Hematology Lab - 11 Fibrinolysis and Mixing Studies

FIBRINOLYSIS AND MIXING STUDIES OUTLINE • • Mixing Studies and Fibrinolysis o Mixing Studies o Pathways: Review o Sample Problems in Mixing Studies o Fibrinolysis o Plasmin o Laboratory Evaluation of Fibrinolysis PATHWAYS: REVIEW MIXING STUDIES AND FIBRINOLYSIS MIXING STUDIES • Used to detect specific coagulation factor deficiencies. o No longer performed due to availability of tests that determines concentration of specific coagulation factor o Considered as an obsolete test • PT & PTT - Tests that alert to factor deficiency o PT - examine extrinsic & common pathway o PTT/APTT - examine intrinsic & common pathway • Also known as substitution test. • The patient’s deficient plasma is diluted 1:1 with a plasma or serum substitute and the APTT or PT is repeated. • A correction if the original prolonged APTT or PT indicates that the deficient factor has been added to the patient’s plasma by substitution solutions as follow: • PT and PTT can only tell whether the problem is in the extrinsic, intrinsic, or common pathway, but cannot pinpoint which specific coagulation factor is deficient. • Specimen – Plasma (Na Citrate/Light blue top) Group Factors Vit K Dependent group 2, 7, 9, 10 Fibrinogen group 1, 5, 8, 13 Contact 11, 12, P, HMWK • Vit. K group - synthetized by liver in the presence of Vit K./Adsorbable factors (BaSO4) o Requires vitamin K o Absent in barium sulfate adsorbed plasma o Activation requires presence of calcium • Fibrinogen group - labile group; readily utilized o Completely consumed in clotting process o Absent in serum • Contact – First factors activated in intrinsic pathway. • Correction Reagents Reagent/Substitute Present Absent solutions Fresh plasma All factors None Aged plasma All factors, except 5, 8 5, 8 Adsorbed plasma All factors, except 2, 7, 9, 10 2, 7, 9, 10 Fresh serum All factor, except 1, 5, 8 1, 5, 8 Aged serum All factors, except 1, 2, 5, 8 1, 2, 5, 8 o Fresh plasma - not 8 hours after collection o Aged plasma - standing for 8 hours after colelction o Adsorbed plasma - plasma treated with BaSO4 ▪ BaSO4 - adsorbs prothrombin group o Plasma – Liquid portion of anti-coagulated blood. o Serum – Liquid portion of coagulated blood. o WE DO NOT MEASURE FACTORS 3, 4, 6, 13 o Factor 3 – Tissue factor (Always present as long as there’s tissue) o Factor 4 – Calcium, always present Factor 6 – Nonexistent Factor 13 – Clot forms at the activation of fibrinogen. There will be visible clot as long as theres fibrin, even if it is not stable. Deficiency is not detected Procedure o If PT & PTT is prolonged, add a substitution reagent. Retest for PT and/or PTT depending on what is originally abnormal. o If there is correction (normalized PT/PTT), that means the substitute contains the missing/deficient factor from the patient’s plasma. o o HEMATOLOGY LABORATORY Pathway Coagulation Factor present Extrinsic 7 Intrinsic 8, 9, 11, 12 Common 1, 2, 5, 10 • Monitoring o Deficiency in Extrinsic - abnormal PT, normal PTT o Deficiency in Intrinsic - abnormal A/PTT, normal PT o Deficiency in Common - abnormal PT & PTT SAMPLE PROBLEMS IN MIXING STUDIES • • • • • Normal Values o APTT = 25 - 35 secs o PT = 10 - 14 secs Notes o If the factor is absent in a substitute that is not able to correct the PT/PTT, then the factors that are absent are possibly the factors deficient Number 1: o APTT – 42.7s (prolonged) o PT – 13.4s (normal) o Corrected by Fresh plasma o Not corrected by Fresh serum (absent: 1, 5, 8, 13) o If not corrected by fresh plasma, there might be presence of circulating antioagulants. (Lupus anticoagulant - LA) o Prolonged APTT - problem with intrinsic pathway o Possible deficiencies - 12, 11, 9, 8:c o The px has Factor 8 deficiency (hemophilia A) Number 2: o APTT – 53.4s (prolonged) o PT – 22.1s (prolonged) o Not corrected by adsorbed plasma (absent: 2, 7, 9, 10) o Corrected by aged serum (1, 5, 8, 2) o Both are prolonged - Problem with common pathway o Possible deficiencies - 10, 5. 