Normal value of platelets is 100,000-300,000
Clotting time
Clotting time is usually involved in the examination of the medical blood routine.
I. Bleeding time measurement and clinical significance
From the rupture or injury of blood vessels, the blood flow out of its own to the platelet embolus formation of temporary hemostasis is called hemostatic process, many pathological factors can affect this process and lead to bleeding. Many pathologic factors can influence this process and lead to bleeding episodes. Many tests have been designed to look for the cause of this pathophysiology, such as endothelin assays, vascular hemophilic factor (vWF) assays, and platelet aggregation assays. These tests are sensitive but complicated to perform, and bleeding time testing is often used as a screening test, followed by the above tests if positive. There are three methods to determine bleeding time, namely, the Duke method, the Ivy method, and the bleeding time meter method. Although the Duke method is simple to operate, it is difficult to standardize the depth and width of the puncture, and is affected by the distribution of capillaries at the puncture site and the degree of vasoconstriction, which results in poor sensitivity of the test. In the literature, the positive rate of patients with platelets less than 3×109/L is only 4 2%, and the positive rate of patients with hereditary pseudohaemophilia (VWD) is 32%, whereas the positive rate of Ivy's method is 92% and 67%, respectively. Ivy's method is more sensitive than the Duke's method, but it is cumbersome to operate, and is also damaging, and it is difficult to standardize the incision, and it is not easy to promote in the field. The Bleeding Time Sedimenter method is easy to control the depth and width of the incision because the blade is embedded inside the instrument and is quickly ejected by a spring, which can be standardized and has a high sensitivity. Therefore, the document stipulates that the bleeding time measurement experiment should use the bleeding time meter method and abolish the Duke method.
The coagulation time measurement and clinical significance
The coagulation time test refers to the time it takes for blood to be withdrawn from a blood vessel and placed into a test tube from a liquid sol state to a semi-solid gel state. It is actually a reflection of the activation of blood (or plasma) E.D. factor, the plasma in the zymogen state of the coagulation factor is activated in turn, successive "amplification", and ultimately the fibrinogen into fibrin. The latter forms a reticular network of platelet plugs and erythrocytes, and finally the reticular fibrin shrinks to form a solid clot. This process requires the participation of coagulation factors such as X, D, Ⅺ, IX, V, Ca2+, prothrombin and fibrinogen, etc. The lack of any one of these factors can cause the above "continuous amplification" process to be impaired, resulting in poor coagulation. Therefore, a prolonged clotting time can reflect the lack of these factors. Although it records the entire coagulation process, this test is sensitive to factors Ⅺ, Ⅸ, and VIII, and only shows prolonged results when there is a significant decrease in prothrombin, factor X, and fibrinogen. There are 6 kinds of coagulation time test methods, Wang Hongli et al. used 6 kinds of coagulation time test to measure 12 cases of heavy hemophilia (factor VIII activity <1%), 4 cases of medium hemophilia (factor VIII activity 1%-5%), the results (detection rate/number of cases) were: capillary method, heavy 1/12, medium 0/4; slide method, heavy 2/12, medium 0/4; common test tube method, heavy 12/12, medium 0/4; and the results of the test tube method, heavy 12/12, heavy 0/4; the results of the test tube method, heavy 12/12, heavy 0/4; the results of the test tube method, heavy 12/12, heavy 0/4; the results of the test tube method, heavy 12/12, heavy 1/12, middle 0/4, middle 0/4, middle 0/4, middle 0/4, middle 0/4, middle 0/4. It can be seen that the capillary method and the slide method are very insensitive and can no longer be used for the screening of factors Ⅺ, Ⅸ, and Ⅷ. Ordinary test tube method is simple and sensitive, the amount of specimen is small, can be used for routine examination, but because of manual operation, attention should be paid to the standardization of experimental equipment. APTT is simple, fast, there are various types of coagulometers available in the market for use in laboratories of different sizes, and there are matching reagents, quality control substances, calibrators, and easy to quality control, which makes it a more desirable routine experimental method. APTT can also be performed manually, but there are many factors affecting it, and it is not suitable for use when the amount of specimen is large. In order to avoid intraoperative bleeding, it is very necessary to check bleeding tendency before surgery. Since there are many factors affecting hemostasis, but it is impossible to do many experiments, therefore, bleeding time and coagulation time were used as diagnostic indexes in the past, and when the results were abnormal, more complicated tests were done to find out the cause of the disease. The results are abnormal, and then more complicated tests are performed to find the cause of the disease. As mentioned before, Duke's method is simple and suitable for routine application, but the sensitivity is very poor and cannot meet the diagnostic requirements, while the bleeding time tester method is complicated and time-consuming to operate, and is not suitable for routine use. Although it reflects pathological changes in blood vessels and platelet quantity and quality, thrombocytopenia is the most common cause of clinical changes. Vascular diseases and platelet quality abnormalities are hereditary diseases with a very low incidence and are basically reflected in clinical manifestations, signs and history, which may prompt some confirmatory tests. In this sense, a preoperative platelet count without a bleeding time is basically sufficient for preoperative screening. The APTT assay is sensitive only to factors Ⅺ, Ⅸ, and VIII of the coagulation phase. For this reason, a PT should be performed in conjunction with the APTT to ensure that bleeding tendencies due to deficiencies of factors Ⅺ, Ⅺ, Ⅺ, and Ⅺ can be detected. Four, activated APTT, PT test methods and quality control (a) blood specimens should be taken to pay attention to the problem of 1. PT, APTT coagulation experiments are using venous blood. The blood collector should be skilled to prevent tissue damage, exogenous coagulation factors into the syringe. 