What are the clinical significances of prothrombin time (PT) measurement and cholinesterase (ChE) activity detection?
Prothrombin is a protein synthesized by the liver. The prothrombin test is a test to understand blood coagulation, which reflects the coagulation function of the liver. Prothrombin time (PT) is related to coagulation factors I, V, VII and X, and these factors are also synthesized in the liver. The normal value of prothrombin time is 11 to 15 seconds (Quiet's method). When liver disease occurs, liver function is poor, and the content of the above-mentioned coagulation factors is reduced due to synthesis disorders, it can cause prolongation of the prothrombin time and cause coagulation disorders. For example, in severe hepatitis, the prothrombin time is significantly prolonged, and the patient is prone to bleeding and has a poor prognosis; in chronic active hepatitis and cirrhosis, the prothrombin time can be slightly prolonged; in extrahepatic obstruction without obvious liver cell damage, Prothrombin time may be normal. Long-term extrahepatic obstruction, cholestasis, and affecting the absorption of vitamin K can 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 within a short period of time after the onset of acute severe hepatitis. Therefore, measuring the prothrombin time is important for the diagnosis, condition and diagnosis of severe hepatitis. The determination of prognosis has important clinical significance.
Serum cholinesterase, also known as "pseudo" or "non-specific" cholinesterase, is a less specific enzyme synthesized by the liver. It can act on acetylcholine and Enzymes acting on other choline esters. Its normal value is 0.80~1.00 or more than 40 units.
Due to the short half-life of serum cholinesterase, it is an extremely sensitive test for intrahepatic damage and reflects disorders of liver enzyme synthesis. The degree of reduced activity is often consistent with the severity of liver disease. For example, the cholinesterase activity value of patients with severe hepatitis is generally lower than normal, and the degree of decrease is closely related to the severity of the disease; in about 80% of patients, their serum cholinesterase activity can drop to 60% of the normal value, and in critically ill patients Even if it drops below 10%, most such patients will die soon. 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, serum cholinesterase is also reduced to varying degrees in patients with severe chronic hepatitis and advanced cirrhosis.
The clinical importance of thrombosis and hemostasis laboratory examinations is increasing day by day, the content of examinations is constantly expanding, and the workload is also increasing; the continuous emergence of method updates, commercialization of reagents, and automation of operations have changed the past manual operations and Self-prepared reagents and low work efficiency. At the same time, method standardization and quality control are also particularly important. However, due to the specificity of thrombosis and hemostasis tests...
(prothrombin time (PT), activated partial thromboplastin time (APTT) and fibrinogen (FIB) Three experiments including measurement)
Abstract: The prothrombin time (PT), activated partial coagulation activity and other internationally published documents in the form of documents such as ICSH, ICTH or the US National Committee of Clinical Biochemistry (NCCLS) The standardization and importance of three experiments including enzyme time (APTT) and fibrinogen determination are briefly described.
Keywords: prothrombin time, activated partial thromboplastin time, fibrinogen standardization
Due to the particularity of thrombosis and hemostasis experiments, so far only prothrombin time ( Three experiments, including 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 reporting forms, etc.; other tests, such as platelet function, anticoagulant factors and fibrinolytic components, still lack mature standardized protocols. This article only focuses on the standardization of the three tests of PT, APTT and Fg published in the form of documents by international organizations such as the International Committee for Standardization of Hematology (ICSH), the International Committee on Thrombosis and Hemostasis (ICTH) or the National Committee for Clinical Biochemistry Standards (NCCLS). An introduction to the problem.
1. The importance of standardization and quality control
The so-called standardization and quality control of thrombosis and hemostasis detection methods refer to the use of statistical principles and the use of standardized physical, chemical, and biological methods. Scientific methods are used to conduct reasonable management, testing and evaluation of the quality level of the technology, operations, instruments, reagents and specimens of thrombosis and hemostasis experiments, and use standardization and quality control to eliminate errors and improve the precision and accuracy of experiments. Accuracy and reliability. The importance of standardization and quality control is:
1. Provide 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 cause misdiagnosis and mistreatment; if it is false negative, it will cause missed diagnosis and missed treatment; if the experimental result is too high or too low, it will affect the patient's diagnosis, differential diagnosis, and the doctor's judgment of the condition and therapeutic 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 has good reliability, it can reveal the objective laws of thrombosis and hemostasis, and can even bring about major theoretical breakthroughs and/or social and economic benefits; if the experiment has poor reliability or errors, it will lead to illusions or wrong theories.
