heparin can be determined. In vitro sensitivity refers to the determination of APTT by adding clinical heparin to normal plasma according to its therapeutic concentration. In vitro sensitivity is different from in vivo sensitivity, but it can be used for reference.
2, wave sore anticoagulant factor: lupus anticoagulant factor is an anti-phospholipid autoantibody, because it is an important component of anticoagulant phospholipids, it can interfere with coagulation. If there is lupus anticoagulant in the blood, APTT will be prolonged. However, the sensitivity of APTT reagents to lupus anticoagulant factors is very different, and the reagent manufacturers should provide sufficient explanations, and the relevant national institutions can also provide information on lupus anticoagulant factors. But we should know that there are also great differences among patients, and no reagent can detect all lupus anticoagulants.
Fourthly, the standardization of fibrinogen determination (Fg)
(A) the standardization of fibrinogen determination
Fibrinogen (death) is synthesized by the liver and exists in plasma and body fluids. Its structure and function have been basically clarified, but there is still no ideal clinical detection method. There are many kinds of determination methods reported in the literature, some of which have good precision and accuracy, but they are too complicated. Some methods are simple and fast, but their precision and accuracy are poor.
1992, the British National Institute for Biological Standards and Control (NIBSC) completed a fibrinogen standard numbered 89/644, which was recommended to the WHO Expert Committee on Biological Standards (ECBS), [15] and approved by ECBC as an international reference (IRP). Since then, all countries have issued this standard to regulate the sub-polar standard produced by their own countries or manufacturers (the clinical laboratory center of the Ministry of Health of China has also issued it). The standardization of fibrinogen determination has taken a key step. Therefore, according to the survey, the fibrinogen content of each standard varies greatly.
(2) Recommended method for determination of fibrinogen (Fg)
When IRP is used to standardize its own standards, it is suggested to adopt Jacobson's improved method, which is introduced as follows.
[reagent]
1, buffer solution: Na2O·2H2O 0.882g；; ; KH2PO4 2.77gl liter, which is the storage buffer; Add 1 part storage buffer and 2 parts physiological saline to obtain application buffer with pH of 6.35.
2. Normal saline: 0. 1.5 mol/L.
3. human or bovine thrombin: 500IU/mL, physiological saline solution.
4. clot dissolving agent: dissolve 400g urea in a small amount of distilled water, 200ml of which is 200mL 1. 0.0 1 mole/liter of sodium hydroxide, and then add water to 1 liter.
[Operation Steps]
Freeze-dried fibrinogen (standard) product (from 89/644) is re-dissolved with 1 ml distilled water, added to plexiglass tray or other container containing 2 ml application buffer, then added with 50 microliters thrombin solution, quickly mixed evenly, and allowed to stand at room temperature for 2 hours. Invert the container on the absorbent cloth (absorbent paper can be placed under it), and then blot the clot with a suitable substance (filter paper). Take out the blood clot and wash it twice with 50 ml of normal saline. After each washing, the clot should be sucked dry (the clot can be squeezed by glass), and the liquid contained in the clot should be removed as much as possible to avoid remaining other plasma proteins in the liquid. If necessary, squeeze in clean cotton cloth.
Carefully add the clot into a container containing 7.5 ml of clot dissolving agent and shake thoroughly until the clot is completely dissolved. Pour it into a cuvette with an optical diameter of 65438±0cm, and read the absorbance (a) at the wavelengths of 280nm and 365438±0.5nm with the blood clot dissolving agent as blank.
(3) Suitable fibrinogen (Fg) determination (Clauss method)
[principle]
Thrombin is added to the plasma to change fibrinogen into fibrin, and the plasma will coagulate. When thrombin is sufficient, it reacts with different fibrinogen content, and the plasma coagulation time is negatively correlated with fibrinogen content.
[reagent]
1, bovine thrombin l00NIHU/mL.
2. Fibrinogen standard (IRP sub-standard)
3. Buffer (choose one of the following two items):
(1) barbital buffer (pH 7.5); Dissolve 2.5g barbital, 2.75g barbital sodium and 7.3g sodium chloride in 750mL deionized water, adjust the pH to PH7.5, and add water to1L.
(2) Imidazole (or glyoxal) buffer solution: 3.4g(0.05mol/L) of imidazole and 5.85g of sodium chloride, added into about 500mL of water; Add O. 1mol/L hydrochloric acid 186mL, adjust the PH to 7.3-7.4, and finally add distilled water to1l.
[Operation Steps]
1, manual mode
(1) Dilute the standard with the above buffer until the fibrinogen concentration is 0.8, 1.6, 2.4 and 4.0g/L, and then dilute each concentration with the buffer of 1: 10.
(2) Dilute the patient's plasma and quality control with a buffer of 1: 10.
(3) Mix 100NIHU/mL with bovine thrombin and store at room temperature. If measured by instrumental method, 100NIHU/mL, bovine thrombin, does not need to be diluted.
(4) In the test tube, add 0.2mL of diluted plasma and put it in a water bath at 37℃ for 4 minutes.
(5) Add 0. 2ml lOONIHU/mL thrombin solution with a pre-temperature of 37℃, start the stopwatch immediately after shaking, and continuously observe the coagulation time. Stop the watch when solidification occurs.
(6) Each sample is measured twice and averaged. At the same time, the standard tube and the control tube were determined by the same method, and the time was accurately recorded.
(7) Calculation: Drawing with double logarithmic paper, taking setting time as the ordinate and fibrinogen concentration as the abscissa, marking the corresponding points of standard tube pairs with corresponding concentrations on the drawing and connecting them into a straight line to make a standard curve. Then according to the coagulation time measured by the patient and the quality control plasma. Find the content of fibrinogen on the graph.
If the plasma fibrinogen content is greater than 4g/L, the plasma should be diluted and re-tested, and the result should be multiplied by the dilution multiple. If the fibrinogen content in plasma is lower than 0.8g/L, it is necessary to dilute the original plasma with 1:2 or 1:5, and divide it by 5 or 2 after finding the result on the standard curve.
2. Instrumental method: This method can measure thrombin time (TT) by automatic or semi-automatic hemagglutination instrument. The results can be printed automatically. The general instrument method is more accurate than the manual method.