Fibrinolysis & Hemostasis Lab Evaluation PDF

Fibrinolysis and Lab Evaluation  Components Plasminogen Plasmin Plasminogen Activators Fibrinogen and Fibrin Inhibitors  Plasminogen Protein derived from liver Inactive in nature Half life: roughl...

Fibrinolysis and Lab Evaluation  Components Plasminogen Plasmin Plasminogen Activators Fibrinogen and Fibrin Inhibitors  Plasminogen Protein derived from liver Inactive in nature Half life: roughly around 2 days Usually circulates in plasma Inactive plasminogen circulates in the plasma until an injury occurs. The activators of plasminogen consist of endogenous and exogenous groups. Plasminogen activation to plasmin is the result of the activity of a number of proteolytic enzymes. These enzymes, the kinases, are referred to as the plasminogen activators.  Plasmin Active form of plasminogen Serine protease that cleaves fibrin Enzyme responsible for the lysis of clot Plasmin is responsible for forming degradation or fibrin split products consisting of intermediate fragments X and Y, and fragments D and E. Plasmin is not normally found in plasma because it is neutralized by an excess of inhibitors. Plasminogen Activator tPA or tissue plasminogen activator; urokinase-PA ; contact phase activators, bacterial products To facilitate the plasminogen activation (plasminogen becoming plasmin) NOTES: Major/Primary activator: TPA or Tissue Plasminogen Activator derived from endothelial cells and once released automatically will convert plasminogen to plasmin. activates plasminogen to become plasmin Initiates fibrinolysis  Plasminogen Activator Urokinase-PA - derived from genitourinary tract - Urinary tract epithelial cells, monocytes,and macrophages contains Urokinase-PA Contact Phase Inhibitors: K, XIa, XIIa. Bacterial components that can destroy a clot by converting plasminogen to become plasmin - Staphylokinase and Streptokinase - enzyme present that can destroy the clot.  Plasminogen Activator Plasminogen activators are found in various sites, such as the vascular endothelium or lysosomal granules, and biological fluids. At least two forms of tissue activators have been described: those that seem related to urokinase, a urinary activator of plasminogen, and those unrelated to urokinase. The activators unrelated to urokinase include thrombin, bacterial products such as streptokinase from beta-hemolytic streptococci, and staphylokinase. Plasma activators of plasminogen include plasma kallikrein, activated plasma thromboplastin antecedents (factor XI), and activated Hageman factor (factor XIIa).  Fibrinogen and Fibrin Act as a substrate Plasmin also binds with Fibrinogen to ensure that no more clot is formed NOTES: Plasmin destroys fibrin, but it also destroys fibrinogen. Why? - Because there are still fibrinogen present even if you destroyed the fibrin to ensure that no more clot is formed. If there’s still fibrinogen = there’s still formation of clot.  Inhibitors TAFI (Thrombin Activatable Fibrinolysis Inhibitor) - TAFI is activated by thrombin in the presence of fibrin. Once activated, it removes carboxy-terminal lysine residues from fibrin and other proteins. This action decreases the binding sites for plasminogen, thereby inhibiting fibrinolysis and helping to stabilize blood clots.  Inhibitors AI-1 (Plasminogen Activator Inhibitor-1) - The primary inhibitor of tissue plasminogen activator (tPA) and urokinase. By inhibiting these plasminogen activators, PAI-1 regulates the conversion of plasminogen to plasmin, thereby controlling the rate of fibrinolysis. Elevated levels of PAI-1 are often associated with thrombotic disorders.  