Page 1 :
c€, , THROMBOPLASTIN REAGENT FOR PROTHROMBIN TIME (PT) DETERMINATION, , , , SUMMARY, The arrest of bleeding depends upon primary platelet plug formed along with the formation of a stable fibrin clot. Formation of, this clot involves the sequential interaction of series of plasma proteins in a highly ordered and complex manner and also the, interaction of hese complexes with blood platelets and materials released from the tissues., , Tissue Thromboplastin, in the presence of calcium, is an activator, which initiates the extrinsic pathway of coagulation, which, includes plasma coagulation factors Vil, X, V, Prothrombin and Fibrinogen. During oral anticoagulant therapy most of the, factors are depressed, as also during the deficiencies of clotting factor activity which may be hereditary or acquired., Prothrombin Time determination is the preferred method for presurgical screening, determination of congenital deficiency of, factors l,V, VIland X and for monitoring of patients on oral anticoagulant therapy and as aliver function test., , , , , , , , , , , , , , , , , , , , , , , , , , PRESENTATION, REF 10610005 | 10610125 | 106100004 | 106100124, LIQUIPLASTIN® Simi 12x5mi_|4mi 12x4ml, INRonversiontable | 4 1 1 1, Packinsert 1 1 1 1, , REAGENT, , LIQUIPLASTIN’ isa liquid ready to use Calcified Thromboplastin Reagent, which is derived from rabbit brain., Each batch of reagent undergoes rigorous quality control at various stages of manufacture forits sensitivity and performance., , REAGENT STORAGE AND STABILITY, , (a) Store the reagent at 2-8°C, DO NOT FREEZE. (b) The shelf life of the reagent is as per the expiry date mentioned on the, reagent vial label. The uncontaminated reagentis stable as per the labeled shelf life at 2-8°C, 1 week at 18-25°C, 2 days at, 37°C., , PRINCIPLE, , Tissue Thromboplastin in the presence of calcium activates the extrinsic pathway of human blood coagulation mechanism., When LIQUIPLASTIN® reagent is added to normal anticoagulated plasma, the cloting mechanism is initiated, forming a solid, gel clotwithin a specified period of ime. The time required for clot formation would be prolonged ifthere isa deficiency of factors, /factor activity in the extrinsic pathway of the coagulation mechanism,, , NOTE, (1) In vitro diagnostic reagent for laboratory and professional use only. Not fr medicinal use. (2) LIQUIPLASTIN’ reagentis not, SYMBOL KEYS from human source hence contamination due to HBsAg and HIV is practically excluded. (3) LIQUIPLASTIN’ reagent contains, 0.01% Thimerosal as preservative. (4) Itis very important that clean and dry micropipette tips be used to aspirate / dispense, , a oc Nite! Thi ew ton the reagent. (6) Avoid exposure ofthe reagent to elevated temperatures and contamination. Immediately replace cap after, , A Se aleneet tt » use and store at recommended temperatures only., , ww!) Date of, oie, , , , , , Manufacturer, , , , , , consult, , Pircserai In vio Diagnostic, , BE, , , , Desetnofreagent ‘SAMPLE COLLECTION AND PREPARATION OF PPP, Though no special preparation of the patient is required prior to sample collection by approved techniques, itis preferable that, hte Represenitve patients are not heavily exercised before blood collection, Fasting or only ight non-fatty meals prior to blood collection provide, ie irppenn oomeety samples with a desirable lower opacity., Withdraw blood without undue venous stasis or frothing into a plastic syringe fitted with a short needle of 19 to 20 SWG. The, veinpuncture must bea ‘clean’ one and, if there is any difficulty take a new syringe and needle and try another vein. Transfer the, blood into anticoagulated tubes, after detaching the needle from the syringe. Do not delay mixing blood with anticoagulant., Avoid foam formation