3. It is helpful for population health surveys and the establishment of normal ranges of hematological parameters: In order to understand the health level of a certain group of people and establish a range of hematological reference values ??with broad significance, a large-scale population survey is required. Health surveys must be guaranteed by the reliability of experimental results, otherwise they will lead to inaccurate or wrong results, so quality control is also of great significance to group medicine.
In short, the quality control of thrombosis and hemostasis laboratories reflects to a certain extent the level of thrombosis and hemostasis medical care and research in a country, a region, and a unit. Therefore, it should be paid more attention to.
2. Standardization of prothrombin time (PT)
Since Quick created the prothrombin time (PT) measurement method in 1935, it is still the method for testing external sources. An important screening test for various factors and related inhibitors of the coagulation system, PT is also the main method of oral anticoagulant treatment at present. However, PT measurement is affected by many factors, and it must be standardized and quality controlled to improve the precision, accuracy, and reliability of PT testing.
(1) Standardization issues of prothrombin time (PT)
1. Standardization of tissue thromboplastin: the international reference preparation IRP used includes the following Species:
(1) Single tissue thromboplastin international reference product: It is a physiological saline suspension preparation of tissue extract. Based on the unified standard "British comparative thromboplastin (BCT)" used by WHO in the UK, WHO's primary thromboplastin reference preparation (primary reference preparation) is prepared, such as human brain tissue thromboplastin, no. 67/40. At the same time, the secondary reference preparation (secondary reference preparation) was standardized based on BCT67/40. For example, bovine brain tissue thromboplastin is numbered 68/434; rabbit brain is 70/178. Later, WHO also announced some secondary tissue thromboplastin reference products, such as human brain or placenta preparations, such as BCT/253; rabbit or rabbit and monkey tissue mixed preparations, such as RBT/79, etc.
(2) Composite tissue thromboplastin international reference product: It is a preparation composed of a tissue extract and a physiological saline suspension added with an appropriate amount of fibrinogen, factor V and calcium chloride, such as bovine tissue Thromboplastin OBT/79, etc.
Currently, countries around the world widely use these WHO reference materials to calibrate tissue thromboplastin reference materials prepared or produced in their own country or region. This enables a variety of tissue thromboplastin reference products to be available worldwide.
2. International sensitivity index (ISI) of tissue thromboplastin working preparation (WP)
Due to the different effects of tissue thromboplastin on coagulation factors, Sensitivities vary.
In order for tissue thromboplastins with different sensitivities to obtain the same results in detecting PT, a unique sensitivity index must be developed. This requires comparing the homemade reagent with the international reference product (IRP) and then obtaining a correction value. The specific methods are as follows:
(1) Use the international reference product (IRP) to calibrate the national, regional or laboratory reference preparation (reference preparation, RP).
(2) Calibrate the working preparation (WP) with the laboratory reference preparation (RP).
1) Specimens: 2 normal human plasmas and 6 patient plasmas that have been on oral anticoagulants for 6 weeks, collected for 10 consecutive days, totaling 60 samples.
2) Measurement: Measurement is carried out in a certain order, and each sample is measured twice. Point the PT measurement results (seconds) on the logarithmic graph paper. The abscissa represents the PT measurement results of WP, and the ordinate represents the PT measurement results of RP. Draw the best fitting straight line for each point to obtain the calibration. Curve, find the slope b through the WP/RP ratio, or the 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 that produce and sell tissue thromboplastin reagents must label their products with ISI.
3. Reporting method of PT measurement results: Theoretically, no matter what kind of tissue thromboplastin, as long as it is marked with ISI, it can be compared and calibrated with the international reference product, and can be reported in the same measurement unit. In 1985, ICSH and ICTH recommended that the reporting method for PT monitoring of oral anticoagulants be the international normalization ratio (INR).