Inhibitors α2-Antiplasmin - α2-antiplasmin is a direct inhibitor of plasmin. It binds to plasmin and inactivates it, preventing excessive fibrinolysis. This helps maintain hemostatic balance by limiting the breakdown of fibrin clots after their formation. Proteins of the Fibrinolysis Pathway  Plasmin will Once there’s a clot, automatically destroy forming Fragment plasminogen automatically attach on your clot. the clot Y and D As it destroys the clot, Fragment Y will be EC will release TPA (with the help of inducers) it will form FDP/FSP further destroyed by (fragments) plasmin TPA binds to the First fragment formed forming Fragment clot is Fragment X D and E (Dead End) TPA will convert plasminogen Fragment X is destroyed to become plasmin again by plasmin Note: Main potent inducer of TPA release: Thrombin Thrombin induces endothelial cells to release TPA Thrombin has the ability to stimulate endothelial cells to release TPA Note: Once FDP is present, they have the ability to inhibit hemostasis. Acts as an anticoagulant. They will be rempoved by WBCs. Primary Fribrinolysis destroys fibrinogen even without fibrin Occurs when you have free plasmin circulating and destroys fibrinogen even when u have no clot/fibrin Secondary Fribrinolysis destroys fibrinogen when there’s fibrin/clot  Plasmin Destroys other clotting factors Factors V, VIII, IX, XI Complement activation Plasmin activates complement by cleaving a portion of C3 Fragments are lysed; or whatever causes possible injury Kinin system - Plasmin activates kinins which induces inflammatory response. Control of Fibrinolysis Protease Inhibitors - the fibrinolytic system is activated when the coagulation cascade is activated, extra fibrin is degraded and eliminated along with some coagulation factors. Enzymes such as plasmin and kallikrein still circulate until they are eliminated by various mechanisms: liver hepatocytes, mononuclear phagocytic cells, or serine protease inhibitors present in the plasma. Serine protease inhibitors attach to various enzymes and inactivate them. Serine Protease Inhibitors 1. α2 – antiplasmin - 4. Plasminogen activator inhibitor-1 (PAI-1) uniquely uniquely designed to cope designed to - Ensures that you don’t convert with copeplasmin. The moreThe with plasmin. plasminogen to plasmin more plasmin plasmin generated, generated, the more 5. Thrombin-activatable fibrinolysis the more alpha-2 alpha-2 the antiplasmin inhibitor (TAFI) - Inhibits further activation of antiplasmin patient the patient consumes. plasminogen to become plasmin consumes. 6. anti-thrombin III 2. α2 – macroglobulin - Ensures fibrinolysis is controlled 3. α1 – antirypsin I. Laboratory Evaluation of Hemostasis Laboratory in hematology section is basically divided into two: 1. Routine Hematology - more of CBC 2. Special Hematology/Coagulation Laboratory - after with hemostasis; most of the time assessing clotting factors. A. Specimen for Hemostasis Blood specimen collection and management are the most vulnerable stages of the hemostasis blood testing process because every stage is manual and thus error-fraught. Contact to glass surface There are certain clotting factors that become activated whenever it comes into contact with a negative charge (Contact Group) Since we are testing for clotting factors, in the end what we want to form is a clot - In vivo: Inside your body, whenever you have an injury you activate your clotting factors so in the end you form a clot. In vitro: If you want to test for coagulation, you activate your clotting factors upon the addition of test reagents and at the end you will form a clot.  Hemolysis Any hemolyzed specimen should be REJECTED. We reject hemolysis because it can cause premature activation of clotting factors - Hemolysis is the release of hemoglobin from ruptured red cells into the plasma. Hemolyzed red cells act like tissue thromboplastin in activating plasma clotting factors.  Temperature: 37°C As much as possible if you test for clotting factors, you need to make sure that the temperature is set at 37°C for clotting factors to be easily activated or for faster activation of proteins. In coagulation testing, you need to incubate the specimen first at 37°C water bath/drybath so you can easily activate clotting factors. When you are testing the specimen (analytical phase), it should be 37°C If you have collected the specimen an your are not yet able to tet for the specimen?  To preserve plasma specimen for a longer period of time, might as well FREEZE it. FFP (Fresh Frozen Plasma) - can be transfused to patients because it contains all proteins of plasma including your clotting factors.  