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Investigation of bleeding disorder || bleeding disorder

  2. Blood vessels Platelets Plasma coagulation factors InhibitorsFibrinolytic system COMPONENTS OF HEMOSTASIS
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  4. Bachelor of Chinese Medicine, The University of Hong Kong Hemostatic response
  5. ABNORMAL BLEEDING This may result from:  Vascular disorders Thrombocytopenia  Platelet Disorder Defective platelet function  Defective coagulation  Defective Fibrinolysis • The pattern of bleeding is relatively predictable depending on the aetiology. • Vascular and platelet disorders tend to be associated with bleeding from mucous membranes and into the skin whereas in coagulation disorders the bleeding is often into joints or soft tissue 5
  6. • GOOD CLINICAL HISTORY/DATA  hereditary or acquired?
  7. Liver disease Malignancies Renal disease Poor nutrition Medical history
  8. • HISTORY OF MEDICATIONS intake of analgesics ,  anticoagulant therapy??? History of blood transfusion
  9. BLEEDING DISORDERS Inherited bleeding disorders Acquired bleeding disorders • Hemophilia A and B • von Willebrand’s disease • Platelet disorders • Fibrinolytic disorders • Other factor deficiencies • Liver disease • Vitamin K deficiency/warfarin overdose • DIC • Thrombocytopenia • Renal Failure 9
  10. VASCULAR BLEEDING DISORDERS • The vascular disorders are a heterogeneous group of conditions characterized by easy bruising and spontaneous bleeding from the small vessels. • The underlying abnormality is either in the vessels themselves or in the perivascular connective tissues. • Most cases of bleeding caused by vascular defects alone are not severe. • IN THESE CONDITIONS THE STANDARD SCREENING TESTS ARE NORMAL. • THE BLEEDING TIME IS USUALLY NORMAL AND THE OTHER TESTS OF HAEMOSTASIS ARE ALSO NORMAL. 10
  11. INVESTIGATIONS OF THROMBOCYTOPENIA • Platelets have proved more difficult to count than either red or white cells. Historically techniques for platelet enumeration fall into four groups: • Direct methods by hemocytometry • Indirect methods in which red cells were counted directly and their proportion to platelets determined in a stained blood film; • Semi-automated methods in which counts were performed electronically on platelet -rich plasma; • Fully automated methods 11
  12. Assessment of the platelet count is essential as numerical deficiency or defect in their function may lead to bleeding. The normal platelet count at all ages is 150–400 × 10 /L of whole blood. The four main recent analytical procedures for platelet counting are Manual counting using phase contrast microscopy.  Impedance analysis,  Optical light scatter/fluorescence analysis using various commercially available analyzers,  And immunoplatelet counting by flowcytometry (anti CD41 and anti CD61) 9 12
  13. MANUAL COUNTING USING PHASE CONTRAST MICROSCOPY In 1953 the manual phase contrast microscopy method was developed, which enabled platelets to be easily discriminated from lysed red cells within a counting chamber or hemocytometer . It may still be necessary to use manual counting methods in the routine laboratory if the platelet count is low or if there are atypical platelets present in the sample. This method became the International Council for Standardization in Haematology (ICSH) reference method in 1988 13
  14. THE FUNCTIONS OF PLATELETS In homeostasis include; Maintenance of vascular integrity Initial arrest of bleeding by platelet plug formation Stabilization of haemostatic plug by contributing to fibrin formation Platelet must be adequate in number and function to participate optimally in homeostasis. 14
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  16. SCREENING TESTS WORK UP FOR PLATELET FUNCTION TEST 16 Bleeding Time Finger Prick Smear PBF (shape and size examination) Clot Retraction Test CONFIRMATORY TESTS Platelet Adhesion Test Platelet Aggregation Test PF-3 Availability Test
  17. CONFIRMATORY PFTs 17 PLATELET ADHESION TEST • Platelet adhesiveness is determined in vitro by measuring the retention of platelets by a column of glass beads. • It is a complex phenomenon depending not only on the adhesion property of platelets but also the ADP released from RBCs, the conc. of plasma fibrinogen , the viscosity of the blood , the rate of flow through the column , the number & size of the glass beads and the type of plastic used to construct to the column.
