Prothrombin time (PT) assay experiments are mainly used for (1) exogenous system ideal and commonly used screening experiments (2) exogenous pathway coagulation fixation to do quantitative tests (3) monitoring of oral anticoagulant therapy.
Operation method
Prothrombin time (PT) measurement assay
Principle
Plasma to be measured by adding excess calcium-containing tissue thromboplastin, recalcified plasma in the presence of tissue factor activation factor X becomes Xa. The latter converts prothrombin to thrombin, and thrombin converts fibrinogen to coagulated insoluble fibrin, determining the time required for coagulation, i.e., plasma thromboplastin time (PT) to be measured, PT assay is the ideal and commonly used screening experiments of exogenous systems, and it can be used as a quantitative test of exogenous pathway coagulation fixation. PT is an ideal and commonly used screening test for exogenous systems, and can also be used as a quantitative test for exogenous pathway coagulation fixation, as well as for the monitoring of oral anticoagulant therapy. Source and preservation of specimen: Blood is collected from vein, placed in a plastic tube or siliconized glass tube containing 1/10 volume of 0.109 mol/L sodium citrate anticoagulant (1 part of anticoagulant + 9 parts of whole blood), gently inverted and mixed. 3000rpm (or 2500) centrifugation for 15 minutes, the upper layer of the liquid (plasma, yellow) is collected, and the specimen is preserved for the following time under different conditions: -80 ℃ preservation, should not exceed 30 days, a 20 ℃ preservation, should not exceed 14 days, 2 a 8 ℃ preservation, should not exceed 6 hours, 22-24 ℃ preservation, should not exceed 2 hours to determine.
Materials and Instruments
Plasma Move 1. Reagent reconstruction: Caveat l. Blood collection must use disposable plastic syringes or silicone glass syringes, blood storage must use plastic test tubes or silicone glass tubes should not use ordinary glass tubes, to avoid the activation of coagulation factors. 2. The tourniquet should not be tied too tightly during blood collection and should not last more than 5 minutes, otherwise it will lead to the activation of coagulation and fibrinolytic factors. 3. blood should be immediately added anticoagulant, plasma preparation must be timely, plasma separation should be as soon as possible to remove platelets plasma separation should be measured as soon as possible. 4. plasma pre-warming should not exceed the temperature of the plasma. 4. Plasma pre-warming should not exceed 10 minutes. 5. Prothrombin pre-warming should not exceed 30 minutes. 6. automated coagulometers with turbidimetric endpoints, hemolysis and more pronounced jaundice and lipemia may affect the results, in which case manual or electro-mechanical or coagulometric determination is preferred. 7. If the hematocrit is more than 55% or less than 20%, the amount of anticoagulant should be adjusted, with anticoagulant volume (ml) = 0.00185 × blood volume (ml) × (100 - hematocrit). 8. It is not advisable to make EDTA-Na2. Heparin, oxalate anticoagulation, should be used 0.109mol / L sodium citrate anticoagulation. 9. 80 ℃ and a 20 ℃ to save the sample, measurement of 37 ℃ rapid thawing, not repeated freezing and thawing. 10. Avoid hemolysis and tissue fluid contamination during sample collection. 11. It is recommended that each laboratory establish its own reference value range. 12. 12. In manual operation, the end point of thromboplastin time is calculated based on the initial coagulation of turbidity. 13. 12. Measurement temperature 36.5-38.5 ℃ too low or too high temperature will cause PT prolongation. Common Problems It is difficult to standardize the reference values because different instruments/reagents will give different results. Each laboratory should determine a batch of healthy persons on its own and establish reference values according to the conditions of its own 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 separate days to minimize differences between days. The results were statistically processed and the standard deviation was 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. For more product details, please visit Aladdin Scientific website.
PT reagents
Distilled water Beakers Disposable injections
In the preservation of each bottle of PT reagent add 2.0 ml of distilled water and shake to dissolve. Note: The reagent can be stored at 2-8℃ for 7 days after dissolution, and at room temperature (15-25℃) for 24 hours, do not freeze.
2. Manual determination:
Take 0.1ml of plasma to be tested (reference plasma), incubate at 37℃ for 2 minutes, add 0.2ml of pre-warmed thrombin at 37℃; mix well and start the stopwatch immediately to record the coagulation time, i.e. PT value.
3. Hemagglutination instrument determination:
Please operate according to the parameters provided by the instrument
Normal values:
1. Expressed in time 11-15 seconds
2. 0.95-1.24 as PTD
3. 0.94-1.30 by INR