Hematology 2 Lecture - Laboratory Diagnosis of Secondary Hemostasis PDF

EMILIO AGUINALDO COLLEGE Gov. D. Mangubat Ave., Brgy. Burol Main, City of Dasmariñas, Cavite 4114, Philippines Tel. Nos. (046) 416-4339/41 www.eac.edu.ph SCHOOL OF MEDICAL TECHNOLOGY HEMATOLOGY 2 LECTURE Laboratory Diagnosis...

EMILIO AGUINALDO COLLEGE Gov. D. Mangubat Ave., Brgy. Burol Main, City of Dasmariñas, Cavite 4114, Philippines Tel. Nos. (046) 416-4339/41 www.eac.edu.ph SCHOOL OF MEDICAL TECHNOLOGY HEMATOLOGY 2 LECTURE Laboratory Diagnosis of Secondary Considerations: Hemostasis Hematology 2 Lecture CLOT BASED SCREENING TESTS Lee and White (1913) WB Coagulation Time Test o first laboratory procedure designed to assess coagulation o first in vitro clot procedure but now obsolete o Principle: time interval from the initiation of clotting to visible clot formation → reflects coagulopathy Standard Clot Based Coagulation Tests: o PT, PTT, fibrinogen assay, and thrombin clotting time (TCT) ▪ uses the principle of Lee-White test 1. Prothrombin Time (PT) Principle: PPP + PT reagent = measure length of time required to form a fibrin clot is measured in seconds via optical or electromechanical sensors. o activates the extrinsic and common pathway Prolonged: Factor Deficiency (VII, X, V, II, I) o Coumadin therapy: suppress VII, X, I Specimen: citrate fresh plasma (PPP) Reagents: 2. Partial Thromboplastin Time (APTT/PTT) o PT reagent: tissue factor suspended in Principle: PPP + PPT reagent = measure length of phospholipids mixed with a buffered 0.025 M time required to form a fibrin clot is measured in solution of calcium chloride. seconds via optical or electromechanical sensors. RI: 12.6 to 14.6 seconds o activates the intrinsic and common pathway Clinical applications: Prolonged: o monitor oral anticoagulant therapy o Factor Deficiency (pre-K; HMWK; o hemorrhagic disorders: factors XII, XI, IX, VIII, X II, I) ▪ DIC ▪ Heparin therapy: bind to AT that ▪ liver disease neutralizes IIa, Xa, & other ▪ vitamin K deficiency coagulation serine proteases. o Presence of lupus anticoagulant: neutralizes reagent phospholipids o Presence of anti-factor VIII antibody Specimen: citrate fresh plasma (PPP) Reagents: o PTT reagent: phospholipid (partial thromboplastin or cephalin) + negatively charged particulate activator such as silica, kaolin, ellagic acid, or celite in suspension 0.025 M calcium chloride RI: 26 to 38 seconds Prepared by: Rosa Mikaela Dumago HEMA2 LEC- Finals  EMILIO AGUINALDO COLLEGE Gov. D. Mangubat Ave., Brgy. Burol Main, City of Dasmariñas, Cavite 4114, Philippines Tel. Nos. (046) 416-4339/41 www.eac.edu.ph SCHOOL OF MEDICAL TECHNOLOGY HEMATOLOGY 2 LECTURE 3. Thrombin Clotting Time (TCT) Principle: bovine thrombin reagent NIH units/mL cleaves fibrinopeptides A and B from plasma fibrinogen to form a detectable fibrin polymer o activates the thrombin and tests for the polymerization of fibrinogen Prolonged: o Factor Deficiency (II,I) ▪ Heparin therapy: bind to AT that neutralizes IIa o Presence of fibrin degradation products RI: 15 to 20 seconds Clinical applications: o Prolonged: Clinical applications: ▪ dysfibrinogenemia, hypofibrino- o Prolonged: genemia, & afibrinogenemia ▪ Hemorrhagic Disorder Before a prolonged TCT Hemophilia A, Hemophilia B, may be considered as and Rosenthal syndrome evidence of diminished or Vitamin K deficiency: factor abnormal fibrinogen, the II, IX, X (less sensitive) presence of antithrombotic substances (heparin, FDPs, ▪ Thrombosis or paraproteins) must be DIC: due to consumption of ruled out. procoagulants ▪ presence of antithrombotic o confirmed using the materials (FDPs, paraproteins, or D-dimer, platelet heparin) count, and eryth- rocyte morphology ▪ presence of the oral direct thrombin inhibitor dabigatran ▪ Autoimmune Disorders TCT provides binary or anti-factor VIII or anti-factor qualitative evidence for IX, lupus anticoagulant dabigatran markedly prolong TCT: presence of dabigatran normal TCT: rules out dabigatran. plasma-diluted TCT: TCT modification, provides a quantitative measure of dabigatran when used with calibrators of specific drug concentrations. Prepared by: Rosa Mikaela Dumago HEMA2 LEC- Finals  