2, 1 o The px has Factor 10 deficiency Number 3: o APTT – 39.8s (prolonged) o PT – 28.1s (prolonged) o Corrected by aged adsorbed plasma (2, 7, 9, 10; 5, 8) o Not corrected by aged serum and aged plasma (absent: 1, 2, 5, 8) o Common pathway ▪ Stypven time - abnormal o Possible deficiencies - 10, 5, 2, 1 o Factor I deficiency (afibrinogenemia, hypofibrinogenemia, dysfibrinogenemia) ▪ Stypven time - abnormal ▪ TT - abnormal (TT is specific for CFI) ▪ (Factor V deficiency) - original answer • • Number 4: o APTT - normal o PT - prolonged o Corrected by aged plasma (1, 5, 8, 13) o Not corrected by adsorbed plasma (absent: 2, 7, 9, 10) o Abnormality in extrinsic pathway o Possible deficiency: 7 o Factor VII deficiency Number 5: o APTT - abnormal o PT - normal o History of menorrhagia (excessive menstrual bleeding) o Corrected by adsorbed plasma (2, 7, 9, 10) o Corrected by aged serum (1, 5, 8, 2) o Not corrected by o Abnormality in intrinsic pathway o Possible deficiency - 12, 11, 9, 8 ▪ Not a mixed deficiency ▪ Factor 11 & 12 deficiency - check history ▪ Factor XII deficiency - negative bleeding history ▪ Factor XI deficiency - positive bleeding history o Factor XI deficiency ▪ Due to menorrhagia FIBRINOLYSIS: PROCESS 1. 2. 3. 4. 5. 6. 7. 8. FIBRINOLYSIS • • • • • • Dissolution of fibrin clot Once the injury is completely healed, there would be no need for the clot to exist. o Continuous existence of clot will cause thrombosis ▪ Cause tissue hypoxia (lack of oxygen), ischemia (lack of blood flow), accumulation of waste Fibrinolysis is a final phase of hemostasis that ensures that the clot will cease to exist after the damage in the vessel wall has been fixed. Revolves around the activity of Plasmin. Usually a localized phenomenon. Thrombosis may cause o Ischemia or ischaemia is a restriction in blood supply to tissues, causing a shortage of oxygen that is needed for cellular metabolism o Tissue hypoxia o Necrosis COMPONENTS OF THE PLASMA FIBRINOLYTIC SYSTEM • • PLASMIN • • Actions: o Digests fibrinogen – Will lead to formation of fibrinogen degradation products (FDPs/FSPs) ▪ Primary fibrinolysis o Digests fibrin – Will lead to formation of fibrin degradation products (FDPs/FSPs), production of D-dimer ▪ Secondary fibrinolysis o Digests stable fibrin clot – Will lead to formation of DDimers o Digests Factors II and XII – Will prevent clot formation o Digests Factors V and VIII – Will prevent clot formation o These will result to a phenomenon called hypocoagulability → hemorrhagic diasthesis hemorrhagic diathesis – Fancy name for increased bleeding tendencies Plasminogen and tPA from endothelial cells in blood vessels tPA will be activated by thrombin tPA and Plasminogen will be attracted to clot or fibrin tPA and Plasminogen will combine to clot or fibrin Plasmin will be formed Fibrin will be cleaved into two forming fragment X which is still clottable Fragment X will be cleaved forming fragment D and fragment Y Fragment D is not clottable and fragment Y is clottable which will be cleaved into fragment D and E. These are called dead end degradation Products • Plasminogen Activators o Endogenous ▪ Tissue plasminogen activator (t-PA) • Comes from the injured tissues. ▪ Urokinase (u-PA) - From Kidneys ▪ XIa, XIIa, Kallikrein o Exogenous ▪ Streptokinase - Certain strains of beta hemolytic streptococcus (S. pyogenes) ▪ Acyl-plasminogen streptokinase activator complex (APSAC) • Human plasminogen + Streptokinase Plasminogen Inhibitors o Alpha2-antiplasmin - primary inhibitor of plasmin. o