during mixing, ull Mix exactly nine parts of freshly collected blood with one part of tr-sodium citrate (0.11 moll, 3.2%) or PROFACT, available from, TULIP Cat. No.: 10660020. For occasional patients with haematocrit less than 20% or greater than 55%, this ratio must be, readjusted to ensure valid results. Centrifuge immediately for 15 minutes at 1500 g on a laboratory centrifuge and transfer the, T. TULIP DIAGNOSTICS (P) LTD. plasma into a clean test tube. It should be ensured that the plasma is free from platelets (PPP). Cap the test tubes to prevent, deterioration of samples. Plasma must be tested preferably immediately. However if the specimens are held at 22-24°C, then they may be tested within 2 hours and if the specimenis held at 2-4°C then they may be tested within 3 hours., , , , Prothrombin, , Ccaaiogue, Numer, , , , , , , , , , , , , , , , , , , , , , , , , , , , es GITANJALL, TULIP LOCK, DR, ANTONIO 00 REGO BAGH, PLOT NOS. 9296, PHASE IC, VERNA IND. EST., z ALTOSANTACRUZ, BAMBOLIMCOMPLEXP.O, GOA403202, VERNA, GOA4T3722 INDIA, , 5| 4 g | NDIA Wobste: ww tuipgrovp.com ADDITIONAL MATERIAL REQUIRED FOR MANUAL AND CALIBRATION CURVE METHODS, , g)a|s|s [EC [REP] 12x75 mm testtubes (plastic tubes are preferred), 0.1 mland 0,2 ml precision pipettes, Stop watch, Water bath or heating block, , E,3/S/5 at37°C, Fresh normal plasmas for establishing MNPT., , (CMC Medical Devices & Drugs SL, C! Horacio Lengo No. 18, CP 29006, Malaga, Spain

Page 2 :
TEST PROCEDURE, , Manual Method, , 1. Aspirate from the reagent vial enough reagent for immediate testing requirements in a thoroughly clean and dry test tube., , (Plastic test tubes are preferred)., , Bring the reagent vial to room temperature (20-30°C) before prewarming at 37°C for testing purposes., , Recap the reagent vial and replace immediately to 2-8°C., , Toa12x75mmtube add 0.1 ml of plasma (PPP) and place the tube in a waterbath for 3 to 5 minutes at 37°C., , To the tube forcibly add 0.2mlof LIQUIPLASTIN’ reagent (prewarmed at 37°C for atleast 3 minutes) and simultaneously, , starta stopwatch. Shake the tube gently to mix contents., , 6. Gently tit the tube back and forth and stop the stopwatch as soon as the first fibrin strand is visible and the gel / clot, formation begins. Record the time in'seconds’., , 7. Repeatsteps 4-6 fora duplicate test on the same sample., , 8. Findthe average ofthe duplicate test values. Thisis the Prothrombin Time (PT),, , Ifa coagulation instrument is being used to perform the tests, the instrument manufacturers instructions must be, , strictly adheredto., , CALCULATION OF RESULTS, Manual Method, The result may be reported directly in terms of the mean of the double determination of PT of he test plasma in'seconds’, Orasaratio'R: R= Mean ofthepatient plasma PTin seconds, , MNPT forthe reagent*, Oras International Normalized Ratio (INR), INR= (R)®, where ISI= International Sensitivity Index of the reagent (Refer reagent, Vial label), * Itis recommended by the WHO that MNPT should be established for each lot of PT reagents by each laboratory, since PT, results are dependent on the combination of reagent lot, instrument and technique followed at each laboratory. Usually plasma, from atleast 20 normal healthy individuals should be used to establish the MNPT. The average of such PT results in seconds =, MNPT., , EXPECTED VALUES, Normal values using LIQUIPLASTIN® are between 10-14 seconds. Between manual and Turbo densitometric instrument, results a variation of 1-2 seconds may be expected. For photo optical instruments, itis recommended that each laboratory must, establish their own normal range. It is mandatory that each laboratory must establish its own MNPT for each lot of, LIQUIPLASTIN®, , Oral Anticoagulant Therapeutic range: