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Approach to diagnosis of bleeding disorders

This document discusses normal hemostasis and bleeding disorders. It begins by describing the normal mechanisms of hemostasis, including the primary and secondary stages. It then discusses various bleeding disorders that can result from defects in blood vessels, platelets, or coagulation factors. Common laboratory tests for evaluation of hemostasis are also presented, including prothrombin time, activated partial thromboplastin time, thrombin time, and fibrinogen levels. Interpretation of laboratory findings and secondary tests like mixing studies are outlined. Causes of abnormal bleeding including qualitative and quantitative platelet and coagulation factor disorders are explored.

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PRESENTED BY –DR. BISWAJEETA SAHA
Normal hemostasis  Mechanism by which bleeding from an injured vessel is arrested by formation of a thrombus.  Functions-  To maintain the blood in fluid state  To prevent clots in intact vessels  To arrest bleeding in injured vessels  Components-  Blood vessels  Platelets  Plasma coagulation factors  Fibrinolytic system
STAGES OF HEMOSTASIS INJURY VESSEL WALL+PLATELET FORMATION OF PLT PLUG ACTIVATION OF PLASMA COAGULATION FACTORS FORMATION OF STABLE FIBRIN CLOT PRIMARY SECONDARY DISSOLUTION OF FIBRIN CLOT BY FIBRINOLYSIS
STAGES OF HEMOSTASIS PRIMARY  Platelet & vessel wall mediated  Occurs within seconds of injury  Forms Platelet plug  Prevent blood loss from capillary,arterioles and venules SECONDARY  Coagulation factors mediated  Takes several minutes for completion  Forms stable fibrin plug  Prevents blood loss from large vessels
COAGULATION PATHWAYS
FIBRINOLYSIS PLASMINOGEN PLASMIN FIBRIN FDP,D-DIMERS INHIBITORY EFFECTS ON CLOT FORMATION PLASMINOGEN ACTIVATORS KALLIKREIN XIIa PLASMINOGEN ACTIVATORS t-PA UROKINASE STREPTOKINASE
CAUSES OF BLEEDING 1.Vessel wall disorders 2.Platelet disorders: quantitative or functional. 3.Coagulation factor: deficiency or inhibitors. 4.Combination of these.
VASCULAR DISORDERS ACQUIRED CONGENITAL •SENILE PURPURA •VASCULAR PURPURA •HENOCH SCHONLEIN PURPURA •HEREDITARY HEMORRHAGIC TELENGIECTASIA •EHLERS DANLOS SYNDROME
PLATELET ABNORMALITIES QUALITATIVE QUANTITATIVE •THROMBASTHENIA •BERNARD-SOULIER SYNDROME •DRUGS(ASPIRIN,TXA2, INDOMATHACIN •THROMBOCYTOPENIA •THROMBOCYTHEMIA
DISORDERS OF COAGULATION  HEREDITARY  HEMOPHILIA A(factor VIII deficiency)  HEMOPHILIA B(factor IX deficiency)  von WILLEBRAND DISEASE  DISORDERS OF FIBRINOGEN- HEREDITARY AFIBRINOGENAEMIA HYPOFIBRINOGENAEMIA DYSFIBRINOGENAEMIA  ACQUIRED  DISSEMINATED INTRAVASCULAR COAGULATION(DIC)  LIVER DISEASE  VIT K DEFICIENCY  MASSIVE TRANSFUSION OF STORED BLOOD  ACQUIRED INHIBITORS OF COAGULATION  HEPARIN OR ORALANTICOAGULANT THERAPY  RENAL DISEASE
DIAGNOSIS OF BLEEDING DISORDERS  HISTORY  CLINICAL EXAMINATION  LABORATORY INVESTIGATIONS
APPROACH TO A PATIENT OF BLEEDING DIATHESIS , 1.Clinical evaluation: History. Clinical features. 2.Laboratory approach:First line screening tests. Second line specific tests. CLINICAL EVALUATION HISTORY: Age of first manifestation, Family history of bleeding, Spontaneous or after trauma, Time of manifestation after injury, Ease with which bleeding is controlled, Drug history.