Note: → Capped specimen: can be used within the next 6 hours ◆ Uncapped specimen: can be used within the next 2 hours → Refrigerated specimen = Clotting time falsely shortened To preserve plasma specimen for a longer period of time, might as well FREEZE it. ◆ Uncapped specimen = Clotting time falsely prolonged  Tourniquet application Tourniquet should be applied only within 1 minute. Coagulation (anticoagulant) CITRATE - most ideal anticoagulant to be used in coagulation testing. Because it can preserve all clotting factors. As an anticoagulant, citrate acts against calcium.  PH pH (alkaline) clotting time will be falsely prolonged Specimens should be tested as fast as we can to make sure that all those factors are present. Note: If you will test for coagulation, make sure that all of your To preserve plasma specimen for a longer period of time, might as well FREEZE it. clotting factors are present. Avoid any possible technical errors to have a reliable and accurate results. B.Centrifugation Platelet Poor Plasma (PPP): heavy spin for 10-15 mins Plasma doesn’t contain enough platelets For those tests that do not require platelets In testing for coagulation, we make use of PPP Platelet Rich Plasma (PRP): light spin for 10-15 mins Plasma contains enough number of platelets For those tests that require platelets  C. Drug Administration If you are taking any of these drugs, your coagulation test (clotting time) is expected to be elevated = clotting time will be prolonged. Aspirin All of these May Heparin cause damage on Warfarin/Coumadin - oral anticoagulant blood vessels if Penicillin taken for a long Sulfonamides period of time = Quinine consume Procaine  Warfarin was first used as an anticoagulant in 1952. It protects against venous thromboembolic disease (VTE). Coudmadin Heparin it is a therapy that is is also used during monitored by the prothrombin percutaneous coronary time (PT) assay and reported as intervention (PCI) or cardiac an international normalize ratio surgery that requires (INR). extracorporeal circulation.  II. LABORATORY EVALUATION OF PRIMARY HEMOSTASIS Platelet response: Quantitative - all about numbers Test for vascular and platelet Qualitative - all about function of response platelets  A. Test for Vascular Integrity We don’t have confirmatory test for blood Our blood vessels play critical role vessels, only more of screening procedures. Mostly our blood vessels become in hemostasis. Everything we need fragile/abnormal because of an underlying for hemostasis are disease. (e.g. diabetes). If you treat the underlying disease, you are also helping your vessels to become normal again.  Capillary fragility test 1. Tourniquet or Rumpel Leede or Hess Test positive 2. Suction Cup / Petechiometer Method / Negative pressure technique Pressure Technique Principle: by partially obstructing the venous Principle: employs the use of modified da Silva blood, the capillary pressure is increased. This Melle Instrument. will give rise to extravasation of blood which The pressure is constant at 200mmHg. Suction will be manifested in the form of small cup is applied only for 1 minute. Then check for hemorrhage called petechiae. petechiae. III. QUANTITATIVE PLATELET EVALUATION A. Direct Platelet Count Platelets are counted in a hemacytometer as in erythrocytes and leukocytes. Counting platelets in a hemacytometer using different diluting fluids: In computing for cell count using hemacytometer:  III. QUANTITATIVE PLATELET EVALUATION A. Indirect Platelet Count Platelets are counted in their relationship to red cells on a fixedstained smear. This method is NOT RELIABLE because the results depend upon the distribution of platelets and on the red cell count. Fonions: Using Wright Stain Dameshek: Using Brilliant cresyl blue and Wright stain also In Indirect/Relative Platelet counting, we will always prepare a blood smear 10 OIO fields are counted To compute for platelet count:  C. Automated Platelet Count Red cell must first be removed from whole blood, Lorem ipsum dolor sit amet, consectetur either by sedimentation adipiscing elit, sed do eiusmod or by controlled tempor centrifugation. incididunt ut labore et dolore magna aliqua. Fastest and easiest way of quantifying platelets. Platelet volume should be around: 2-20 fL.  