INR=2.0-3.5, , , , REMARKS, , 41. Itis recommended that controls (PLASMATROL H-IIl Available from TULIP Cat. No. 11040061, 11041061) with known, factoractivty should be run simultaneously with each test series to validate test run, , 2. Incorrect mixture of bload and tri-soaium citrate, insufficient prewarming of plasma and reagent, contaminated reagents,, , glassware etc, are potential source of errors., , Oxalated plasma may induce prolonged clotting times, , Since the PT test functions correctly only at 37+ 0.5°C, temperature ofall equipment mustbe calibrated dally., , Clotting time of patients on anticoagulant therapy depends upon the type and dosage of anticoagulant and also the time, , lag between the specimen collected and the last dose., , Turbid, icteric, lipemic or grossly hemolysed samples may generate erroneous PT results, , Glasswares and cuvettes used in the test must be scrupulously clean and free from even traces of acids! alkalies or, , detergents, , Plasma samples held at4-8°C may undergo 'cold activation’ leading to a marked shortening of the PT., , The PT may be shortened during acute inflammatory conditions which are accompanied by increase in Fibrinogen levels, , and also by agents such as antihistamines, butabarbital, phenobarbital, caffeine, oral contraceptives and vitamin K. The, , PT may be prolonged by corticosteroids, EDTA, asparaginase, clofibrate, ethanol, tetracycline, aspirin and anticoagulants, , such as heparin and warfarin,, , 410. tis important that each laboratory express the results in terms of INR for patients on oral anticoagulant therapy for the, clinician to adjust the dosage based on INR., , 411. Since the test uses platelet poor plasma, each laboratory must calibrate the necessary force and time required during, centrifugation to yield the PPP. Contamination of plasma with excess platelets could falsely elevate levels of some of the, factors,, , 42. Homogenisation of LIQUIPLASTIN’ reagent suspension before use is important to achieve accurate and consistent, results, , NO fae, , om, , , , , , PERFORMANCE CHARACTERISTICS, © The Precision of Prothrombin time determination is highly dependent on the method used. Precision studies were, performed on Hemostar-XF coagulometer by assaying normal and abnormal control plasmas with LIQUIPLASTIN’. One, ‘normal control plasma and one abnormal control plasma in replicates of 10 were used to determine inter assay and intra-assay, precision of the clotting times (seconds)., , , , , , , , , , , , , , , , , , Inter-assay precision Intra-assay precision, Mean sD cv) | Mean sD cw), , Normal control plasma 11.6 0.17 147 11.8 0.13, 1.10, , ‘Abnormal control plasma 21.3 0.17 0.79 215 0.44 2.04, , , , , , , , , , , , © LIQUIPLASTIN’ is useful for measuring the deficiencies of factors of the extrinsic pathway. The factor sensitivity of, LIQUIPLASTIN’ was performed on Hemostar-XF (coagulometer based on turbodensitometric principle of clot detection), by diluting pool normal plasma with factor deficient plasmasiin the range corresponding to 3.12-100 % activities., , , , , , , , , , , , , , , , , , , , , , - Clotting time with LIQUIPLASTIN’ with factor deficient plasmas (seconds), , Activity of Factor (%), Factor VII FactorX Factoril FactorV, , 100 203 15.5 16.1 209, 50 26.0 198 184 24.2, 25 327 22.0 224 276, 125 425 26.7 25.0 309, 625 620 308 276 33.9, 3.12 65.9 36.0 29.0 36.0, , , , , , , , , , , , The above values should only be used as guidelines. Each laboratory should establish sensitivity to individual factors using, instruments, reagents and techniques used in their laboratory., , WARRANTY, This product is designed to perform as described on the label and package insert. The manufacturer disclaims any implied, warranty of use and sale