Prothrombin Time
PROTHROMBIN TIME
(By ELITE PRO analyzer)
Purpose of examination:
The prothrombin time is a coagulation screening test. It measures as a whole the activity of the coagulation factors II, V, VII, X and I
The prothrombin test measures the clotting time of plasma in the presence of an optimal concentration of tissue extract (thromboplastin) and indicate overall efficiency of the extrinsic clotting system.
Principle of examination:
The principle of the test consists of the use of calcium thromboplastin to measure the clotting time of the patient’s plasma and to compare it with that of a normal standard.
The test measures as a whole, the activity of the coagulation factor II, V, VII, X, I
Principle of instrument:
Optical Measuring System
The Loading and Analysis area also houses the optical system for analysis on two channels:nephelometric and absorbance.
Nephelometric channel: the light source for this channel is a light emitting diode (LED); the light (λ = 660 nm) is directed to the reaction cuvettes in the rotor by a fiber optic system. The scattered light is read at a 90o angle with respect to the incident beam using a solid state detector located below the rotor holder.
Absorbance channel: the light source is a halogen lamp, from which the radiation is directed to the reaction cuvettes in the rotor via a quartz optic fibe and a focusing system. The selection of the wavelength for analysis is effected by a narrow-band interference filter centered at λ = 405 nm. The optical detector is mounted in the cover of the loading/analysis area; therefore the readings are made at an 180oangle from the light beam.
The optical path width for the absorbance channel is 0.5 cm (cuvette height).
The absorbance values provided by the analyzer are normalized to 1 cm.
These values are generally twice those ones obtained on other ACL models,for which the absorbance values are not normalized and are thus exactly the ones obtained for the 0.5 cm cuvette path.
Performance specification:
Sample type required:
Citrated plasma
Preservative needed:
Sample collected In Sodium Citrate bulb. 9 volume of blood is collected in 1
volume of 3.2% trisodium citrate anticoagulant
Reagents required:
Recombiplastin 2G
Hemosil cleaning solution A
Hemosil cleaning agent B
Calibration method: Calibration is done by manufacture annually and calibration report is received in the laboratory . Calibration reports are filed in HI: C\ Records\File\21\Calibration Records
Detailed work bench instruction:
Switch on the main power supply.
Switch on the ELITE Pro switch.(Back side of machine)
Switch on the monitor.
Monitor will display starting
ACL, please wait and then system load in progress.
For log in enter user and password.
Database view appear on screen.
Menu contains: ‘Analysis’ ‘QC’ ‘Calibration’ ‘Diagnostic’ ‘Setup’ ‘utility’
Select option ‘Diagnostic’ then move to maintenance and everyday change cleaning cycle date.
Select option ‘Diagnostic’ then move to cleaning and start.
After compeletion of cleaning select ‘Analysis’ and move to multitest session.
Multitest preanalysis displayed on monitor then click on circle (Number) and enter sample ID then select test like PT or APTT then confirm test.
Daily controls are run first.
Then for second sample just like first sample select position and enter sample ID.
After loading all samples click on √ box then press on ‘RUN’.
Then monitor will display ‘Pre Analysis in progress’ then pre analysis result control pannel is displayed then select ‘Continue’.
Samples are tested automatically. Results are displayed on screen.
After compeletion select clening and in last shut down.
Quality control procedure:
Control plasma (Freeze dried pooled plasma of 20 normal individual) is run in duplicate as normal control with every batch and mean is derive and recorded.
Daily level 1 and level 2 controls run. Results of control samples are recorded in C\Records\File\9\Internal Quality Control Records
Comparison done once a day by running a pooled normal plasma on Elite Pro coagulation analyzer and then by Manual method. Results are compared and documented.
Inter instrument comparison done once a day by running a random sample on Elite Pro and STAGO. Results are compared and documented.
External quality assessment: Participate in external quality assessment program ( ISHTM-CMC VELLORE EQAS PROGRAM- HAEMOSTASIS ) every 4 mthly and maintain data in C\Records\File\2\Results of EQA and interlaboratory comparison
Interference:
In order to maintain the activity of various coagulation factors, the samples should be taken with care and following the professional standards in tubes with a specified concentration of citrate.
Quality of centrifugation and storage temperature of the sample should also be carefully insured before the analysis.
Plasma that is hemolysed, partially coagulated (presence of microclot), damaged by temperature changes or with bubbles on its surface may cause inaccurate results
Plasma that has been frozen may contain interfering precipitates when thawed. These precipitates should be removed before measurement is performed
Poor preparation of the reagent regarding reconstitution volume, stabilization time, stirring as well as the presence of bubbles or the accidental presence of a magnetic rod may lead to incorrect result.