The calculation formula of INR is as follows: After using INR, various tissue thromboplastins with different sensitivities can obtain the same INR value.
4. Instrument-specific ISI: The above PT is suitable for manual method (test tube tilting method) measurement of PT according to the reporting method. However, there are still differences between the manual method and the instrumental method and the instrumental method and the instrumental method in determining the INR of PT. Research shows that on three automated instruments, ACL, Cobas Fibro and Coaga-Pet, using the same Thromborel S reagent with an ISI of 1.12, the average PT values ??were 10.7 seconds, 12.1 seconds and 11.1 seconds respectively. However, if the INR is calculated using the same ISI value, the resulting values ??will also differ to varying degrees. For this reason, experts have suggested that when the same tissue thromboplastin reagent is used in different instruments, the so-called "instrument specific ISIs" (instrument specific ISIs) or regional ISI (Local ISI) can be used to calculate the INR. The thromboplastin reagent used in each instrument should have a specific ISI value. The way to recalibrate the ISI value is to purchase freeze-dried plasma labeled with INR, and then recalibrate the ISI of the thromboplastin reagent used on your own instrument. value so that patients' INRs can be compared.
(2) Recommended method for prothrombin time (PT)
[Principle]
The tissue thromboplastin (mainly containing tissue factor and lipid ) and calcium ions are added to citrate anticoagulated plasma, incubated at 37°C, and the plasma coagulation time is measured, 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 silicone 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 to 38.5°C. 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. Once 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.
So far, ICSH and ICTH have not yet come up with a feasible plan to standardize 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.
[Operating 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-removed 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.
[Reference value] Refer to PT
[Special explanation]
1. Sensitivity to heparin: APTT is often used to monitor heparin treatment, usually in patients The ratio of APTT to normal plasma APTT is 1.5 to 2.5 as the therapeutic control range: but different reagents/instrument systems have different sensitivities to heparin. The sensitivity of its reagent/instrument system to heparin can be determined. In vitro sensitivity refers to adding the same type of heparin used clinically to normal plasma at its therapeutic concentration to measure APTT.
In vitro sensitivity is not the same as in vivo sensitivity, but it can be used as a reference.
2. Lupus anticoagulant factor: Lupus anticoagulant factor is an autoantibody against phospholipids. Because it resists phospholipids, an important component of blood coagulation, it can interfere with coagulation. If lupus anticoagulant factor is present in the blood, APTT will be prolonged. However, the sensitivity of APTT reagents to lupus anticoagulant factors varies greatly. The reagent manufacturer should provide sufficient instructions, and relevant national agencies can also provide information on lupus anticoagulant factors. But be aware that individual patients vary greatly, and no single reagent can measure all lupus anticoagulant factors.
IV. Standardization of fibrinogen measurement (Fg)
(1) Standardization of fibrinogen measurement (Fg)
Fibrinogen (Fg) ) is synthesized by the liver and exists in plasma and body fluids. Its structure and function have been basically understood, but there is still no ideal clinical detection method; there are more than 10 measurement methods reported in the literature, and some have relatively low precision and accuracy. Good, but too complicated and cumbersome; some are simple and fast, but have poor precision and accuracy.
In 1992, the British National Institute of Biological Standards and Control (NIBSC) completed research on a fibrinogen standard, numbered 89/644, and recommended it to the WHO Expert Committee on Biological Standards (ECBS). [15] and approved by ECBC as an International Reference Product (IRP). Since then, various countries have introduced this standard to standardize sub-standard standards produced by their own countries or manufacturers (the Clinical Testing Center of the Ministry of Health of my country has also introduced it). This has taken a critical step towards the standardization of fibrinogen determination. Therefore, according to the survey, the fibrinogen content of each standard product varies greatly.
(2) Recommended methods for the determination of fibrinogen (Fg)
When each family uses IRP to standardize their own standards, it is recommended to use Jacobsson’s improved method. This method is now introduced as follows .
[Reagent]
1. Buffer: Na2O·2H2O 0.882g; KH2PO4 2.77gl, this is the storage buffer; add 1 part of the storage buffer to 2 saline This is the application buffer, with a pH of 6.35.