for any other purpose., , BIBLIOGRAPHY, , 4. Biggs R.and R.G. McFarlane: Human Blood Coagulation and its disorders, Blackwell Scientific Publications, Oxford 1962., 2. QuickA.J., Hemorrhagic diseases and thrombosis, 2nd Ed., Philadelphia, Lee and Febiger, 1966., , 3. CRC Handbook Series in Clinical Laboratory, Science, Section 1: Haematology, Volume Ill, 1980., , 4. _E.A. Loeliger, A.M.H.P Van den besselaar and S.M. Lewis, Reliability and Clinical Impact of Normalization of Prothrombin, , Times in Oral Anticoagulant Control-F.K. Schattauer verlag Gmbh (1985)., , 5. Hull R, Hirsh H., Jay R., et al., Different intensities of oral anticoagulant therapy in the treatment of proximal-vein, thrombosis, N. Engl. J. Med, 1982, 307, 1676-81, , 6. WHO Expert Committee on Biological Standardization, No.687, 1983., , NCCLS guideline H21-A3, Vol. 18, No. 20., , 8. Dataonfile: Tulip Diagnostics (P) Ltd

Page 3 :
MONITORING ORAL ANTICOAGULANT THERAPY USING ISI AND INR, , © Oral anticoagulant drugs derived from coumarin and, sometimes indandiones are widely used in the, Prophylaxis and treatment of thrombotic disorders, Adjustment of the dose of these drugs is periodically, required to ensure that adequate and not excessive, degree of anticoagulationis achieved, , © The ICSH and the international committee for, hemostasis and thrombosis have agreed to the, reporting of Prothrombin ime (PT) results based on the, ISI (International Sensitivity Index) of the, thromboplastin reagents and INR (International, Normalised Ratio), , © LIQUIPLASTIN’is assigned an ISI value by calibration, against an international preparation, which by definition, has a preassigned sensitivity (ISI). LIQUIPLASTIN’ is, assigned an ISI value based on the method as, recommended by the WHO. The ISI value assigned to, LIQUIPLASTIN® defines its comparative slope or, relative sensitivity as compared to the IRP/standard, product., , © In general the lower the ISI value of a thromboplastin, reagent the more sensitive tis, , INR is calculated from the PT ratio (R) using a, thromboplastin with a known ISI. The INR may be, interpreted as the prothrombin time ratio that would, have been obtained ifthe same plasma had been tested, using the primary international reference preparation, IRP67/40 or IRP RBT/79., , © These of INR's enables direct comparison to be made, between all results on patient plasmas regardless of, interlab variations or reagentin question., , The INRis calculated as INR=(R)", , where ISI=Lot specific ISI forthe reagent, Patient PT, , Mean normal PT., , Mean normal PT = Mean of the normal range that is, specifically determined by each user laboratory for each lot, of thromboplastin reagent with specific instrument and, techniques routinely used for patient testing, , And, R=, , Example:, PatientPT result = 21 seconds, MNPT, ISlofreagent, 21.0, Re 515, 135, INR=(1.5)" =18, , = 13.5seconds, =18, , , , Lot No,, , , , MANUAL, , IS|_ | HEMOSTART, Value | HEMOSTAR XF, COASTAT-1/, COASTAT DUO, , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , INR Conversion Table, Altermatively the INR value can be read off directly from the, LIQUIPLASTIN® INR conversion table (see reverse) as, follows:, isi ==>, Interpolate the reading intersecting the ratio row (R), as R [44 [44s 45 [455 46 [405 a7 [47s 48 [18s [45, obtained for the patient sample with the reagent ISI value mT to T4010) 401 407 107 4071 10°) 40 1 10 1 4.0, from the IS! cohumn 11 [44 [41 ) 42 [42 | 12) 42 | 42 | 12) 42 | 12 | 12, © MNPT: Each laboratory should establish its own MNPT > 2113 [43 [13 | 43 [13 | 14 [14 | 14 [14 | 14 | 14, as per the procedure given in the LIQUIPLASTIN® & [a3 [44 [415 [45 [15 [15 [45 | 16 | 16 | 16 | 16 | 16, package insert. It is recommended to approximately & [44 [16 [46 [47 [47 [17 [47 | 18 | 48 | 18 | 1.9 | 1.9, include equal number of males and females in the age 15 | 18 | 18 | 18 | 19] 19 | 20 | 20 | 20 | 21 | 21 | 22, group of 18-45 by following the sample collection 16 | 19 | 20 | 20 | 21 | 24 | 22 | 22 | 23 | 23 | 24 | 24, procedures diigently and carefully excluding out donors J 17 | 21) 22 | 22) 2323 | 24) 25) 25 | 26 | 27 | 27, oninterfering medications. 