  18. Platelet Aggregation Tests Principle:- • Platelet functions are determined either in whole blood or platelet rich plasma. • It is proportional to changes either in turbidity or impedance. • The lesser the turbidity or the greater the impedance, better the platelets in responding to various aggregating agents. • The aggregation rxns can be measured and plotted using one of several instruments that measure and record the optical density. Method:- Platelet Rich Plasma 18
  19. 1.Base line 2.Addition of agonist 3. Shape change 4.Primary wave 5.Secondary wave
  20. THE ‘CLOTTING SCREEN’ Basic tests of coagulation are often performed with no specific diagnosis in mind and in the absence of any clinical indication of a haemostatic disorder. • OUR CURRENT PRACTICE IS TO PERFORM • PT, • APTT, • FIBRINOGEN ASSAY • Platelet Count • D-Dimer 22
  21. PROTHROMBIN TIME Principle; • The PT test measures the clotting time of recalcified plasma in the presence of an optimal concentration of tissue extract (thromboplastin) and indicates the overall efficiency of the extrinsic clotting system. • It is sensitive to changes in factors V, VII, and X, and less so to factor II (prothrombin). 23
  22. Activated partial thromboplastin time Principle • The test measures the clotting time of plasma after the activation of contact factors and the addition of phospholipid and CaCl2, but without added tissue thromboplastin, and so indicates the overall efficiency of the intrinsic pathway. • To standardize the activation of contact factors, the plasma is first preincubated for a set period with a contact activator such as kaolin, silica or ellagic acid. • During this phase of the test, factor XIIa is produced, which cleaves factor XI to factor XIa, but coagulation does not proceed beyond this in the absence of calcium. • After recalcification, factor XIa activates factor IX and coagulation follows. A standardized phospholipid is provided to allow the test to be performed on PPP. • The test depends not only on the contact factors and on factors VIII and IX but also on the reactions with factors X, V, prothrombin. • It is also sensitive to the presence of circulating anticoagulants (inhibitors) and heparin 24
  23. THROMBIN CLOTTING TIME PRINCIPLE • The thrombin time reflects the reaction between thrombin and fibrinogen. Thrombin Fibrinogen Fibrin • It is prolonged when the fibrinogen level is very low (less than 1.0 g/l); in the presence of heparin and heparin-like substances; in the presence of other inhibitors, such as fibrin degradation products (FDPs); and when fibrinogen is qualitatively abnormal (dysfibrinogenemia), including both congenital and acquired. NORMAL RANGE • A patient’s TT should be within the control (i.e. 15–19 s). Times of 20 s and longer are definitely abnormal. 25
  24. MEASUREMENT OF FIBRINOGEN CONCENTRATION • Numerous methods of determining fibrinogen concentration have been devised, including clotting, immunological, physical and nephelometric techniques, and all tend to give slightly different results, because of the heterogeneous nature of plasma fibrinogen. 26
  25. CLAUSS ASSAY PRINCIPLE • Dilutions of standard normal plasma with known fibrinogen content are prepared in glyoxaline buffer. • The clotting time is measured after the addition of thrombin, and a graph is constructed. • The clotting time is proportional to the concentration of fibrinogen, and the 1/10 dilution is taken to represent the value in the standard preparation. • The test plasma is diluted 1/10, and the result read from the standard line. • Normal Range: 2.0 – 4.0 g/l 27
  26. SECOND-LINE INVESTIGATIONS If all the first-line investigations are normal in a patient who continues to bleed from the site of an injury or after surgery (or has a history of such bleeding), there are several possible diagnoses:  von Willebrand disease (VWD) in which the factor VIII is not sufficiently low to cause prolongation of the APTT. This is quite common in mild cases  A mild coagulation disorder that is below the sensitivity of the routine tests to detect or that has been masked by the administration of blood products or by high levels of other factors: for example, mild factor VIII deficiency of 30 iu/dl  Factor XIII deficiency  Bleeding from a severely damaged vessel or vessels with normal haemostasis  A disorder of fibrinolysis such as antiplasmin or PAI-1 deficiency  Administration of LMWH. 28