2. Normal saline: 0.15mol/L
3. Human or bovine thrombin: 500IU/mL, normal saline solution.
4. Clot dissolving agent: Dissolve 400g of urea in a small amount of distilled water, 200mL of 1.0mol/L NaOH, and add water to 1 liter.
[Operating steps]
Reconstitute the fibrinogen lyophilized (standard) product (derived from 89/644) with 1 mL of distilled water, and add it to an organic solution containing 2 mL of application buffer. Add 50 μL of thrombin solution to a glass shallow dish or other container, mix quickly, and let stand at room temperature for 2 hours. Place the container upside down on an absorbent cloth (absorbent paper can be placed under it), and then use an appropriate substance (plus filter paper) to absorb the clots. Remove the clot and wash it twice in 50 mL of normal saline. After each washing, the clot should be sucked dry (glass can be used to squeeze the clot), and the liquid contained in the clot should be removed as much as possible to avoid contamination of the liquid. Other plasma proteins remain. Squeeze on a clean cotton cloth if necessary.
Carefully add the clot to a container containing 7.5 mL of clot dissolving agent and shake well until the clot is completely dissolved. Pour it into a cuvette with a light path of 1cm, use the clot dissolving agent as the blank, and read the absorbance (A) at 280nm and 315nm wavelengths.
(3) Applicable fibrinogen (Fg) determination (Clauss method)
[Principle]
Add thrombin to plasma to make fibrin Proteinogen changes to fibrin, and coagulation occurs in the plasma. When there is a sufficient amount of thrombin, it reacts with different contents of fibrinogen, and the time for plasma coagulation is negatively correlated with the fibrinogen content.
[Reagent]
1. Bovine thrombin l00NIHU/mL
2. Fibrinogen standard (IRP secondary standard)
< p>3. Buffer (choose one of the following two):(1) Barbiturate buffer (PH7.5); barbiturate 2.5g, barbiturate sodium 2.75g, Dissolve 7.3g of Lu sodium chloride in 750mL of deionized water, calibrate to pH 7.5, and add water to make it 1L.
(2) Imidazole (or Glyoxaline) buffer: 3.4g imidazole (0.05mol/L), 5.85g sodium chloride, add to about 500mL of water; add 186mL of 0.1mol/L hydrochloric acid , adjust the pH to 7.3-7.4, and finally add distilled water to 1 liter.
[Operation steps]
1. Manual method
(1) Use the above buffer to dilute the standard to 0.8, 1.6, 2.4, and 4.0 g/L fibrinogen concentration, and each concentration was diluted 1:10 with buffer.
(2) Patient plasma and quality control substances are diluted 1:10 with buffer.
(3) Mix the solution containing 100 NIHU/mL and bovine thrombin and store it at room temperature. If measured by instrumental method, use 100 NIHU/mL, bovine thrombin, without dilution.
(4) In a test tube, add 0.2mL of diluted plasma and place it in a 37°C water bath for 4 minutes.
(5) Add 0.2 mL of lOONIHU/mL thrombin solution pre-warmed at 37°C, shake well and start the stopwatch immediately to continuously observe the coagulation time. Stop the watch when solidification occurs.
(6) Measure each specimen twice and calculate the average. Measure each standard tube and control (quality control) tube at the same time using the same method and record the time accurately.
(7) Calculation: Use logarithmic paper to draw a graph, with the coagulation time as the vertical axis and the fibrinogen concentration as the abscissa, and compare the standard tubes of each corresponding concentration with the coagulation time on the graph. Mark the corresponding points and connect them with straight lines to make a standard curve. Clotting times were then measured based on patient and control plasma. Find the fibrinogen content on the chart.
If the plasma fibrinogen content is greater than 4g/L, the plasma should be diluted and retested, and the result should be multiplied by the dilution factor. If the plasma fibrinogen content is lower than 0.8g/L, the original plasma needs to be diluted to 1:2 or 1:5, and the result is checked on the standard curve and divided by 5 or 2.
2. Instrumental method: This method can be measured by the thrombin time (TT) method using an automatic or semi-automatic coagulometer. Results can be printed out automatically. Generally, the instrument method is more precise than the manual method.