18 | 23 | 23 | 24 | 25 | 26 | 26 | 27 | 28 | 29 | 3.0 | 34, © The individuals included for establishing MNPT should ea 2 2 a es, represent the population routinely tested in the 20 | 26 | 27 | 28 | 29 { 30 | 31 | 32 { 34 | 35 | 3.6 | 3.7, laboratory, with blood collection methods, testing 21 | 28 | 29 | 30 | 32 | 33 | 34 | 35 | 37 | 38 | 39 | 41, techniques and instrumentation routinely used in the 22 | 3.0 | 3.1 | 33 | 34 | 35 | 37 | 38 | 40 | 41 | 43 | 45, specific laboratory. 23 | 32) 33 | 35 | 36/38 | 40| 41] 43 | 45 | 47 | 49, 2.4 3.4 3.6 3.7 3.9 41 42 44 46 48 5.1 5.3, ©. ‘In case of major ‘changes In type. of reagents, 25 [36 | 38 | 40 | 41/43 | 45 | 47 | 50 | 52 | 54 | 57, antcoogan NPT shoul eat bet be vrs fe ee, aleve lle ati sama ace 27 | 40 | 42 | 44 | 47 | 49 | 51 | 54 | 57 | 60 | 63 | 66, 28 | 42 | 45 | 47 | 49 | 52 | 55 | 58 | 61 | 64 | 67 | 7.4, 29 | 44 | 47 | 49 | 52 | 55 | 58 | 61] 64 | 68 | 7.2 | 7.6, 3.0 47 49 5.2 5.5 5.8 6.1 6.5 6.8 7.2 7.6 8.1, Recommended Therapeutic Range for Oral 31 | 49 | 52/55 | 58 | 61 | 65 | 68 | 72 | 7.7 | 84 | 86, Anticoagulant Therapy 32 | 51 | 54 | 57 | 61 | 64 | 68 | 7.2 | 7.7 | 61 | 86 | 9.1, 33 | 53 | 56 | 60 | 64 | 68 | 72 | 76 | 81 | 86 | 91 | 97, Indication [ INR 34 | 5.5 | 59 | 63 | 67 | 74 | 7.5 | 80 | 85 | 9.1 | 9.6 | 10.2, © Prophylaxis of venaus thrombosis 35 | 58 | 62 | 65 | 7.0 | 74 | 7.9 | 84 | 9.0 | 95 | 10.2 | 108, (High risk surgery) 36 | 60 | 64 | 68 | 7.3 | 7.8 | 83 | 88 | 9.4 | 10.0 | 10.7 | 11.4, © Treatmentof venous thrombosis ar [ez [er [ra [re [84 | o7 [sz | e9 [tos [113 [20, 8 5, 6. 7 7.9 5 0 9.7 | 10.3 | 11.1 1.8 | 12.6, Tresimentot pulmonary embotiem 39 | 67 | 72 | 7.7 | 82 | 88 | 94 | 10.1 | 10.8 | 11.6 | 12.4 | 13.3, #; Prevention ofsystemmcembolism 209.0 40 | 7.0 | 75 | 80 | 86 | 92 | 9.8 | 10.6 | 11.3 | 12.1 | 13.0 | 13.9, Se eee ercion 41 | 72 | 77 | 83 | 89 | 96 | 10.3 | 11.0 | 11.8 | 127 | 13.6 | 14.6, (isoreventaietnicenbler) 42 7.5 8.0 8.6 9.2 9.9 10.7 | 11.5 | 12.3 | 13.2 | 14.2 | 15.3, 43 | 77 | 83 | 89 | 96 | 10.3 | 11.1 | 11.9 | 128 | 13.8 | 14.9 | 16.0, © Valvularheart disease 44 | 80 | 86 | 9.2 | 99 | 10.7 | 11.5 | 12.4 | 13.4 | 14.4 | 15.5 | 16.7, Atrial fibrillation 45 | 82 | 89 | 95 | 10.3 | 11.1 | 12.0 | 12.9 | 13.9 | 15.0 | 162 | 17.4, = 46 | 35 | 91 | 9.9 | 106 | 11.5 | 12.4 | 13.4 | 14.4 | 15.6 | 168 | 18.2, ean eran [ee Tieel wears Cae] eso [ieee as, © [icenionotrecamanimyocarda 49 [93 | 100 [408 [11.7 [427 [138 [149 | 164 [17.5 | 18.9 | 20.5, 5.0 | 9.5 | 10.3] 11.2 | 121) 13.1 | 14.2 | 15.4 | 16.7 | 18.1 | 19.6 | 21.3, 51 | 98 | 10.6 | 11.5 | 12.5 | 13.6 | 14.7 | 16.0 | 17.3 | 18.8 | 20.4 | 22.1, 5.2 | 10.1 | 10.9 | 11.9 | 12.9 | 14.0 | 15.2 | 16.5 | 17.9 | 19.4 | 21.1 | 22.9, 53 | 10.3 | 11.2 | 122 | 13.3 | 144 | 15.7 | 17.0 | 18.5 | 20.1 | 219 | 23.8, 5.4 10.6 | 11.5 | 12.5 | 13.7 | 14.9 | 16.2 | 17.6 | 19.1 | 20.8 | 22.6 | 24.6, 55 | 109 | 11.8 | 129 | 14.0 | 15.3 | 16.7 | 18.1 | 19.8 | 21.5 | 23.4 | 25.5, 5.6 11.2 | 12.2 | 13.3 | 14.4 | 15.7 | 17.2 | 18.7 | 20.4 | 22.2 | 24.2 | 26.4, 57 | 11.4 | 125 | 13.6 | 14.8 | 162 | 17.7 | 19.3 | 21.0 | 22.9 | 25.0 | 27.3, 5.8 11.7 | 12.8 | 14.0 | 15.3 | 16.7 | 18.2 | 19.9 | 21.7 | 23.7 | 25.8 | 28.2, 59 | 12.0 | 13.1 | 143 | 15.7 | 17.1 | 187 | 20.4 | 22.3 | 244 | 26.7 | 29.1, 6.0 12.3 | 13.4 | 14.7 | 16.1 | 17.6 | 19.2 | 21.0 | 23.0 | 25.2 | 27.5 | 30.1, , 09t7IVER-01