INHERITED DISORDERS  Early age of presentation  Family history positive  More sever  Bleeding is the dominant feature  Single factor defect ACQUIRED DISORDERS  Later age of presentation  Family history usually negative  Less sever  Clinical picture is dominated by the underlying disorder e.g.DIC  Multiple hemostatic defect
Findings Disorders of Platelet Disorders of Coagulation i) Petechiae ii) Superficial ecchymosis iii) Deep dissecting hematomas iv) Haemarthrosis v) Bleeding from the superficial cuts & scratches. vi) Positive family history vii) Bleeding from mucous membrane Characteristic Characteristic, usually small & multiple Rare Rare Persistent often profuse Rare Prominent Rare Common, usually large & solitary Characteristic Characteristic Minimal Common May occur
LABORATORY METHODS PREANALYTICAL VARIABLES Collection of Sample  Plastic or polypropylene syringes should be used.  Venous blood is preferred over capillary blood.  Don`t use pressure cuff or use for <1 min.  Blood sample from a indwelling line or catheter should not be used.  Mix thoroughly with anticoagulant by inverting the container several times.  Keep the sample on crushed ice until delivered to the laboratory. Anticoagulation  Most commonly: Trisodium citrate (1:9).  Unacceptable: Oxalate,Heparin, EDTA.
Centrifugation: Preparation of Platelet Poor Plasma(PPP)  Centrifuged at 4000 rev/min at 4 C (for most of the tests) and at room temp. for Platelet function testing, LAC and APCR tests.  PPP should be kept at room temp. for PT,LAC & factor VII assay,and at 4 C for other assay.  Platelet count should be <10,000/mm, it is best achieved by double centrifugation or filtration through a 0.2micron filter.
End-point detection  Detecting clot formation as the end-point depends on the rate of its formation: the shorter the clotting time the more opaque is the clot and the easier to detect.  A slowly forming clot appear as mere fibrin wisp, which are difficult to detect by eye or machine.  Always try to adopt a uniform convention in detecting the end point.  Automated analyser are not always correct, they may be spurious.  The Coagulometer must detect long clotting times reliably and reproducibly. Different techniques used:  Electromechanical  Photo optical  Electrochemical etc.
LABORATORY INVESTIGATIONS TESTS FOR COAGULATION FACTORS I. Prothrombin Time(PT) II. Activated Partial Thromboplastin Time(APTT) III. Thrombin Time(TT) IV. Fibrinogen assay  TESTS FOR PLATELET I. Platelet count II. Bleeding Time(BT)
PROTHROMBIN TIME(PT) Significance  Reflects overall activity of the Extrinsic Pathway.  Most sensitive to changes in Factor V,VII,X.  Lesser to Factor I & II. Principle Platelet poor plasma+Tissue Thromboplastin+Calcium In Presence of F VII Extrinsic pathway is activated & clot formed Normal Range 11 to 16 seconds(with rabbit thromboplastin) 10-12 seconds(with human thromboplastin)
Interpretation Causes of prolonged PT 1. Deficiency of Factor VII,X,V,II,I 2. Vit K deficiency 3. Liver disease esp.Obstructive Jaundice 4. Oral anticoagulants 5. DIC
ACTIVATED PARTIAL THROMBOPLASTIN TIME (APTT) Significance  Reflects efficiency of Intrinsic Pathway.  Sensitive to changes in Factor VIII,IX,XI,XII.  Also sensitive to heparin & circulating anticoagulants. Principle The test measures the clotting time of plasma after the activation of contact Factors(Kaolin/Silica/Ellagic acid) and the addition of phospholipid and CaCl2, but without added tissue thromboplastin. So it indicates the overall efficiency of the Intrinsic pathway Normal range 26 to 40 seconds.
Interpretation Causes of prolonged APTT 1. Deficiency of Factor VIII(Hemophilia A). 2. Deficiency of Factor IX(Hemophilia B). 3. DIC. 4. Heparin therapy. 5. Circulating anticoagulants. 6. Liver disease. 7. Massive transfusion of plasma depleted stored blood.