Machines will base it as to the volume of the cells: 1. Voltage-pulse counting 2. Electro-optical counting  IV. THROMBOCYTOPENIA Low platelet count (platelet count of fewer than 100,000/mL) Lorem ipsum dolor sit amet, consectetur More of ending up with bleeding because if platelets is not enough, adipiscing elit, sed do eiusmod tempor youincididunt are at risk in bleeding. ut labore et dolore magna aliqua. Thrombocytopenia is the most common cause of clinically important bleeding. True thrombocytopenia has to be differentiated from the thrombocytopenia artifact that can result from poorly prepared blood films or automated cell counts when platelet clumping or platelet satellitosis is present.  IV. THROMBOCYTOPENIA Thrombocytopenia results from decreased platelet production increased Lorem ipsum dolor sit amet, consectetur destruction, or abnormal distribution of platelets and manifests with small-vessel adipiscing elit, sed do eiusmod tempor bleeding in the skin. incididunt ut labore et dolore magna aliqua. Decreased production of platelets can be attributed to megakaryocyte hypoplasia, ineffective thrombopoiesis, or replacement of marrow by abnormal cells.  IV. THROMBOCYTOPENIA Presumably caused by megakaryocyte suppression also has been reported to follow the Lorem ipsum administration dolordoses of large sit amet, consectetur of estrogen or estrogenic drugs such as diethylstilbestrol. Certain adipiscing elit, sed doantibacterial agents (e.g., chloramphenicol), tranquilizers, eiusmod tempor and anticonvulsants incididunt alsoethave ut labore beenmagna dolore associated with thrombocytopenia caused by bone aliqua. marrow suppression. Is a usual feature of the megaloblastic anemia’s (pernicious anemia, folic acid deficiency, and vitamin B12 deficiency). Quantitative studies indicate that, as with erythrocyte production in these disorders, platelet production is ineffective.  V. THROMBOCYTOSIS MDS: PV, ET Lorem ipsum dolor sit amet, consectetur Reactive: after hemorrhage, IDA, malignancy, epinephrine adipiscing elit, sed do eiusmod tempor administration incididunt ut laboreToo muchmagna et dolore platelet count aliqua. Too much platelet count (typically 450,000/mL.) If platelet count is too much, you are at risk in forming unwanted and unnecessary clots.  V. THROMBOCYTOSIS Is defined as an abnormally high platelet count. The term reactive Lorem ipsum dolor sit amet, consectetur thrombocytosis is used to describe an elevation in the platelet count adipiscing elit, sed do eiusmod tempor secondary to inflammation, trauma, or other underlying and seemingly incididunt ut labore et dolore magna aliqua. unrelated conditions. In reactive thrombocytosis the platelet count is elevated for a limited period and usually does not exceed 800,000/mL, although platelet counts greater than 1 million/mL are occasionally seen.  V. THROMBOCYTOSIS A marked and persistent elevation in the platelet count is a hallmark of Lorem ipsum dolor myeloproliferative sit amet, disorders suchconsectetur as polycythemia vera, chronic myelogenous adipiscing elit, sed do eiusmod tempor leukemia, and myelofibrosis with myeloid metaplasia (or primary incididunt ut labore et dolore magna aliqua. myelofibrosis). In these conditions the platelet count often exceeds 1 million/mL. In the myeloproliferative disorder known as essential thrombocythemia, platelet counts typically exceed 1 million/mL and may reach levels of several million. VI. Qualitative Platelet Evaluation Test for specific platelet function Adhesion Aggregation Clot retraction Factor assays  A. Test for Platelet Adhesiveness First step in forming a plug is platelet adhesion with the help of VWF, GP-Ib, GP-IX. Bleeding Time In vivo Standard skin wound: 2-4 mm depth In vivo platelet function and number Bleeding time would be completely affected if the platelet count is