Laboratory investigations in coagulation disorders

Laboratory investigations in coagulation disorders

• 1.
  LABORATORY INVESTIGATIONS IN COAGULATION DISORDERS PRESENTER :DR. HAJRA K. MEHDI MODERATOR : DR. S. DAS
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  CONTENTS  INTRODUCTION  SPECIMENCOLLECTION AND PROCESSING  LABORATORY INVESTIGATIONS OF COAGULATION SYSTEM  SCREENING TESTS  CONFIRMATORY TESTS  TESTS TO EVALUATE CIRCULATING INHIBITORS  LAB INVESTIGATIONS FOR FIBRINOLYTIC SYSTEM  AUTOMATION  SUMMARY  REFERENCES
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  INTRODUCTION  Hemostasis isa process by which the body prevents loss of blood from the vascular system and maintains it in a fluid state.  Involves a series of complex and regulated events linking platelets , vascular endothelial cells and coagulation factors. Hemostasis includes  Primary hemostasis  Secondary hemostasis
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  Coagulation factors Phospholipids CA2+ Factor XIIa, XIa Kallikrein T-PA U-PA FIBRINOLYSISCLOTTING ACTIVATORS INHIBITORS AT ProteinC, S TFPI α2Macroglobulin α1Antitrypsin C1 Inactivators Heparin Cofactor PAI- 1,2,3 α2- antiplasmin α2- macroglobulin TAFI THE COAGULATION SYSTEM
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  COAGULATION DISORDERS CONGENITAL DISORDERSACQUIRED DISORDERS Hemophilia A and B Disseminated intravascular coagulation Von Willebrand’s disease Deficiency of vitamin K dependent factors Hypofibrinogenemia Liver disease Afibrinogenemia Massive transfusion syndrome Dysfibrinogenemia Heparin therapy Inhibitors of coagulation-fVIII inhibitor, APLA
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  SPECIMEN COLLECTION AND PROCESSING Whole blood is aseptically collected by venipuncture.  Anticoagulant of choice is 3.2% sodium citrate.  Volume of anticoagulant necessary for a 5 ml blood sample adjusted according to the hematocrit value.  One may also choose to keep the anticoagulant volume of 0.5 ml constant and adjust the volume of added blood according to the hematocrit.  The volume of blood to be added (to 0.5 ml of 0.109M citrate) is calculated from the formula: (60 - hematocrit)/100 × 4.5
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  HAEMATOCRIT (%) VOLUME OF ANTICOAGULANT (ML) VOLUMEOF BLOOD (ML) 25 – 55 0.5 4.5 20 0.7 4.3 60 0.4 4.6 70 0.25 4.75 80 0.2 4.8
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  SPECIMEN PROCESSING  Citratedwhole blood is centrifuged to obtain plasma for coagulation testing. Platelet Poor Plasma:  Blood sample is centrifuged at a minimum of 1700rpm for at least 10 minutes (2500rpm for 15min) preferably at 4°C in a refrigerated centrifuge.  Open the samples at RT, separate and store the plasma sample at 4°C as quickly as possible.  When RT >25°C then labile coagulation proteins may be affected, a refrigerated (4°C) centrifuge should be used.  Platelet poor plasma <10× 109/l
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  Samples for ImmediateTesting:  Samples should be tested within 4 hours of sample collection when possible.  Longer storage should be avoided for screening tests and clotting factor assays.  Samples for factor assays should be stored at 4°C testing.  Samples for screening tests and assay of factor VII should be maintained at room temperature to avoid the possibility of cold activation. SPECIMEN PROCESSING
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  Deep Freezing ofPlasma:  Samples can be frozen for testing at a later stage.  Storage at -70°C or lower is preferable (better) but at -35°C is adequate for most tests.  Storage at -20°C is usually inadequate.  Double centrifugation should be used if samples are frozen prior to analysis for lupus anticoagulant.  Freezing and thawing is best avoided before APTT determinations, since results obtained by some can be affected. SPECIMEN PROCESSING
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  1. SCREENING TESTS Prothrombin time  Activated partial thromboplastin time  Thrombin time 2. CONFIRMATORY TESTS FOR FACTOR ABNORMALITIES  Reptilase time  Mixing tests.  Fibrinogen assay (modified Claus Assay) LABORATORY INVESTIGATIONS OF COAGULATION SYSTEM
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   Platelet neutralizingprocedure  Lupus anticoagulant & antiphospholipid antibodies TESTS TO EVALUATE CIRCULATING INHIBITORS
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   Detection ofFDPs using latex agglutination method  D-dimer test  Euglobin clot lysis LAB INVESTIGATIONS OF FIBRINOLYTIC SYSTEM
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  SCREENING TESTS
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  PROTHROMBIN TIME Principle:  Measuresthe time for fibrin formation.  Reflects the overall efficiency of the extrinsic system.  Sensitive to changes in factor V, VII and X, and less so to factor II (prothrombin).  It is also unsuitable for detecting minor changes in fibrinogen level  May be abnormal if the fibrinogen level is very low or if there is an inhibitor present.  The sensitivity of the test is influenced by the reagent and technique used and it is important to establish a reference range locally.