THROMBIN TIME(TT) Significance Asses the final step of coagulation i.e. conversion of fibrinogen to fibrin in presence of thrombin. Bypasses Extrinsic & Intrinsic pathway. Principle Thrombin is added to plasma and the clotting time is measured.TT is affected by the concentration and reaction of fibrinogen and by the presence of inhibitory substances including fibrinogen/fibrin degradation products(FDPs) and heparin. Normal range A patient’s TT should be within 2 s of the control (i.e. 15–19 sec). Times of 20 s and longer are definitely abnormal.
Interpretation Causes of prolonged TT 1. Disorders of fibrinogen- Afibrinogenaemia. Hypofibrinogenaemia. Dysfibrinogenaemia. 2. Presence of FDP- DIC or Liver disease. 3. Unfractioned heparin therapy. 4. Hypoalbuminaemia. 5. Paraproteinaemia. .
Fibrinogen Assay Usually done by CLAUSS TECHNIQUE. Principle Diluted plasma is clotted with a strong thrombin solution. The plasma must be diluted to give a low level of any inhibitors(e.g. FDPs and heparin). A strong thrombin solution must be used so that the clotting time over a wide range is independent of the thrombin concentration. Normal range 1.8 to 3.6 g/l Interpretation Sensitive to inherited Dysfibrinogenaemia. Insensitive to Heparin unless the level is very high(>0.8µ/µl). High level of FDP(>190µg/ml)may also interfare with the result.
Platelet count & Bleeding Time Platelet count must be done in a suspected bleeding disorder. PBS must be examined for size & staining properties of plt. BLEEDING TIME(BT) Significance Assess Primary Hemostatic defect(vessel wall or platelet). Dependent on adequate functioning of plt. & Bl.Vs. Methods I. Ivy’s II. Duke’s-not recommended. III. Template Range Ivy’s method: 2 to 7 mins. Template method: 2.5 to 9.5 mins.
Interpretation Causes of prolonged BT I. THROMBOCYTOPENIA. II. VWD. III. PLATLET FUNCTION DISORDER. IV. DISORDERS OF BLOOD VESSEL.
DIAGNOSIS OF VESSEL WALL DISORDER  LABORATORY TESTS: TPC,PT, APTT,TT are usually normal. The only test of any use is BT. BT may be normal or increased. HESS` CAPILLARY FRAGILITY TEST: Cuff is wrapped in upper arm and pressure is maintained midway b/w systolic and diastolic BP for 15 minutes. 4 cm below the elbow joint, a circle of 2.5 cm diameter is drawn on the anterior aspect of forearm. Upto 10 new hemorrhagic spots are normal. But >20 new spots are always pathological.  This is positive in increased capillary fragility, ITP.
THROMBOCYTOPENIA
SECOND LINE INVESTIGATIONS Relevant second line investigations are carried out with each of the patterns of abnormalities in first line tests.
1 PT-N APTT-N TT-N FIBRINOGEN-N PLATELET-N Interpretation 1. Normal hemostasis. 2. Disorders of platelet function(cong or acquired). 3. Vascular disorders of hemostasis. 4. Factor XIII deficiency. 5. Mild von Willebrand disease. 6. Disorder of fibrinolysis. 7. Administration of LMWH.
SECOND LINE INVESTIGATIONS  Clot solubility test.  Specific factor assay for suspected factor deficiency(factor XIII).  PFA 100 system.
CLOT SOLUBILITY TEST PRINCIPLE— Fibrin clot in presence of factor XIII & thrombin is stable as A result of cross- linking. But in absence of factor XIII clot dissolves rapidly. Interpretation— CLOT NOT DISSOLVED IN 24HRS- F XIII PRESENT CLOT DISSOLVES – F XIII ABSENT
PLATELET FUNCTION ANALYZER(PFA)100  In vitro system for measuring plt- vwf fuction.  Assesses both plt adhesion & aggregation  More sensitive than BT to asses Primary hemostasis.  The membrane is coated with collagen & epinephrine or collagen & ADP.  It reproduces platelet vwf function under high shear rate.  Time req. for closure of full aperture is closure time  Normal closure time: 1 to 3 mins.  Interpretation:- Thrombocytopenia. von Willebrand Disease. Plt. Function abnormality.
