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BLEEDING DISORDER BY Dr AKS IMS BHU KGMU LKO .pptx

This presentation explores the essential aspects of bleeding disorders, focusing on their definition, classification, and underlying pathology. Bleeding disorders are conditions characterized by abnormal or excessive bleeding, often due to defects in the vascular system, platelet function or number, or coagulation factors. The presentation begins with a clear definition, followed by a classification into three main categories: vascular disorders, platelet disorders, and coagulation disorders. Vascular disorders involve structural defects in blood vessels, such as in hereditary hemorrhagic telangiectasia or scurvy. Platelet disorders are further subdivided into quantitative (e.g., thrombocytopenia) and qualitative (e.g., Glanzmann thrombasthenia) defects, often resulting in mucocutaneous bleeding. Coagulation disorders, such as Hemophilia A, Hemophilia B, and von Willebrand disease, typically present with deep tissue bleeding and hemarthroses.

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Bleeding disorder Dr. Abhishek Singh
CONTENT  Introduction  Disorder of coagulation  Diagnosis of bleeding disorder  Von willebrand disease  Bernard soulier syndrome  Glanzmann’s thrombasthenia  Hemophilia
INTRODUCTION  A group of disorders characterized by defective haemostasis with abnormal bleeding.  These causes are due to : 1. Vessel wall abnormalities 2. Platelet deficiency or dysfunction 3. Derangement of coagulation
DISORDERS OF COAGULATION HEREDITARY  Hemophilia A (factor VIII deficiency)  Hemophilia B(factor IX deficiency)  Von willebrand disease  Fibrinolytic disorder  Bernard-soulier syndrome  Glanzmann’s thrombasthenia  Wiscott-Aldrich syndrome  Chediak-Higashi syndrome  Hereditary hemorrhagic telengectasia  Ehlers-Danlos syndrome  Osteogenesis imperfecta ACQUIRED  Disseminated intravascular coagulation (DIC)  Liver disease  Vit K deficiency  Massive transfusion of stored blood  Acquired inhibitors of coagulation  Heparin or oral anticoagulant therapy  Renal disease  Idiopathic thrombocytopenic purpura  Thrombotic thrombocytopenic purpura  Hemolytic uremic syndrome  Henoch-schonlein purpura
DIAGNOSIS OF INHERITED BLEEDING DISORDERS  HISTORY  CLINICAL EXAMINATION  LABORATORY INVESTIGATIONS
CLINICAL HISTORY  Age of first manifestation  Family history of bleeding  Past history  Spontaneous or after trauma  Site of bleeding  Time of manifestation after injury  H/o defective wound healing  H/o menorrhagia/PPH  Drug history
Clinical examination Platelet disorder Coagulation disorder Site of bleeding Skin, mucous membranes (epistaxis, gum, vaginal, GI tract) Deep in soft tissues (joints, muscle) Petechiae Yes No Ecchymoses("bruises") Small, Superficial Large, Deep Hemarthrosis/ Muscle bleeding Extremely rare Common Bleeding after cut & scratches Yes No Bleeding after surgery or trauma Immediate, usually mild Delayed(12-24hrs), often severe
Laboratory approach 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. Anticoagulation  Most commonly: Trisodium citrate (1:9)
Centrifugation Preparation of Platelet Poor Plasma(PPP)  Centrifuged at 2000 rpm for 10 min.  PPP should be kept at room temp. for PT, lupus anticoagulant & factor VII assay, and at 4 C for other assay.  Platelet count should be <10,000/mm, it is best achieved by double centrifugation.
End-point detection  Refers to clot formation.  Early forming clot is more opaque and easier to detect.  Slowly forming clot appears as fibrin wisp.  Different techniques used in Coagulometer : Electromechanical Photo optical Electrochemical etc.
TESTS FOR BLEEDING DISORDER First line screening Test  Prothrombin Time(PT)  Activated Partial Thromboplastin Time(APTT)  Thrombin Time(TT)  Clotting Time(CT)  Platelet count  Bleeding Time(BT) Second line specific test for factor abnormalities  Mixing study  Factor assay  Fibrinogen assay  Clot solubility test  PFA-100  D-dimer
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 & Method Platelet poor plasma(100µl) + Tissue Thromboplastin(200µl) In Presence of FVII Extrinsic pathway is activated & clot formed Normal Range  11 to 16 seconds INR- PTtest/PTnormal
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. DIC
ACTIVATED PARTIAL THROMBOPLASTIN TIME (aPTT) Significance  Reflects efficiency of Intrinsic Pathway  Sensitive to changes in Factor VIII,IX,XI,XII Principle The test measures the clotting time of plasma after the activation of contact Factors(Kaolin/Silica) and the addition of CaCl2, but without added tissue thromboplastin. Method- Plasma(100µl) + aPTT reagent (100µl) mix & incubate for 3-5 min add prewarm Cacl2(100µl) Clot formation Normal range 26 to 40 seconds (+/-5 of control)
Interpretation Causes of prolonged APTT  1. Deficiency of Factor VIII(Hemophilia A)  2. Deficiency of Factor IX(Hemophilia B)  3. DIC  5. Circulating anticoagulants  6. Liver disease
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. Method – Pt plasma(100µl) + thrombin(200µl) clot formation Normal range 15–19 sec A patient’s TT should be within 2 sec of the control
Interpretation Causes of prolonged TT  Disorders of fibrinogen- Afibrinogenaemia. Hypofibrinogenaemia. Dysfibrinogenaemia.  Liver disease
CLOTTING TIME To measure the time required for a sample of blood to coagulate in vitro under standard conditions. Normal value 5-10 minute The clotting time is prolonged, if there is a marked deficiency in any of the coagulation factors of the extrinsic/ intrinsic pathway.