EMILIO AGUINALDO COLLEGE Gov. D. Mangubat Ave., Brgy. Burol Main, City of Dasmariñas, Cavite 4114, Philippines Tel. Nos. (046) 416-4339/41 www.eac.edu.ph SCHOOL OF MEDICAL TECHNOLOGY HEMATOLOGY 2 LECTURE 4. Venom Activated Assays a. Reptilase Time Principle: Thrombin-like enzyme (isolated from the venom of Bothrops atrox) catalyzes the conversion of fibrinogen to fibrin. In contrast to thrombin, this enzyme cleaves only fibrinopeptide A from the ends of the fibrinogen molecule Clinical Applications: o not affected by heparin and factor XIII deficiency o Prolonged: ▪ dysfibrinogenemia & hypofibrinogenemia ▪ presence of FDPs and paraproteins b. Russell Viper Venom Test Principle: Russell viper venom (RVV) from the (Daboia russelii viper) triggers coagulation at the level of factor X once used as an alternative to the PT named the Stypven time, but is now obsolete Clinical Applications: o prolonged buffer-diluted RVV: LAC ▪ routinely employed to detect LAC 5. Mixing Study (PT/PTT) Principle: Mixing studies may either be immediate (PT or aPTT measured immediately after mixing) or incubated (aPTT measured after mix incubated for 1 – 2 hours at 37 °C) Patient plasma and normal pooled plasma are mixed using 1:1 ratio. clotting time corrects after mixing → Factor Deficiency clotting time does not correct after mixing → presence of inhibitor Prepared by: Rosa Mikaela Dumago HEMA2 LEC- Finals  EMILIO AGUINALDO COLLEGE Gov. D. Mangubat Ave., Brgy. Burol Main, City of Dasmariñas, Cavite 4114, Philippines Tel. Nos. (046) 416-4339/41 www.eac.edu.ph SCHOOL OF MEDICAL TECHNOLOGY HEMATOLOGY 2 LECTURE COAGULATION FACTOR ASSAYS 2. Single-Factor Assays Using the PTT prolonged PTT 1. Fibrinogen Assay normal PT Principle: normal TCT o Clauss method, a modification of the TCT o recommended procedure for estimating o factor VIII deficiency (hemophilia A) fibrinogen function o factor IX deficiency (hemophilia B) o factor XI deficiency (Rosenthal syndrome) o PPP is diluted 1:10 with Owren buffer + bovine thrombin at 50 to 100 NIH units/mL → convert fibrinogen to fibrin polymer prolonged PTT o interval to clot formation (∝) is proportional concentration of functional fibrinogen prolonged PT normal TCT o diluting the PPP minimizes the antithrombotic effects of heparin, FDPs, and paraproteins o factor V deficiency o heparin levels less than 0.6 units/mL; o factor X deficiency o FDP levels less than 100 mg/dL ▪ Does not affect the fibrinogen assay 3. Factor XIII Assay provided the fibrinogen is > 150 chromogenic factor XIII assay (Technochrom mg/dL Factor XIII) RI: 220 to 498 mg/dL Principle: quantitation of factor XIII activity is based on the measurement of ammonia released during an in-vitro transglutaminase reaction Clinical applications: o Prolonged: plasma factor XIII activated by reagent thrombin ▪ Hypofibrinogenemia: fibrinogen than NADPH consumption is measured by the decrease 498 mg/dL of absorbance at 340 nm. ▪ Congenital afibrinogenemia: seen in absorbance (∝) is proportional to factor XIII prolonged clotting times & activity associated anatomic hemorrhage o normal PTT o normal PTT ▪ Dysfibrinogenemia: resemble o normal TCT hypofibrinogenemia by the Clauss method, as abnormal fibrinogen o normal platelet count species are hydrolyzed more slowly by o normal fibrinogen level thrombin than is normal fibrinogen. ▪ PT-derived fibrinogen assay: normal results for dysfibrinogenemia Prepared by: Rosa Mikaela Dumago HEMA2 LEC- Finals  EMILIO AGUINALDO COLLEGE Gov. D. Mangubat Ave., Brgy. Burol Main, City of Dasmariñas, Cavite 4114, Philippines Tel. Nos. (046) 416-4339/41 www.eac.edu.ph SCHOOL OF MEDICAL TECHNOLOGY HEMATOLOGY 2 LECTURE Laboratory Diagnosis of Fibrinolysis Hematology 2 Lecture D-Dimer Immunoassay Overview Fibrin Cross-Linking: During coagulation, fibrin polymers are cross-linked by Factor XIIIa, binding plasma, plasminogen and tissue plasminogen activator (TPA). Plasmin Activation: Over time, TPA activates nearby plasminogen to form plasmin, which cleaves fibrin into fibrin degradation products (FDPs) such as D, E, X, Y, and D-dimer. After the activation process, the fibrin clot is needed to be dissolved, that’s why there is a balance