Calculation of results and uncertainty:
The slightest coagulation (micro clot) will induce considerable shortening of the times measured (autocatalytic activation of all the factors) whereas extensive coagulation will prolong the clotting times because of consumption of factors and fibrinogen
Plasma should not be kept at 2-8’C because in this temperature range the factor VII may be activated by the kallikrein system
Maintain the correct anticoagulant / blood sample volume ratio of 1:9. If there is any considerable variation in hematocrit, quantity of anticoagulant should be modified accordingly
The test is insensitive to unfractionated heparin levels upto 1IU/ml and to low molecular weight heparin levels up to 1.5 anti- Xa IU/ml
Thrombin inhibitors present in the sample to be tested may lead to a prolonged prothrombin time
Biological reference interval:
11- 16 seconds
Reportable interval for examination results:
5-170 seconds
Critical values:
one minute
Interpretation by the laboratory:
Results given by the analyzer must always be analyzed according to the patient’s history, clinical examination and to any other biological results.
The common causes of prolonged one stage PT are as follow:
Administration of oral anti-coagulant drugs (vitamin K antagonists); PT is commonly used for monitoring vitamin K antagonist therapy because of its sensitivity to variations in the concentration of vitamin K dependent factors II, VII, and X.
Liver disease, particularly obstructive
vitamin K deficiency
Disseminated intravascular coagulation
Rarely, a previously undiagnosed factor VII, X, V, or prothrombin deficiency or defect
Potential sources of variability:
Pre analytic variables:
Whenever possible venous samples should be collected without a pressure cuff. Venous occlusion causes hemoconcentration and activation of some clotting factors
Faulty collection of sample resulting in it undergoing partial clotting
Blood samples from indwelling line or catheter should not be used for test as they are prone to dilution or heparin contamination.
Order of draw should be taken care of while blood collection
Underfilling or overfilling of tube or high or low hematocrit ( can cause the volume of citrate in relation to the plasma volume to be incorrect)
Testing should be completed within 2 hours of collection; undue delay in sample analysis can cause inaccuracy
Reference:
Reference Manual of ELITE PRO.
Practical Haematology (Dacie and Lewis)- twelfth edition
(By STAGO analyzer)
Purpose of examination:
The prothrombin time is a coagulation screening test. It measures as a whole the activity of the coagulation factors II, V, VII, X and I
The prothrombin test measures the clotting time of plasma in the presence of an optimal concentration of tissue extract (thromboplastin) and indicate overall efficiency of the extrinsic clotting system.
Principle of examination:
The principle of the test consists of the use of calcium thromboplastin to measure the clotting time of the patient’s plasma and to compare it with that of a normal standard.
The test measures as a whole, the activity of the coagulation factor II, V, VII, X, I
Principle of instrument:
The STA compact system performs in vitro tests for diagnosis and monitoring of pathologies linked to hemostasis.
The principle consist in measuring the variation of the ball oscillation amplitude through inductive sensors. The ball has a pendular movement obtained to the two curveted rail tracks of the cuvettes and an alternate electro-magnetic field created by two independent coils. The intensity of the magnetic field varies depending on the test to be carried out and on the expected clot.
The oscillation amplitude is constant when the environment has constant viscosity and decreases when the environment viscosity increases.
The detection system of the oscillation amplitude variation is based on two measurement coils. The transmitting coil emits an electro- magnetic field. The signal received by the receiver coil depends on the ball position in the cuvette. An algorithm uses these magnetic field variation to calculate the oscillation amplitude variation and to accurately determine the clotting time.
Performance specification:
Sample type required:
Citrated plasma
Preservative needed:
Sample collected In Sodium Citrate bulb. 9 volume of blood is collected in 1
volume of 3.2% trisodium citrate anticoagulant
Reagents required:
STA DESORB U
STA Neoplastine CI plus 5: reagent 1- lyophilized thromboplastin prepared from fresh rabbit cerebral tissues
Reagent 2- solvent containing calcium
Cleaner solution
Coolant (Ethylene Glycol)
Calibration method: Calibration is done by manufacture annually and calibration report is received in the laboratory . Calibration reports are filed in HI: C\ Records\File\21\Calibration Records
Detailed work bench instruction:
Switch on the main power supply.