Alpha2-macroglobulin - inactivates plasmin not inhibited by alpha2-antiplasmin o Thrombomodulin - released by damaged endothelial cells. o Plasminogen activator inhibitor 1 and 2 (PAI-1 & PAI-2) - Comes from endothelial cells and platelets ▪ PAI-1 – Secreted by the endothelium, platelets and adipose tissues ▪ PAI-2 – Secreted by the placenta Principles: o Increased Activator = Increased fibrinolytic activity = Increased risk of thrombosis o Increased Inhibitor = Decreased fibrinolytic activity = Increased risk of bleeding ACTIVATORS • Tissue Plasminogen Activator o Main converter of Plasminogen to plasmin o It is secreted by Endothelial cells o It is bound with thrombus and PAI-1 • Urokinase Plasminogen Activator o It is secreted by Kidneys, UT epithelial cells, Monocytes and Macrophages o Circulates in plasma o uPA- Same function as tPA. LABORATORY EVALUATION OF FIBRINOLYSIS • INHIBITORS • • • • Plasminogen Activator Inhibitor 1 o Principal inhibitor of plasminogen activation (in tPA and uPA) o Produced by ECs, Megakaryocytes, smooth muscle cells, Fibroblasts, Monocytes, Adipocytes, Hepatocytes o PAI-1 has high concentration than tPA o PAI-1 Deficiency → Chronic Mild Bleeding o It also functions as Acute Phase Reactant o Acute phase reactants - inflammation markers (Increases during inflammation) α2-Antiplasmin o Primary inhibitor of Free Plasmin o Synthesized in the liver a2-Macroglobulin o Inhibits plasmin and prekallikrein Thrombin Activatable Fibrinolysis Inhibitor o Also synthesized in the liver o Functions as anti-fibrinolytic enzyme o Inhibits the binding of TPA and plasminogen to fibrin o Also plays a role in inflammation and wound healing WHOLE BLOOD CLOT LYSIS TIME • • • • • • PAI-1 – Inhibits conversion of plasminogen to plasmin a2-antiplasmin – Inhibits free plasmin Thrombin Activatable Fibrinolysis Inhibitor – Inhibits the binding of TPA and plasminogen to fibrin • • • • • • Protein C and S - Inhibitors of factor V and VIII Antithrombin III - Inhibitor of thrombin Heparin - Amplifies the activity of antithrombin III C1 inhibitor - Inactivate/inhibitors kallikrein (activated form of PK) Alpha-1-antitrypsin - Inhibits thrombin, Xa, XIa Thrombin + Thrombomodulin -> Thrombin Thrombomodulin Complex AT3 = Also factors Ixa and Xa Same as whole blood clot lysis time but blood is diluted. More sensitive NV: Lysis of clot after 6-10 hours EUGLOBULIN LYSIS TIME • • • • OTHER INHIBITORS OF COAGULATION • • • • Allow to clot in a test tube @ 37°C Place in water bath to 48 hours Form a clot Normal: clot is intact in 24 hours Abnormal: clot is lysed before 24 hours Clot is lysed before 28 hours, meaning there is excessive fibrinolysis or fibrinolytic system DILUTE WHOLE BLOOD CLOT LYSIS TIME • • • • Determination of Lysis Time o Whole Blood Clot Lysis Time o Dilute Whole Blood Clot Lysis Time o Plasma Clot Lysis Time o Euglobulin Lysis Time Plasma is treated with an acid to precipitate certain clotting factors in a complex called euglobin fraction. Euglobin fraction will be allowed to clot at 37degC and incubated. Normal Value: o Lysis in about 300 mins or longer - normal. o Lysis in 120 mins – increased lytic activity. o Lysis in 60 mins or less – strong lysis. Precipitate – More chances of contact between the substances. Euglobin fraction – Composed of Fibrinogen, plasminogen, all plasminogen activators, and traces of a2antiplasmin. QUANTITATIVE ASSAY OF FIBRIN/FIBRINOGEN DEGRADATION PRODUCTS • • Based on agglutination (Hemagglutination, Staphylococcal, Latex agglutination) Uses monoclonal antibodies specific for FDPS D-DIMER TEST • • • • Superior test; Same principle as FPD TEST but more sensitive Uses monoclonal antibodies specific for D-Dimers Kind of serologic test – Labeled antibody that is specific for FDPs or D-Dimers