between coagulation and fibrinolysis. Fibrin Monomer is the conversion of Fibrinogen by our FIIa, known as Thrombin. Fibrin Monomer can still be stabilized into cross-link fibrin with the action of Fibrin Stabilizing Factor (FXIII). But before FSF can do that, it still needs to be activated by the preceding enzyme, Thrombin. Primary Digester of Product: Plasmin Plasmin has this small portion in the blood called Free Plasmin which means we already have an activated plasmin in our blood flow. But the free plasmin isn’t enough to dissolve a product. Free Plasmin has one zymogen called Plasminogen → activated by the TPA/UPA → Plasmin Fibrin Monomer is the fibrin clot with the action of Thrombin → degraded → turns into FDPS. Cross-link Fibrin has the action of FSF → degraded → turns into F D-d (D-dimer). D-Dimer Immunoassay and Quantitative D-Dimer Assay Prepared by: Rosa Mikaela Dumago HEMA2 LEC- Finals  EMILIO AGUINALDO COLLEGE Gov. D. Mangubat Ave., Brgy. Burol Main, City of Dasmariñas, Cavite 4114, Philippines Tel. Nos. (046) 416-4339/41 www.eac.edu.ph SCHOOL OF MEDICAL TECHNOLOGY HEMATOLOGY 2 LECTURE FDPs & D-Dimer: assay’s monoclonal antibody target. FDPs are fragments from the original fibrinogen structure. D-Dimers are a subset of cross-linked D domains, indicative Fibrin Degradation Product (FDP) of Factor XIIIa cross-linking in fibrin production. Immunoassay Clinical Significance: Overview of FDP Immunoassay: Replacement by D-Dimer Assay: The FDP immunoassay D-Dimer assays are used to detect active fibrinolysis, is largely replaced by automated quantitative D-dimer indirectly indication thrombosis. assays for routine testing. Detection Method: Qualitative visible agglutination Normal D-dimer/FDP concentration: 200 ng/mL 1. Thrombo-WellCoTest Elevated levels suggest acute/chronic disseminated intravascular coagulation (DIC), Systemic Fibrinolysis, Deep Method: A slide agglutination test where: Vein Thrombosis (DVT), Pulmonary Embolism, or predict o Sample Mixing: One drop of sample is mixed Stroke. with one drop of latex suspension on a slide. o Latex Particles: Polystyrene latex particles in FDPs, including D-dimer, are also present after thrombolytic buffered saline are coated with polyclonal therapy. antibodies specific for D and E fragments. o Detection Sensitivity: Calibrated to detect FDPs Principle of the Quantitative D-Dimer Assay: at concentrations of 2 mg/mL or higher. Assay Format: Plasma D-dimer immunoassays are widely Result: Visible agglutination indicates the presence of available, with automated quantitative options providing FDPs at or above the threshold concentration. results in approximately 30 minutes. Sample Requirements: Mechanism: o Performed on urine and serum. o Microlatex particles in buffered saline are coated o Samples are collected in special tubes to promote with monoclonal anti-D-dimer antibodies. clotting and prevent in vitro fibrinolysis. o D-dimer in patient plasma causes agglutination of coated particles. Clinical Application: o Resultant turbidity is measured using turbidimetric Sensitivity: Limited sensitivity at low concentrations. or nephelometric methods. Usage: Primarily used in diagnosis and monitoring of disseminated intravascular coagulation (DIC). Plasminogen Assay and Chromogenic Substrate Assay Plasminogen Overview: Definition: Plasminogen is an inactive precursor of plasmin, a trypsin-like enzyme essential to the fibrinolytic pathway, produced in the liver. Activation Mechanism: When plasminogen binds to fibrin, it is activated to plasmsin by tissue Sensitivity: plasminogen activator (TPA). Varies based on the monoclonal antibody’s avidity and detection method. Most assays detect concentrations as low as 10 ng/mL. Reporting Units: Results may be reported as D-dimer units (DDU) or Fibrinogen Equivalent Units (FEU) depending on the Prepared by: Rosa Mikaela Dumago HEMA2 LEC- Finals  EMILIO AGUINALDO COLLEGE Gov. D. Mangubat Ave., Brgy. Burol Main, City of Dasmariñas, Cavite 4114, Philippines Tel. Nos. (046) 416-4339/41 www.eac.edu.ph SCHOOL OF MEDICAL TECHNOLOGY HEMATOLOGY 2 LECTURE Bound Plasmin: Degrades fibrin directly at the site. Substrate Options: Chromogenic and