2 PT—LONG APTT—N TT—N FIBRINOGEN—N PLATELET COUNT-N Interpretation 1. Factor VII deficiency. 2. Liver disease. 3. Vit K deficiency. 4. At the start of oral anticoagulant therapy. 5. Mild deficiency of Factor II, V, X.
SECOND LINE INVESTIGATIONS 1. Mixing test. 2. Factor VII assay. 3. Liver function test.
3 PT—N APTT—LONG TT—N FIBRINOGEN—N PLT COUNT--N Interpretation 1. Congenital deficiency of F VIII, FIX,prekallikrein or HMWK. 2. von Willebrand disease. 3. Heparin-either pt. Is on t/t or contaminated sample. 4. Circulating anticoagulants- Specific (Anti factor VIII). Non-specific(Antiphospholipid Ab). Second line investigations 1. Mixing test. 2. Factor VIII & factor ix assay.
Mixing study CORRECTION TEST USING PT or APTT PRINCIPLE  Unexplained prolongation of PT or APTT can be investigated with simple correction test by mixing the pt`s plasma with normal plasma.  Correction (should be within few seconds) indicates a possible factor deficiency, whereas failure to correct suggests the presence of an inhibitor. METHOD  Perform a PT and/or APTT on control, patient`s,and a 50:50 mixture of the control and pt`s plasma.
PT IS PROLONGED TREAT WITH HEPARINASE PT NORMALIZES HEPARIN IS THE CAUSE PT REMAINS PROLONGED PERFORM MIXING STUDY 1:1 MIX OF PATIENT’S AND NORMAL PLASMA PT NORMALIZES PT INITIALLY NORMALIZES,SUBSEQUENTLY PROLONGED PT REMAINS PROLONGED FACTOR I,II,V,VII,X DEFICIENCY FACTOR V INHIBITOR INHIBITORS
APTT IS PROLONGED TREAT WITH HEPARINASE APTT NORMALIZES HEPARIN IS THE CAUSE APTT REMAINS PROLONGED PERFORM MIXING STUDY 1:1 MIX OF PATIENT’S AND NORMAL PLASMA APTT NORMALIZES APTT INITIALLY NORMALIZES,SUBSEQUENTLY PROLONGED APTT REMAINS PROLONGED FACTOR VII,IX,X,XI,XII DEFICIENCY FACTOR VII INHIBITOR INHIBITORS(LAC)
CORRECTION TEST USING TT PRINCIPLE  The tests use certain physiochemical properties of reagents to bind to inhibitors or abnormal molecules and normalise the prolonged TT. INTERPRETATION TT of test plasma corrected with Interpretation Saline Normal plasma ProtaminSO4 Toluidine blue No Yes No No Deficiency No Variable No Yes Dysfibrinogenemia of liver disease No Variable Yes No High level of FDP
DETECTION OF CIRCULATING ANTICOAGULANT PRINCIPLE  Circulating anticoagulants or inhibitors may act immediately or be time dependent.  To detect both type of inhibitors, normal plasma and test plasma samples are tested immediately after mixing and also after incubation together at 37C for 120 min.
4 PT—LONG APTT—LONG TT-N FIBRINOGEN—N PLT COUNT—N Interpretation 1. Vit k def. 2. On oral anticoagulants. 3. Liver dis. 4. Rare congenital or acq. Deficiency of factor v,x,ii . 5. Combined factor V+VIII deficiency. Second line investigations 1. Mixing test. 2. Specific factor assay. 3. Liver function test.
5 PT—LONG APTT—LONG TT—LONG FIBRINOGEN—N/ABNORMAL PLT COUNT--N Interpretation 1. Unfractionated heparin 2. Hypofibinogenaemia 3. Afibrinogenemia 4. Dysfibrinogenemia 5. Systemic hyperfibrinolysis with increased FDP e.g. DIC 6. Some cases of liver disease. Second line investigations 1. Replitase or ancord time 2. D-dimer level.