2. Coagulation specimens should preferably not be collected together with other experiments, otherwise the blood will remain in the syringe for a longer period of time due to specimen distribution and filling. In general, the shorter the time between the collection of blood, its entry into the container, and the performance of the experiment, the better the protection of the analyzed coagulation factors. It is not advisable to remove blood from the infusion tee. 3. When removing blood, the patient should be relaxed, the environment should be warm to prevent venous contracture, the pressure of the tourniquet should be as small as possible, high pressure and long binding time can affect the concentration of local blood and endothelial cells to release tissue plasminogen activator (t-PA), the latter will cause the enhancement of plasminolysis, the platelet shortness and inner diameter should be standardized, and the international recommendation is to use the 21G1.5 or 20G1.5 needles. During blood sampling, the speed of pulling the needle plug should be slow and even, so that the blood can enter the syringe smoothly and prevent the generation of air bubbles. 4. Once sampling is complete, mix the blood with the anticoagulant, and generally advocate the use of vacuum blood collection tubes. This tube has sufficient transparency, according to the design of the blood volume, there are 2.0, 5.0, 10 ml of various types of blood collection tubes, vacuum negative pressure and a quantitative anticoagulant, can ensure that the anticoagulant and the ratio of the volume of blood, and can have sufficient space to facilitate the mixing of blood and anticoagulant. 5. Blood collection with siliconized glass or plastic syringes. Considering the accuracy of the results, the possible error in the graduations of the tubes should be limited to 10% of the established volume. 6. the specimen is stored at low temperature for a certain period of time after blood collection. The author has investigated the changes of plasma factors stored under different conditions, and the results showed that plasma stored at 32℃ for 6, 12, 24h factor VIII activity can be lost 50%, 60%95%, and even stored at 4℃ also lost 5%, 55%, 70%, respectively. Factor V activity at 32℃ was lost 25, 40, 80%, and at 4℃ was lost only 0%, 5%, 10%. Therefore, plasma for the determination of APTT can be stored for at least 1 month at -80°C, 6h at 4°C, 6h at 20°C, and only 2h at 32°C. For PT plasma, 24h at 4°C, 6h at 20°C, and 2h at 32°C. 7. The International Committee for Standardization of Haematology (ICSH), and the International Committee on Thrombosis and Haemostasis (ICTH) recommend the use of 3.2g/dl (with 2H2O) or 0.109mo l/L of blood. The International Committee on Thrombosis and Haemostasis (ICTH) recommends the use of 3.2 g/dl (with 2H2O) or 0.109 mo l/L sodium citrate as an anticoagulant for coagulation factor testing. In addition, in recent years, many laboratories have adopted buffered anticoagulants, which are more suitable for analyzing specimens after cryopreservation. Because plasma pH increases over time without buffering agents, coagulation times can be prolonged, particularly for factor VIII, which has a 50% reduction in coagulant activity even when stored at -20 °C. Retarders are used to maintain a constant plasma pH and to prevent inactivation of labile factors. The absolute amount of anticoagulant in a blood specimen can alter the plasma calcium concentration, which in turn affects the results of the test, so it is important to pay attention to the ratio of blood to anticoagulant at the time of blood collection. It should be noted that the 9:1 ratio of blood to anticoagulant actually refers to the ratio of anticoagulant to normal blood pressure. Therefore, the amount of anticoagulant should be adjusted according to the patient's Hct. (II) Application of reagents should pay attention to the problems 1. Prothrombin should pay attention to the problems: (1) Tissue thromboplastin standardized application of the international reference preparation (international reference preparation, IRP), there are the following kinds of: a single tissue thromboplastin international reference product: is a tissue extract of saline suspension preparation. WHO in the United Kingdom uses the unified standard "British Comparative Thromboplastin", No. 67/40, and at the same time standardizes the secondary reference products based on BCT67/40. For example, bovine brain tissue thromboplastin, No. 68/434; rabbit brain, No. 70/178, and so on. Compound Tissue Thromboplastin International Reference Material: It is a preparation consisting of a saline suspension of tissue extract with appropriate amount of fibrinogen, factor V and calcium chloride, such as Bovine Tissue Thromboplastin OBT/79. (2) International sensitivity index (ISI) of tissue thromboplastin working preparations, due to the different sensitivities of different tissue thromboplastins to coagulation factors. In order to obtain the same results