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  REAGENTS - Platelet poorplasma from the patient. - Control plasma sample. - Thromboplastin (this may contain calcium chloride). - Calcium chloride – 0.025 mol/lit (only required if thromboplastin reagent does not contain calcium).
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  INR  INR –International Normalized Ratio.  Was established by WHO and the international committee on thrombosis and hemostasis on reporting the result of PT.  INR = (PT of Patient/Control PT)ISI  Anticoagulant therapy control = 2.0-3.0 ISI  International Sensitivity Index.  Given by manufacturer.  Reflects reagent sensitivity to different Vitamin K dependent factor Deficiency.
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  RESULTS Results can beexpressed in 1. Mean of duplicate recordings in secs 2. INR = (PT of Patient/Control PT)ISI NORMAL RANGE  With rabbit thromboplastin : 11-16 secs  With recombinant human thromboplastin : 10- 12 secs.
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  CAUSES OF PROLONGEDPT  Administration of oral anticoagulants drugs (vitamin K anticoagulants).  Liver diseases  Vitamin K deficiency  DIC  Previously underdiagnosed factor VII, X, V or prothrombin deficiency or defect.
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  ACTIVATED PARTIAL THROMBOPLASTIN TIME Principle: Measures the clotting time of plasma after the activation of contact factors but without added tissue thromboplastin.  Indicates the overall efficiency of the intrinsic pathway.  It is also sensitive to circulating anticoagulants (inhibitors) and heparin.  Depends on contact factors, factors VIII, IX as well as X, V, prothrombin and fibrinogen.
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  REAGENTS  Platelet poorplasma  Kaolin 0.5gm in 100ml barbitone buffered saline, pH 7.4  Other can be used like silica, celite or elagic acid can be used.  Phospholipid  Calcium chloride 0.025M
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  METHOD 1. Mix equalvolumes of phospholipid reagent and the kaolin kaolin suspension and leave in a glass tube in the water bath at 37 degree. 2. Place 0.1ml of plasma into a new glass tube. 3. Add 0.2ml of kaolin-phospholipid solution, mix the contents and start the stopwatch simultaneously. 4. Leave at 37 degrees for 10mins with occasional shaking. 5. At exactly 10mins, add 0.1ml of pre-warmed CaCl2 and start a second stopwatch. 6. Record the time taken for the mixture to clot. 7. Repeat the test on both the patient and the control plasma atleast once.
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  INTERPRETATION Normal range: 30– 40 seconds  A prolonged APTT with a normal PT indicates a possible deficiency of factor VIII, IX, XI, XII, high molecular weight kininogen, prekallikrein or the presence of an inhibitor.
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   Example: APTTcontrol : 35 sec test : 60 sec  If 50:50 mix : 42 sec – this is a good correction, i.e. factor deficiency 60 – 42 = 18, 60 – 35 = 25, 25/2 = 12.5, 18 > 12.5  If 50:50 mix : 52 sec – this is a poor correction, i.e. probably an inhibitor is present. 60 – 52 = 8, 8 < 12.5
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  CAUSES OF PROLONGEDAPTT  DIC  Liver diseases  Massive transfusion with stored blood.  Administration of heparin or contamination with heparin.  A circulating anticoagulant.  Deficiency of coagulation factor other than factor VII.