REPTILASE OR ANCORD TIME  REPTILASE & ANCORD are purified enzymes from snake.  May be used to replace Thrombin in TT test.  Snake venom is not inhibited by heparin-  Normal time for clotting in presence of Heparin.  Clotting time will be prolonged in presence of raised FDPs/decreased Fibrinogen.
6 PT-LONG APTT-LONG TT-N FIBRINOGEN-N/LOW PLT COUNT-LOW Interpretation 1. Massive transfusion of stored/plasma depleted blood. 2. DIC. 3. Chronic liver disease esp.cirrohis. Second line investigations 1. Specific factor assay. 2. Peripheral blood smear. 3. Bone marrow aspirate.
7 PT—N APTT—N TT—N FIBRINOGEN—N PLT COUNT—LOW Interpretation 1. Thrombocytopenia. 2. Heparin use. Second line investigations 1. Peripheral blood smear. 2. Bone marrow aspirate.
8 PT—LONG APTT—LONG TT—LONG FIBRINOGEN—LOW PLT COUNT—LOW Interpretation 1. DIC 2. Acute liver necrosis with DIC. Second line investigations FDP or D-Dimer assay.
FDP ASSAY Plasminogen Plasmin Fibrinogen/ FDP X,Y,D,E Fibrin Principle A Suspension of latex particles sensitized with specific Ab to FDP fragments D & E. Suspension mixed on a glass slide with a dilution of test serum. Agglutination indicates presence of FDPs.
Interpretation of FDP Assessment Agglutination with 1in 5 dilution:-FDP >10µg/ml Agglutination with 1 in 20 dilution:-FDP>40µg/ml Normal range- <10 µg/ml 10-40 µg/ml- Acute myocardial Infarction. Acute venous thromboembolism. Acute pneumonia. >40 µg/ml- DIC. Thrombolytic therapy with Streptokinase .
D-DIMER ASSAY Identical to FDP except latex particles are coated with a Monoclonal Antibody specifically directed against Fibrin D-dimer in human. Normal range <200mg/l.
DIAGNOSIS OF vWD . TESTS DONE 1.Factor VIII:C concentration, 2.vWF:Ag concentration, 3.Ristocetin Cofactor Assay(vWF:RCo), 4.Collagen binding activity(vWF:CB), 5.Multimeric analysis of vWF:Ag. vWF:RCo Assay: It measures the ability of the Ristocetin(developed as an antibiotic) to promote the interaction b/w vWF & Platelet membrane gp Ib-IX. Multivalent ristocetin-dependent binding of vWF creates interplatelet bridges leading to platelet clumps(agglutination) which is measured by an Aggregometer. vWF:CB Assay: Is complementary(rather than alternative) to vWF:RCo assay. It is an ELISA based system, giving a greater precision.
DIAGNOSIS OF DIC • Finding a prolonged PT and APTT with a reduced TPC & Fibrinogen level is usually DIC, until proven otherwise. • The diagnosis of DIC is made by the presence of a confirmatory test that shows the simultaneous presence of thrombin and plasmin formation. D-dimer Assay: • It is the best test for diagnosing DIC. Value >2000ng/ml is consistent with DIC. • It is the confirmatory test that shows that both thrombin and plasmin have been formed. • It measures plasmin-cleaved, insoluble,cross-linked fibrin that originally arose from thrombin cleavage of fibrinogen. • D-dimer assay is characteristic but not pathognomonic for DIC. FDP Assay: • FDPs only indicate plasmin-cleaved fibrinogen, soluble fibrin or insoluble fibrin. • It does not indicate plasmin-cleaved,insoluble,cross-linked fibrin. Fibrin monomer assay: • Fibrin monomer is the large molecular mass of fibrinogen that remains after release of fibrinopeptide A & B. • Elevated in early stage, but can be absent in sever DIC. Hence unreliable.