in PT for tissue thromboplastin with different sensitivities, it is necessary to develop a ****similar sensitivity index. Homemade reagents are compared to an international reference product (IRP) and a calibration value (i.e., ISI) is derived. The closer the ISI value is to 1.0, the more sensitive the tissue thromboplastin reagent is, and therefore, products must be labeled with an ISI when they are manufactured and sold. (3) Instrument-Specific ISI: The above PTs are applicable to the manual method (tube tilt) for the determination of PTs based on the mode of reporting. However, there are still differences in the International Normalized Ratio (INR) between manual and instrumental methods and between instrumental and instrumental PT determinations. For this reason, it was recommended that the INR be calculated using the so-called "instrument-specific ISI" or regional ISI when the same tissue thromboplastin reagent is used in different instruments. Each instrument should have a specific ISI for the thromboplastin reagent used, and recalibration of the ISI should be performed by purchasing lyophilized plasma labeled with the INR and then recalibrating the ISI of the thromboplastin reagent used on one's own instrument. To recalibrate the ISI, purchase lyophilized plasma labeled with the INR and then recalibrate the ISI of the thrombin reagent used on your own instrument so that the INRs of the patients are comparable. (4) Certain thromboplastins and partially activated thromboplastins are not necessarily compatible with this instrument. Turbid or particulate prothrombin and partially activated thrombin cannot be detected by an instrument that determines the endpoint by means of an optical system. (5) Prothrombin and partially activated thrombin must be used in accordance with the instructions. As a rule, the reagents must be well mixed before use to resuspend the particles evenly in the liquid. Store the reagents at the temperature specified in the instructions and do not leave the reagents at 37°C for longer than the temperature specified in the instructions during the test. (6) The sensitivity to heparin, lupus anticoagulant and Factor VIII/IX deficiency in blood varies with the activator used in the APTT test, so the APTT reagents with different activators should be selected according to the test items. Similarly, PT prolongation is associated with pathological phenomena caused by defects in a single or several coagulation factors. Since different diseases may reflect changes in only one factor, it is not possible for a specific reagent to have a consistent sensitivity to changes in these diseases. Due to the different quality of PT reagents produced by different manufacturers, the results of the same specimen with different PT reagents can be significantly different. Therefore, it is preferable to use the INR reporting method for PT experiments. 2. 2. Deionized water should be used for reagent preparation. If water with elevated pH is not properly buffered, it will prolong the results. If ammonia-containing water is used to prepare the anticoagulant, it will result in a rapid decrease in Factor V activity, thereby prolonging the prothrombin time. Thrombin, partially activated thrombin, buffers, and anticoagulants should be stored at 4-10°C, except for calcium chloride and water, which should be stored at room temperature. 3. 3. Normal control plasma is usually prepared by mixing multiple healthy human blood specimens to compensate for individual errors. Normal control plasma should be obtained from 20 or more healthy individuals of either sex, 18-55 years of age, who are not taking medication, and who are calm and at rest. The samples should be centrifuged to remove the plasma, mixed, vialed, frozen at -80°C, or freeze-dried. 4. 4. Reference plasma: Standardization is an important part of quality control, and method standardization is difficult for some laboratories due to equipment limitations. In order to make the results comparable over time in the same laboratory, it is necessary to use standards or reference plasma. WHO has established more than 10 international standards. (C) equipment and instruments should pay attention to the problem 1. glassware requirements: storage and testing should be clean, no scratches in the test tube. Acid cleaning method (hydrochloric acid 3mol / L) is recognized as the best method for coagulation studies to clean glassware. Glassware must be rinsed thoroughly to remove residual acid and to avoid altering the pH of the container surface. Keep disposable glass and plastic products separate. For manual PT or in vitro whole blood clotting time determinations, test tubes should be 8 mm in caliber; the larger the diameter, the longer the CT. 2. 2. Temperature requirements: The manual endpoint method requires that the temperature of the water bath must be maintained at (37±1)°C and that the surrounding illumination should be good enough to facilitate reading of the endpoint. If in order to read the test tube out of the water bath, tilted observation, this action should be rapid, otherwise the temperature of the plasma in the test tube will quickly drop. 3. 3. Choice of instrument: There are two types of automated endpoint assays, semi-automated and fully automated, the difference being that in the former the reagent and plasma pipetting steps are still manual, while in the latter the instrument is fully automated. The repeatability of the results obtained by the automated endpoint method greatly improves the comparability of results between different laboratories. The choice of instrument should be based on precision, reliability, ease of use and maintenance. There are many reports on the use of synthetic substrates instead of the biological assay with solidification as the endpoint. However, the clinical significance of these two methods is not the same. (IV) Problems to be noted in experimental operation 1. Many experimental errors are due to technical errors. Small changes in technique, reagents, temperature and pH can lead to significant changes in the results. Incubation time and temperature are parameters that should be strictly controlled for the determination of prothrombin time in the phase I assay. Plasma should not be left at 37°C for more than 10 min, and thrombin should not be left for longer than the specified time. 2. 