General Approach in Investigation of Hemostasis The Islamic University – Gaza Faculty of Health Science Medical Technology Department General Approach in Investigation of Hemostasis Lecture 5 : Prothrombin Time (PT) Test

Protime /Prothrombin (PT) Prothrombin time is the time required for the plasma to clot after an excess of thromboplastin and an optimal concentration of calcium have been added. Although the PT was originally described as a specific, one-stage assay of prothrombin (FII), it is sensitive to a quantitative or qualitative abnormalities of any of the factors involved in the extrinsic and common pathways of the coagulation system (Factors II, V, VII, X, and Fibrinogen). Prothrombin is one of the coagulation factors produced by the liver

The PT used to determine the clotting tendency of blood, in the measure of warfarin dosage, liver damage, and vitamin K status. Occasionally, the test may be used to screen patients for any previously undetected bleeding problems prior to surgical procedures. Warfarin Comidin and other antigogulant durgs act by inhibiting the synthesis of vitamin K-dependant clotting factors II, V, VII, X also anticoagulant protiens C, S

When is it ordered? Used to monitor oral anticoagulant therapy (Warfarin / Coumadin). When a patient who is not taking anti-coagulant drugs has signs or symptoms of a bleeding disorder When a patient is to undergo an invasive medical procedure, such as surgery, to ensure normal clotting ability.

An elevated Prothrombin time may indicate the presence of Vitamin K deficiency DIC, Liver disease, Presence of FSP’s A deficiency in one or more of the Concerning factors: Factor I (Fibrinogen), Factor II (Prothrombin), Factor V (Proaccelerin, Labile Factor), Factor VII (Proconvertin, Stable Factor, Factor X (Stuart-Prower Factor, Factor XIII (Fibrin Stabilizing Factor) In addition, inhibitors can cause prolonged PT’s. FSP’s : Fibrinogen split products

The Test The Standard operating Procedure Procedure name Clinical significance Principle of method Specimen of choice Reagents and equipments Procedure Reference values Comments References Principle

Principle When reagent thromboplastin--to which calcium has been added--is mixed with plasma (derived from sodium citrated whole blood), the time (in seconds) it takes for the formation of a clot is reported as the Prothrombin time (PT). Calcium is necessary for the correct orientation and binding of a number of complexes including : tissue factor-VIIa, IXa-VIIIa, and Va-Xa.

PT Reagent Thromborel® S Reagent: lyophilized human placental thromboplastin, calcium chloride, stabilizers Thromboplastin may be obtained from other sources like Rabbit brain or lung tissue Neoplastine® C1 Plus - lyophilized fresh rabbit brain thromboplastin with a specific heparin inhibitor hydrated with a solvent containing calcium with stabilizers, polybrene, buffer and preservatives. (Tissue Factor, coagulation factor III ) protein-lipid complex found in tissues outside blood vessels. It is the combination of both phospholipids and tissue factor, both needed in the activation of the extrinsic pathway. However, partial thromboplastin is just phospholipids, and not tissue factor. Tissue factor is not needed to activate the intrinsic pathway. Tissue(TF) is the cell-surface transmembrane receptor that initiates both the extrinsic and intrinsic blood coagulation cascades.

Recombinant thromboplastin has been produced using human tissue factor in Escherichia coli. These synthetic phospholipids do not contain any other clotting factors such as Prothrombin, factor VII, and factor X. Therefore they are highly sensitive to factor deficiencies and oral anticoagulant–treated patient plasma samples and have an International Sensitivity Index (ISI) close to 1. Recombinant Thromboplastin: producing a sensitive human thromboplastin reagent from a non-recombinant source: cultured human cells. Cloning and expression of recombinant human tissue factor (TF) has enabled production of a new generation of thromboplastin reagents whose performance and utility are under active investigation.

Calibration of Reagent Each Thromboplastin Reagents must be calibrated against standard PT reagent established by the WHO. ISI = International Sensitivity Index. ISI is assigned by the manufacturer for each lot of reagent using reference material from WHO. The lower the ISI the more sensitive the Reagent ISI of 1.8 to 2.4 = Low sensitivity ISI of 1.4 to 1.8 = Average sensitivity ISI 1.0 to 1.4 = High Sensitivity

SPECIMEN Citrated plasma: 1 part of sodium citrate solution (0.11 mol/ L) to 9 part of venous blood, avoiding the formation of foam. Control: normal plasma (Commercial, Pooled Plasma) Pooled Normal Plasma Collection Minimum 20 normal healthy individuals not taking medications that interfere with clotting factors and coagulation reaction. It is acceptable to include women taking oral contraceptives. An approximately equal number of males and females is desirable. The age range should be 20 to 50 years.