CAPILLARY METHOD PRINCIPLE-  puncture the skin, blood is taken to a plain capillary tube and stop watch started.  Formation of fibrin strings is noted by breaking the capillary tube at regular intervals.  The time taken for the first appearance of the fibrin string is noted.
OTHER METHOD whole blood clotting Slide method
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)  Time required for bleeding to cease following skin puncture  Assess primary hemostatic defect(vessel wall or platelet) Methods Ivy’s- On volar surface of forearm with lancet 2mm deep under venous pressure of 40mmHg Blood oozing is blotted with filter paper & time taken to stop bleeding is noted. Template- same as ivy’s with 6-9mm depth of incision Duke’s – lobes are punctured Normal 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 VESSELS
SECOND LINE INVESTIGATIONS  Relevant second line investigations are carried out with each of the patterns of abnormalities in first line tests.
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 Prolonged PT/aPTT in pt suspected bleeding disorder Repeat PT/aPTT after adding normal plasma ( patient plasma 50% + normal plasma 50%) PT/aPTT Normal PT/aPTT still prolonged Incubate at 37deg and repeat Due to immediate acting inhibitor PT/aPTT still Normal PT/aPTT prolongs Coagulation factor deficiency Delayed acting inhibitor Perform TGT
Thromboplastin Generation Test(TGT) Sample + Phospholipid + calcium Fxa-V complex (aka prothrombinase) prothrombin thrombin fibrinogen fibrin Estimation of CT Method stage1 Stage2 If clotting time is abnormal, PT and aPTT are done and substitusions studies are carried out to determine deficient factors in plasma and serum Prepared serum +sample+Phospholipid+calcium Adsorbed plasma +Sample+Phospholipid+calcium Estimation of CT Estimation of clotting time Interpretation Coagulation Screen TGT Interpretation PT aPPT N P Plasma defect F VIII deficient P N Plasma defect F V deficient N P Serum defect F IX deficient P N Serum defect F X deficient N P Both defect F XI deficient N P Both defect F XII deficient
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 24 hrs - F XIII PRESENT  Clot dissolves – F XIII ABSENT
Fibrinogen Assay Usually done by CLAUSS TECHNIQUE Principle  Diluted plasma is clotted with a strong thrombin solution (independent of CT)  Thrombin time of diluted plasma is inversely proportion to concentration of fibrinogen. Normal range 1.8 to 3.6 g/l Interpretation  Sensitive to inherited Dysfibrinogenaemia
FDP ASSAY Plasminogen Plasmin Fibrinogen/Fibrin FDP X,Y,D,E 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 Normal range- <10 µg/ml 10-40 µg/ml- Acute myocardial Infarction Acute venous thromboembolism >40 µg/ml- DIC
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 1. Latex agglutination D-dimer assay 2. Ultrasensitive D-dimer assay
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 von willebrand Disease Platelet Function abnormality
Blood aspirated through Capillary into machinee collagen Epinephrin/ ADP Blood passes throughAperture in machine Which is coated with platelet Agonist such as Collagen/epinephrine/ADP Platelet adhesion, activation and Aggregation closes the aperture overtime This time known as closure time is indicator of platelet function Abnormals: Closure time is prolonged in 1. Platelet disorder 2. Von willebrand disease Not prolonged in vascular disorder The PFA 100 ANALYSER
1) PT-N,APTT-N,TT-N,FIBRINOGEN-N,PLATELET-N Interpretation  1. Normal hemostasis  2. Disorders of platelet function  3. Vascular disorders of hemostasis  4. Factor XIII deficiency  5. Mild von Willebrand disease  6. Disorder of fibrinolysis SECOND LINE INVESTIGATIONS  Clot solubility test.  Specific factor assay for suspected factor deficiency(factor XIII).  PFA 100 system.
2) PT—PROLONG,APTT—N,TT—N,FIBRINOGEN—N,PLATELET COUNT-N Interpretation  Factor VII deficiency  Liver disease  Vit K deficiency  Mild deficiency of Factor II, V, X SECOND LINE INVESTIGATIONS  Mixing test.  Factor VII assay.  Liver function test
3) PT—N,APTT—PROLONG,TT—N,FIBRINOGEN— N,PLT COUNT-N Interpretation  1. Congenital deficiency of F VIII, FIX  2. von willebrand disease  3. Circulating anticoagulants- Specific (Anti factor VIII) Non-specific(Antiphospholipid Ab) Second line investigations  Mixing test.  Factor VIII & factor IX assay
4)PT—PROLONG APTT—PROLONG TT-N FIBRINOGEN—N PLT COUNT—N Interpretation 1. Vit k def 2. Liver dis. 3. Rare congenital or acq. deficiency of factor V,X,II 4. Combined factor V+VIII deficiency. Second line investigations 1. Mixing test. 2. Specific factor assay. 3. Liver function test.