2. When performing manual PT or tube CT, the tube should be tilted gently and at a small angle to minimize the contact area between the blood and the wall of the tube. Two tubes of the same specimen should be performed (three tubes for CT in vitro have been reported in the literature) and the mean value should be reported. Normal controls must be available for each batch of experiments. 3. 3. Blood coagulation is primarily an enzyme-catalyzed reaction, so the ideal pH environment and ionic strength of the solution should be strictly controlled. Most routine experiments are performed in buffer systems that are in the buffering range of physiologic blood pH. The pH of the reaction mixture must be between 7.2 and 7.4. 4. 4. Accurate test results also depend on the amount of reactants used, the order of addition, and the incubation time of the different reactants. For example, the incubation time of the activated partial thromboplastin reagent and the plasma mixture must be the same for each APTT test. The mixing process determines the amount of exposure to activation in the APTT test, and poor technique in mixing the activated fraction of thromboplastin with the plasma during incubation will alter the results of the assay. 5. Platelet plasma is required for APTT and PT. The plasma should be separated by centrifugation at 3000 r/min for 10 min. Pay attention to the amount of specimen before centrifugation and the amount of plasma (Hc t) after centrifugation to ensure the optimal ratio of anticoagulant to specimen. 6. Check plasma for hemolysis, jaundice, lipemia, and clots before testing. Red cell membranes contain phospholipids, and hemolyzed specimens have similar clotting activity to platelet factor III (phospholipids), which shortens the APTT of hemolyzed plasma. The presence of a clot in the specimen, no matter how small, will affect the result. 7. Reporting: (1) Reported in seconds (s) of PT. (2) Reported as patient PT(s)/normal control(s) ratio (PTR). (3) Reported as INR during monitoring of oral drug therapy. (4) No longer reported as a percentage (activity) according to ICSH. (5) APTT is reported in seconds. (E) Drawing quality control charts to monitor the instruments and reagents Drawing quality control charts and analyzing their trends at any time is an important means of quality control. In the daily work, normal and normal control plasma (commercial or homemade) were measured at the same time, and the Le vey-Jeuny quality control chart was used to check whether there was any loss of control. The procedure was to take at least 10 measurements of the QC under normal experimental conditions (including highly qualified laboratory personnel, standardized operation, and stable instrumentation), calculate X and the standard deviation, and then plot the QC chart, with the X value of the QC as the baseline, and the vertical axis having X ± 2 s. The QC was tested with the specimen for each test, and the results of the day were plotted on the chart. The following graphs of quality change are useful for the operator to determine if a result is out of control. In the event of a loss of control, the following procedure (Figure 1) is used to investigate the cause. Interlaboratory QA: Laboratories must actively participate in interlaboratory QA activities organized by the Laboratory Center in order to understand the accuracy of their own results. Since different results can be obtained from the same QC plasma due to differences in instrumentation and reagents, the results should be grouped and compared according to the instrumentation and reagents used. The reference values for APTT by manual method are reported to be 31.5-43.5 s in the literature and 32-43 s in women. There is no significant difference between the two. There is no significant difference between the two. Only the prolongation of the examined measurements by more than 10 s compared with the normal control is pathologically significant. The reference value of PT by manual method is 11-14s, and it is abnormal if it is more than 3s than the normal control, and the reference value of PT R is 1±0.15. It is difficult to set a uniform reference value for the instrumental method because different results can be obtained by different instruments and reagents, and each laboratory should measure a batch of healthy people according to its own instruments, reagents and other conditions, and establish the reference value. At least once a year thereafter, or when conditions change, the reference values should be re-established according to the new conditions. At least 40 men between the ages of 18 and 55 years and non-pregnant, non-menstruating women (half men and half women) in good health, with no history of drug use within half a month, blood collected in a calm, resting state, and measured on several separate days (in order to minimize batch differences). Blood was collected and measured on separate days (to minimize lot-to-lot variation) in a calm and rested state. The test was performed under the usual specimen-testing conditions. The results were processed statistically, standard deviations were calculated, and 95% confidence limits were used as reference ranges.
The results were statistically processed and standard deviations were calculated.