  27. INVESTIGATION OF A BLEEDING DISORDER RESULTING FROM A COAGULATION FACTOR DEFICIENCY • When the screening tests indicate that an individual has a coagulation defect, the plasma concentration of the coagulation factors should be assayed. • Such assays establish the diagnosis of the deficiency or defect and they assess its severity; they also can be used to monitor replacement therapy and to detect the carrier state in families in which one or more members are affected by a congenital bleeding disorder. 29
  28. Correction Tests Using the PT or APTT • Unexplained prolongation of the PT or APTT can be investigated with simple correction tests by mixing the patient’s plasma with normal plasma. • Correction indicates a possible factor deficiency, whereas failure to correct suggests the presence of an inhibitor, but interpretation should initially be cautious 30
  29. Assays Based on PT & APTT • Essentially, the assay consists of comparing the ability of dilutions of a standard or reference plasma and test plasma to correct the prothrombin time & APTT of a plasma known to be totally deficient in the clotting factor being measured. • In a factor VIII assay, the plasma is deficient in factor VIII. • Clotting factors INTRINSIC, EXTRINSIC & COMMON PATHWAY may be assayed in a similar way. • Plot results on semi log graph paper, with percent factor activity on the x axis and seconds on the y axis. Draw a best-fit line. 31
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  31. Urea clot solubility test PRINCIPLE  Fibrin clots formed in the presence of factor XIII and thrombin are stable (as a result of crosslinking) for at least 1 h in 5 mol/l urea, whereas clots formed in the absence of factor XIII dissolve rapidly.  0.2 ml patient plasma + 0.2 ml thrombin.  Incubate at 37°C for 20 min and add 3 ml urea solution (5 molar) for 24 hour.  In absence of factor XIII, the clot is dissolved. Test for factor XIII
  32. VON WILLEBRAND FACTOR  VWF is produced in endothelial cells and megakaryocytes. It has two roles It promotes platelet adhesion to subendothelium. it is the carrier molecule for factor VIII, protecting it from premature destruction. Th e latter property explains the reduced factor VIII levels found in VWD.  To diagnosis of von willebrand disesae(VWD),probably the most common congenital bleeding disorders, requires a number of special test at the lab level. among them vwf-ag determination is essential and must be performed on every pateint to reach proper diagnosis. depending upon the lab findings. 34
  33. VW -types • Type I – most frequent, quantitave defect (decreased VWf) • Type II – qualitative defect (abnormal VWf ) • Type III – severe, rare, (absence of VWf )
  34. Laboratory findings  The PFA - 100 test .This has largely replaced the bleeding time test Factor VIII levels are often low. If low, a factor VIII/VWF binding assay is performed.  The APTT may be prolonged.  VWF levels are usually low.  There is defective platelet aggregation by patient plasma in the presence of ristocetin (VWF: Rco). Aggregation to other agents (adenosine diphosphate (ADP), thrombin or adrenaline) is usually normal. Collagen - binding function (VWF: CB) is usually reduced.  The platelet count is normal except for type 2B disease (where it is low). 36
  35. In the PFA - 100 test • Citrated blood is aspirated through a capillary tube onto a membrane coated with collagen/ADP or collagen/adrenaline. • Blood flow is maintained. Platelets begin to adhere and aggregate, primarily via VWF interactions with GPIb and GPIIb/IIIa, resulting in occlusion of the aperture. • Full platelet function tests and VWF screening may be required to exclude abnormal platelet function, even if the PFA - 100 test is normal. 37
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  37. Treatment Approaches to the Bleeding Patient • Red blood cells • Platelet transfusions • Fresh frozen plasma • Cryoprecipitate • Amicar • DDAVP • Recombinant Human factor VIIa
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