5) PT—PROLONG APTT—PROLONG TT—PROLONG FIBRINOGEN—N/ABN PLT COUNT-N Interpretation 1. Hypofibinogenaemia 2. Afibrinogenemia 3. Dysfibrinogenemia 4. Systemic hyperfibrinolysis with increased FDP e.g. DIC 5. 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. DIC 2. Chronic liver disease esp. cirrohis Second line investigations 1. Specific factor assay 2. Peripheral blood smear 3. Bone marrow aspirate
7) PT—PROLONG APTT—PROLONG TT—PROLONG FIBRINOGEN—LOW PLT COUNT—LOW Interpretation  DIC  Acute liver necrosis with DIC Second line investigations  FDP or D-Dimer assay.
Von willebrand disease  Most common congenital bleeding disorder characterized by deficiency or functional defect of vwf. Type1  Most common & AD  Partial quantitative deficiency of vwf  vwf multimers (small, intermediate & large) are mildly deficient Type2  AD & AR  Qualitative defects in vwf with absence of large vwf multimers  4 types- 2A, 2B, 2M & 2N(AR) Type3  AR  Complete deficiency of vwf  Severe deficiency of all forms of vwf multimers
CLASSIFICATION OF vWD ON THE BASIS OF LAB TEST Type1 Type2A Type2B Type2N Type2M Type3 Mild decreased/ Normal multimeric pattern Loss of high and intermedia te vWF multimer Loss of high molecular weight multimer Reduce affinity for FVIII Decreased vWF platelet adhesion without deficiency of HMW multimer Complete deficiency of vWF vWF:RCo/ vWF:Ag >0.6 <0.6 0.5-0.7 <0.6 <0.6 <0.03 - Collagen binding defect Loss of platelet binding activity normal Collagen binding defect -
LABORATORY TEST FOR DIAGNOSIS OF vWD  H/o bleeding, Family history or similar past history  Prolong PTT & BT First line tests 1. Factor VIII binding(vWF : FVIIIB): assessed first by FVIII assay if reduced then by an assay of vwf FVIII binding capacity using ELISA based technique. 2. Ristocetin Cofactor Assay(vWF:RCo) : dilution of pt plasma are tested for their ability to promote plt agglutination in the presence of ristocetin. Automated, latex beads method also available. 3. Collagen binding activity(vWF:CB) : ELISA based, in which well coated with collagen is used to capture vwf. 4. vWF:Ag concentration
SECONDARY TESTS for classification of vWF 1. Ristocetin induced platelet aggregation (RIPA) – Inducing plt aggregation by binding of vwf with GpIb receptor. 2. vWF multimer analysis 3. MOLECULAR ANALYSIS  Sanger analysis  Next generation testing
MULTIMER ANALYSIS
Bernard – soulier ( Giant Platelets) Syndrome  PBS - Giant platelets  Thrombocytopenia Platelet aggregation test  Normal aggregation responses ADP, epinephrine, collagen, and arachidonic acid  Impaired aggregation with ristocetin Platelet aggregation curves in von Willebrand disease and Bernard-Soulier syndrome (absent aggregation with ristocetin, normal aggregation with ADP, epinephrine, and arachidonic acid)
Glanzmann Throbasthenia  PBS shows discrete and small platelets  BT increases  Poor clot retraction Platelet aggregation test  Lack of platelet aggregation in response to all platelet-activating agents (ADP, collagen and epinephrine)  Normal aggregation in response to ristocetin
HEMOPHILIA A(CLASSICAL)  Hereditary deficiency of FVIII  X-linked recessive affecting males Classification  Mild- FVIII level >5% , excess bleeding after minor trauma  Moderate- 1-5%, excess bleeding after mild to moderate trauma  Severe- <1%, frequent and spontaneous hematomas & hemarthroses
Diagnosis  Male with h/o spontaneous bleeding in joints  Positive family history in other male on maternal side  Excess bleeding following circumcision & tooth eruption Screening tests PT-nomal, APTT- Prolonged, CBC Second line test  Factor VIII assay  Genetic testing
HEMOPHILIA B (CHRISTMAS DISEASE)  F IX deficiency Screening Test APTT – Prolonged PT – Normal TT – Normal Second line test  Factor IX Assay  DNA Analysis
REFERENCES  Dacie and Lewis practical hematology, twelfth edition  Shirish M Kawthalkar, essentials of clinical pathology, second edition  Robbins & Cotran, pathologic basis of disease, tenth edition
BLEEDING DISORDER BY Dr AKS IMS BHU KGMU LKO .pptx