Switch on the STAGO switch
Switch on the monitor
Global verification is done automatically and on completion GLOBAL VERIFICATION DONE is displayed on monitor
To continue press ‘ENTER’
Text box is displayed on screen: START OF TEST; Press ‘Esc’ to continue
“Test Panel” is displayed on monitor; Press ‘Esc’ for MENU
MENU contains: status/ loading/ files/ calibration- control/ set up/ maintenance/ Halt
Select option ‘loading’ and move on products to load the reagents. Product reagent drawer opens: reagents for PT ( STA DESORB U; STA Neoplastine CI plus 5) are first read by barcode and then product is kept at appropriate place according to size of vial; details are entered ( reagent ID, reagent name, volume, stability, lot number and position);
Press ‘Esc’ to return to main MENU
Select option ‘calibration- control’; select the test option from the list
Press ‘Esc’ for the option and select “change range” and enter the reference range and save (F10)
Press ‘Esc’ to return to the option ; enter the access code and select option “RUN”
Press ‘Esc’ to return to the option and select “result list”
Press ‘Esc’ to return to main MENU and select the option “Loading” ; select samples (F1); sample drawer will open. Samples are loaded by giving ID and sample position; Test is selected and validated by pressing F10
Samples are tested automatically. When the results are displayed on screen printout taken
Quality control procedure:
Control plasma (Freeze dried pooled plasma of 20 normal individual) is run in duplicate as normal control with every batch and mean is derive and recorded.
Inter instrument comparison done once a day by running a random sample on Elite Pro and STAGO. Results are compared and documented.
Interference:
In order to maintain the activity of various coagulation factor, the samples should be taken with care and following the professional standards in tubes with a specified concentration of citrate.
Quality of centrifugation and storage temperature of the sample should also be carefully insured before the analysis.
Plasma that is hemolysed, partially coagulated (presence of microclot), damaged by temperature changes or with bubbles on its surface may cause inaccurate results
Plasma that has been frozen may contain interfering precipitates when thawed. These precipitates should be removed before measurement is performed
Poor preparation of the reagent regarding reconstitution volume, stabilization time, stirring as well as the presence of bubbles or the accidental presence of a magnetic rod may lead to incorrect result.
Calculation of results and uncertainty:
The slightest coagulation (micro clot) will induce considerable shortening of the times measured (autocatalytic activation of all the factors) whereas extensive coagulation will prolong the clotting times because of consumption of factors and fibrinogen
Plasma should not be kept at 2-8’C because in this temperature range the factor VII may be activated by the kallikrein system
Maintain the correct anticoagulant / blood sample volume ratio of 1:9. If there is any considerable variation in hematocrit, quantity of anticoagulant should be modified accordingly
The test is insensitive to unfractionated heparin levels upto 1IU/ml and to low molecular weight heparin levels up to 1.5 anti- Xa IU/ml
Thrombin inhibitors present in the sample to be tested may lead to a prolonged prothrombin time
Biological reference interval:
11- 16 seconds
Reportable interval for examination results:
1-120 seconds
Critical values:
one minute
Interpretation by the laboratory:
Results given by the analyzer must always be analyzed according to the patient’s history, clinical examination and to any other biological results.
The common causes of prolonged one stage PT are as follow:
Administration of oral anti-coagulant drugs (vitamin K antagonists); PT is commonly used for monitoring vitamin K antagonist therapy because of its sensitivity to variations in the concentration of vitamin K dependent factors II, VII, and X.
Liver disease, particularly obstructive
vitamin K deficiency
Disseminated intravascular coagulation
Rarely, a previously undiagnosed factor VII, X, V, or prothrombin deficiency or defect
Potential sources of variability:
Pre analytic variables:
Whenever possible venous samples should be collected without a pressure cuff. Venous occlusion causes hemoconcentration and activation of some clotting factors
Faulty collection of sample resulting in it undergoing partial clotting
Blood samples from indwelling line or catheter should not be used for test as they are prone to dilution or heparin contamination.
Order of draw should be taken care of while blood collection
Underfilling or overfilling of tube or high or low hematocrit ( can cause the volume of citrate in relation to the plasma volume to be incorrect)
Testing should be completed within 2 hours of collection; undue delay in sample analysis can cause inaccuracy
Reference:
Reference Manual of STA compact (STAGO)
Practical Haematology (Dacie and Lewis)-tenth edition
Reagent insert of STAGO reagent( Neoplastine CI plus)
(Manual Method)
Purpose of examination:
The prothrombin time is a coagulation screening test. It measures as a whole the activity of the coagulation factors II, V, VII, X and I
The prothrombin test measures the clotting time of plasma in the presence of an optimal concentration of tissue extract (thromboplastin) and indicate overall efficiency of the extrinsic clotting system.