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  THROMBIN TIME Principle:  Thethrombin time reflects the reaction between thrombin and fibrinogen. Thrombin Fibrinogen Fibrin  Thrombin is added to the plasma and the clotting time is measured.  The Thrombin time is affected by the concentration and reaction of fibrinogen, and by the presence of inhibitory substances, including fibrinogen/FDP and heparin.
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  REAGENTS  Platelet poorplasma.  Thrombin solution which induces clotting of normal plasma in about 15 seconds. Normal range: 15 – 19 secs
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  METHOD – MANUAL 1.Pipette 0.2 ml test and control plasma into separate glass clotting tubes. 2. Warm to 37oC. 3. Add 0.1 ml thrombin solution to each tube. 4. Start stopwatch for each tube. 5. Measure the clotting time and observe the nature of clot. 6. Repeat procedure for patient and control plasma.
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  CAUSES OF PROLONGEDTT  Hypofibrinogenaemia as found in DIC and, more rarely, in a congenital defect or deficiency.  Raised concentrations of FDP, as encountered in DIC or liver disease.  Extreme prolongation of the TT is nearly always a result of the presence of unfractionated heparin.  Dysfibrinogenaemia, either inherited or acquired, in liver disease or in neonates.  Hypoalbuminaemia.  Paraproteinemia.
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  CONFIRMATORY TESTS
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  REPTILASE OR ANCRODTIME  Reptilase: Bothrops atrox  Ancrod: Agkistrodon rhodostoma Principle  It is a serine protease, a thrombin like enzyme.  Cleaves fibrinopeptide A from fibrinogen (thrombin cleaves both A &B). Method  Addition of reptilase to platelet poor plasma initiates clot formation.  Clot formation detected by optical/ electromechanical methods.
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  CAUSES OF PROLONGEDRT/AT Reference interval- 18-22 seconds  Hypofibrinogenemia  Dysfibrinogenemia  FDPs  Other circulating anticoagulants
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  DIFFERENTIATION OF CONDITIONS ASSOCIATEDWITH PROLONGED TT USING RT Condition Thrombin time Reptilase time Heparin contamination Prolonged Normal Dysfibrinogenemia Prolonged More Prolonged Presence of FDP More Prolonged Prolonged
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  MIXING TESTS  Plasmasamples found to have abnormal screening tests, i.e. PT/APTT, can be further investigated to determine the cause of the abnormality.  To differentiate a factor deficiency from the presence of a circulating inhibitor.  Information on the nature of the defect can usually be obtained by mixing experiments.
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  The agents whichcan be used for mixing tests are as follows  Normal plasma  Aged plasma  Adsorbed plasma  Factor VIII deficient plasma  Factor IX deficient plasma
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  INTERPRETATION OF RESULTSFROM MIXED TESTS Defect in Tested Plasma APTT Aged plasma Adsorbed plasma Normal plasma VIII Abnormal No correction Correction Correction IX Abnormal Correction No correction Correction XI/XII Abnormal Correction Correction Correction Inhibitor Abnormal No correction No correction No correction
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  Defect in plasma APTT Aged plasma Adsorbed plasma Normal plasma II AbnormalCorrection No Correction Correction V Abnormal No Correction Correction Correction VII Normal Correction No Correction Correction X Abnormal Correction No Correction Correction
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  FIBRINOGEN (MODIFIED CLAUSS ASSAY) Principle Dilutions of a standard normal plasma with known fibrinogen content are prepared in glyoxaline buffer.  The clotting time is measured after the addition of thrombin.  A graph of clotting times against the fibrinogen concentration is constructed.
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  Reagents  Reference plasmawith known fibrinogen concentration  Thrombin >30u/ml (concentration may vary according source)  Imidazole buffer (glyoxaline) pH 7.35  Normal range : 1.5 – 3.5 g/L
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  TESTS TO EVALUATECIRCULATING INHIBITORS
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  CLOTTING FACTOR INHIBITORSCREEN BASED ON PT/APTT Principle  Coagulation inhibitors affecting the PT/APTT may be immediate acting or time dependent.  Test plasma containing an immediate-acting inhibitor will, when mixed with normal plasma, show little or no correction of the clotting time.  Plasma with time-dependent inhibitors, on the other hand, requires a period of incubation with normal plasma before inhibitors can be detected
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  LUPUS ANTICOAGULANT AND ANTIPHOSPHOLIPIDANTIBODIES  The criteria for presence of lupus anticoagulants are as follows: 1. Prolongation of a phospholipid-dependent coagulation test; 2. Evidence of an inhibitor demonstrated by mixing studies; 3. Phospholipid dependency of the inhibitor.