EQUIPMENTS 100 μL micropipettes (0.10 mL) Stop Watch. Thromborel® S Reagent Racks Test tubes Waterbath (37ºC) Waste containers/ Biohazard bags

Procedure Reconstitute tissue thromboplastin according to instructions. Label the thromboplastin with the time, date and initials. The thromboplastin reagent is stabile for 7 days after reconstitution. Allow to sit 10-15 minutes, then invert gently several times. Mix well prior to pipetting any of this reagent at any step in this procedure. Prewarming of the reagent by pipetting 1-2 mls, using a plastic pipette, of the tissue PT reagent into a glass test tube and place in a 370 C water bath incubator.

Pipette 100 µL of normal control, Patient PPP into each of the test tubes. Allow at least one (1) minute to reach 37°C. Pipette 200 µL of PT reagent into the tube containing the control. Start the stop watch simultaneously. Mix the tube and leave in the water bath for a minimum of 7-8 seconds. Then remove, wipe the exterior, tilt back and forth gently until a visible clot is formed. As the clot forms, the mixture will gelatinize and may turn cloudy. Stop the stop watch and record the result. If the results from run 1 and run 2 are within + 10% second from each other, average the two results and report with appropriate units.

If results are not within required limits, a third run should be performed and average the two that match within acceptable limits. Be sure and cross out any values you are not using for the final calculation. Include measurement unit of seconds on report sheet. Carry out 1 significant figure passed the decimal point. For example, if your result is 12.23 seconds, report as 12.2 seconds. Repeat the procedure for the samples and Record the time.

Interpretation of Result A Normal Plasma is used to evaluate routine result. The INR is not used to evaluate Routine PT results. For Patients who are on oral anticoagulant therapy such as Coumadin INR result must be reported. Standardization of Report from lab to lab, by using INR results. Patients with lupus anticoagulants are not be requested for PT as they have antiphospholipid

Results are expressed as the mean of the duplicate reading in: Seconds Prothrombin ratio International Normalized Ratio (INR)

Reference ranges Critical Values* PT: 11.0 – 13.0 seconds. Therapeutic levels are at a P/C ratio of 2.0 – 3.0 Critical Values* PT critical value changed to > 42.1 seconds; INR changed to > 4.5 PCS; Pathology Clinical Services * (Refer to PCS Policy 7.01.05 Reporting Critical Values for reporting guidelines

Interfering Factors Diet: ingestion of excessive green, leafy vegetables will increase the absorption of vit-K, which promotes blood clots. Alcoholism, Prolonged PT levels Diarrhea and vomiting decrease PT because of dehydration. Quality of Vein puncture. Medication : Antibiotics , Aspirin, Cimethidine…. Prolonged Storage of plasma at 4o C.

Sources of Error Associated with specimen (Preanalytical) Inappropropriate ratio of anticoagulant to blood Failure to correct citrate volume if hematocrit > 55% Clotted, hemolyzed or lipemic samples Lack of PPP Delay in testing or processing Inappropriate storage

Associated with Reagent (Analytical) Incorrect preparation of reagents Use of reagents beyond reconstituted stability time or expiration date Contaminated reagent. Associated with procedure (Analytical) Incorrect temperature Incorrect incubation times Incorrect volumes of sample, reagents or both

Notes If thromboplastin reagent does not contain calcium, the test procedure is ml plasma, 0.1 ml thromboplastin, and clot with 0.1 ml pre-warmed 25mM calcium chloride. Activation of FIX by tissue factor:FVII occurs in vivo. Under the conditions of most PT tests, FX is so strongly activated that the assay is insensitive to deficiency of FIX or FVIII. Clotting times are normally influenced by the use of different coagulometers, depending on how and when the end point is detected. This further emphasizes the importance of establishing normal ranges for the method currently in use in the laboratory.

In the presence of mild deficiencies of factor II, V, VII, or X, the degree of prolongation may be minimal. In the case of FII deficiency, the PT may be within the normal range. Some PT reagents can be affected by the presence of lupus anticoagulants/anti-phospholipid antibodies, and some rare types of antibody may prolong the PT without any prolongation of APTT. Reagents with lower phospholipid concentrations are more likely to be affected, including some reagents that are constructed by lipidating recombinant tissue factor.

The presence of activated FVII, either following therapy with recombinant VIIa or when native FVII has been activated, can shorten the PT. The effect is dependent on the tissue factor reagent used. Reagents containing bovine tissue factor are particularly susceptible to this effect. Blood samples should not be stored at 2°C–8°C prior to determination of PT, since cold activation of FVII may occur. PTs determined with reagents containing human tissue factor may be different from those obtained with reagents containing tissue factor from other species, such as rabbit. In such cases, the result obtained with human tissue factor reagents may be more indicative of bleeding risk.

Heparin Extracted Prothrombin Time to better serve dialysis patients on oral anticoagulant therapy, Spectra Laboratories has validated the use of heparin extraction reagent to neutralize heparin-contaminated samples for PT testing. An anion exchange resin is added to the plasma and binds the heparin. The heparin-cellulose complex is removed from plasma by centrifugation. Prothrombin time results equal to or greater than 60 seconds on initial testing, will be retested utilizing the heparin extraction procedure. This procedure effectively absorbs and removes up to 2U of heparin per ml of plasma, producing accurate PT results without residual heparin interference.