Principle of examination:
The principle of the test consists of the use of calcium thromboplastin to measure the clotting time of the patient’s plasma and to compare it with that of a normal standard.
The test measures as a whole, the activity of the coagulation factor II, V, VII, X, I
Principle of Method:
Tissue thromboplastin in presence of calcium activates the extrinsic pathway of human blood coagulation mechanisum. When LYOPLASTIN-LS reagent is added to normal citrated plasma, the clotting mechanism is initiated, forming a solid gel clot within specified period of time.
Performance specification:
Sample type required:
Citrated plasma
Preservative needed:
Sample collected In Sodium Citrate bulb. 9 volume of blood is collected in 1
volume of 3.2% trisodium citrate anticoagulant
Reagent required:
Lyoplastin (lyophilised calcified thromboplastin reagent, derived from rabbit
brain)
Equipments :
Water bath, Thermometer, Bulb, Stopwatch, Pipette (100 ul & 200 ul), Test
tubes (plastic tubes)
Detailed work bench instructions:
Bring the reagent to the room temperature.
Prewarm the reagent to 370 C.
Add 0.1 ml of plasma to the test tube and place test tube and place the tube in waterbath for 3-5 mins at 370 C.
Forcibly add 0.2 ml of LYOPLASTIN reagent and simultaneously start the stop watch. Shake tube to mix content.
Record the time in seconds.
Quality control procedure:
Control plasma (Freeze dried pooled plasma of 20 normal individual) is run in duplicate as normal control with every batch and mean is derive and recorded.
Comparison done once a day by running a pooled normal plasma on Elite Pro coagulation analyzer and then by Manual method. Results are compared and documented.
Interference:
In order to maintain the activity of various coagulation factor, the samples should be taken with care and following the professional standards in tubes with a specified concentration of citrate.
Quality of centrifugation and storage temperature of the sample should also be carefully insured before the analysis.
Plasma that is hemolysed, partially coagulated (presence of microclot), damaged by temperature changes or with bubbles on its surface may cause inaccurate results
Plasma that has been frozen may contain interfering precipitates when thawed. These precipitates should be removed before measurement is performed
Poor preparation of the reagent regarding reconstitution volume, stabilization time, stirring as well as the presence of bubbles or the accidental presence of a magnetic rod may lead to incorrect result.
Calculation of results and uncertainty:
The slightest coagulation (micro clot) will induce considerable shortening of the times measured (autocatalytic activation of all the factors) whereas extensive coagulation will prolong the clotting times because of consumption of factors and fibrinogen
Plasma should not be kept at 2-8’C because in this temperature range the factor VII may be activated by the kallikrein system
Maintain the correct anticoagulant / blood sample volume ratio of 1:9. If there is any considerable variation in hematocrit, quantity of anticoagulant should be modified accordingly
The test is insensitive to unfractionated heparin levels upto 1IU/ml and to low molecular weight heparin levels up to 1.5 anti- Xa IU/ml
Thrombin inhibitors present in the sample to be tested may lead to a prolonged prothrombin time
Biological reference interval:
11- 16 seconds
Reportable interval for examination results:
1-120 seconds
Critical values:
one minute
Interpretation by the laboratory:
Results given by the analyzer must always be analyzed according to the patient’s history, clinical examination and to any other biological results.
The common causes of prolonged one stage PT are as follow:
Administration of oral anti-coagulant drugs (vitamin K antagonists); PT is commonly used for monitoring vitamin K antagonist therapy because of its sensitivity to variations in the concentration of vitamin K dependent factors II, VII, and X.
Liver disease, particularly obstructive
vitamin K deficiency
Disseminated intravascular coagulation
Rarely, a previously undiagnosed factor VII, X, V, or prothrombin deficiency or defect
Potential sources of variability:
Pre analytic variables:
Whenever possible venous samples should be collected without a pressure cuff. Venous occlusion causes hemoconcentration and activation of some clotting factors
Faulty collection of sample resulting in it undergoing partial clotting
Blood samples from indwelling line or catheter should not be used for test as they are prone to dilution or heparin contamination.
Order of draw should be taken care of while blood collection
Underfilling or overfilling of tube or high or low hematocrit ( can cause the volume of citrate in relation to the plasma volume to be incorrect)
Testing should be completed within 2 hours of collection; undue delay in sample analysis can cause inaccuracy