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  PROCEDURE : 3STEP  1st step- Clotting assay like APTT screening tests like Dilute Russell's Viper Venom Time (DRVVT) Low-phospholipid APTT  2nd step- Mixing studies This distinguishes a factor deficiency from presence of inhibitor.  3rd step - Accomplished by either reducing or adding an excess of phospholipid to the test system This is done using Platelet neutralizing procedure.
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   The confirmatorytest cut off is the value corresponding to the mean of the individual percentage corrections as follows:  % correction = [(screen CT – Confirm CT)/Screen CT] x 100  The result is confirmation of LA if the % correction is above the cutoff value obtained.
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  LAB INVESTIGATIONS OF FIBRINOLYTICSYSTEM
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  DETECTION OF FIBRINDEGRADATION PRODUCTS USING A LATEX AGGLUTINATION METHOD Principle  A suspension of latex particles is sensitized with specific antibodies to purified FDP.  The suspension is mixed on a glass slide with a dilution of the serum to be tested.  Aggregation indicates presence of FDPs.  Hence, by testing different dilutions of the unknown sample , a semi quantitative assay can be performed.
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  Conditions with therange between 10 – 40μ g /ml are:  Acute venous thromboembolism  Acute MI  Severe pneumonia Very high levels are seen in:  DIC  Thrombolytic therapy with streptokinase
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  D-DIMER TEST Principle  D-dimeris a marker specific for plasmin degradation of fibrin.  Its an FDP generated from FXIIIa crosslinked fibrin.  Latex beads are coated with a monoclonal antibody directed specifically against fibrin D dimer in human plasma or serum. Reagents  Latex suspension  Dilution buffer  Positive & negative control
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  METHOD  Undiluted plasmais mixed with one drop of latex suspension on a glass slide. Gently rock the slide for the length of time mentioned in the kit.  If agglutination is observe then dilute the plasma until agglutination can no longer be seen.
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  INTERPRETATION  Agglutination withundiluted plasma - D-dimer > 200mg/L  The D-dimer value can be quantified by multiplying reciprocal of the highest dilution showing a positive result by 200 to give a value in mg/L. NORMAL RANGE : <200 mg/L.
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  EUGLOBIN LYSIS TEST Principle When plasma is diluted & acidified, the precipitate (euglobin) that forms contains plasminogen activator, plasminogen & fibrinogen.  Plasminogen inhibitors are left out in the solution.  The precipitate is redissolved, fibrinogen is clotted with thrombin & the time for clot lysis is measured. Reagents  Acetic acid – 0.01%  Bovine thrombin – 10 U/ml  Fresh PPP from patient & control  Glyoxaline buffer
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   NORMAL RANGE: 90 – 240 Min  Abnormal values Increased Decreased Pregnancy Disseminated malignancies Obese Cirrhosis. Post- op MI
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  AUTOMATION IN COAGULATION STUDIES Introduced in early 20th century.  Initial instruments were based on detection of clot formation: Coaguloviscometer Nephelometer Photoelectric technique
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   The 2principles of clot based detection currently used are electromechanical and optical density. Electromechanical instruments  BBL Fibrosystem  American Bioproducts STArt 4 Clot Detection Instrument Optical Density Instruments  Organon Teknika Coag-A-Mate XM  Chromogenic / Clot detection instruments
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  ADVANTAGES  Faster andmore accurate  In case of an abnormal result can cross check  Can avoid the problem of inter-observer variation DISADVANTAGES  Availability of reagents  High cost of reagents  Strict Quality Control  Availability of well trained technicians
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  QUALITY CONTROL
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  REFERENCES 1. McKenzie ShirlynB. Clinical laboratory Hematology; 2nd edition,2004. 2. Wintrobe’s hematology,10th edition,2nd volume 3. Text book of Hematology- Tejinder Singh 4. Robbins and Cotrans;Pathologic basis of diseases, 8th edition 5. Dacie and Lewis Practical Hematology 11th edition.