fluorogenic Free Plasmin: Rapidly inactivated by circulation a2- substrates are available for plasminogen measurement. antiplasmin. Reference Interval: Typical plasminogen reference Clinical Relevance: interval: 5 to 13.5 mg/dL. Hyperfibrinolysis: Excessive fibrinolysis, often due to Conditions Associated with Decreased trauma, inflammation, bone trauma, or surgery, can lead to Plasminogen: hemorrhage. Hypofibrinolysis: Reduced fibrinolysis occurs with depleted Thrombolytic Therapy: Plasminogen is consumed. TPA or Plasminogen levels, or excess plasminogen activator Disseminated Intravascular Coagulation (DIC): inhibitor-1 (PAI-1), potentially leading to thrombosis. Depletion occurs due to active fibrinolysis. Hepatitis and Cancer: Reduced production or increased Plasminogen Deficiencies: consumption of plasminogen. Hereditary Deficiency: Can lead to lower plasminogen Congenital: Linked with familial thrombosis. levels, associated with an increased risk of thrombosis. Acquired: Seen in conditions like DIC and acute Systemic Fibrinolysis: Elevated levels during extensive promyelocytic leukemia. fibrinolysis. Acute Inflammation: Plasminogen levels may arise as Thrombolytic Therapy: Ineffective when plasminogen levels part of the inflammatory response. are low. Pregnancy: Increased plasminogen levels, which may be associated with hemorrhage risk. Principle of the Plasminogen Chromogenic Substrate Assay: Purpose: Assesses plasminogen by mimicking its natural Tissue Plasminogen Activator (TPA) activation to plasmin. Assay Activation Mechanism: o Activator: Streptokinase, derived from B-hemolytic Overview of Physiologic Plasminogen streptococci, serves as the plasminogen activator. Activators: o Synthetic Substrate: Uses chromogenic substrate Types: Two primary plasminogen activators: S-2251 (H-D-Val-Leu-Lys-pNA) to stimulate o Tissue Plasminogen Activator (TPA): fibrinogen. ▪ Produced in vascular endothelial cells. Process: ▪ Half-Life: Approximately 3 minutes. o Streptokinase is added to patient platelet-poor plasma (PPP), activating plasminogen to plasmin. o The streptokinase-plasmin complex hydrolyzes the substrate (chromogenic substrate S-2251: H-D- Val-Leu-Lys-pNA), releasing para-nitroaniline (pNA), which produces a color change (yellow). Color Intensity: Directly proportional to plasminogen concentration, measured against a calibration curve. ▪ Concentration: ~5 ng/mL in plasma. o Urokinase: ▪ Produced in kidneys and endothelial cells. ▪ Half-life: ~7 minutes ▪ Concentration: 2-4 ng/mL in plasma. Function: Both are serine proteases that form complexes Prepared by: Rosa Mikaela Dumago HEMA2 LEC- Finals  EMILIO AGUINALDO COLLEGE Gov. D. Mangubat Ave., Brgy. Burol Main, City of Dasmariñas, Cavite 4114, Philippines Tel. Nos. (046) 416-4339/41 www.eac.edu.ph SCHOOL OF MEDICAL TECHNOLOGY HEMATOLOGY 2 LECTURE with plasminogen on fibrin surfaces, converting it to TPA Activity. plasmin to degrade thrombi. Inhibition: Both activators are inactivated by Plasminogen (PPP) + Plasminogen → Plasmin (activated by TPA) → Activator Inhibitor-1 (PAI-1) secreted by endothelial cells. Chromogenic Substrate (has substance that will result into → Color Reaction Specimen Collection for TPA Assay: Considerations: Clinical Significance of TPA: o TPA activity varies diurnally and increases with Reference Intervals: exercise. o TPA Activity: Upper limit is 1.1 units/mL. o TPA is unstable in vitro as it binds quickly to PAI- o TPA Antigen: Upper limit is 14 ng/mL. 1. Role in Fibrinolysis: TPA is the main mediator of Procedure: fibrinolysis and is the basis for synthetic TPA therapies o Patients should be at rest with minimal tourniquet (e.g., Activase, alteplase.) use. o Collection time should be recorded. Clinical Implications: o Specimen should be immediately acidified using Low TPA Levels: Associated with increased risk of acetate buffer to stabilize TPA. myocardial infarction, stroke, and deep vein thrombosis o Storage: Supernatant platelet-poor plasma (PPP) can (DVT). be frozen at -70C. Impaired Fibrinolysis: TPA Deficiency or excess PAI-1 is linked with DVT and myocardial infarction. Plasminogen Activator Inhibitor-1 (PAI-1) Assay Overview of PAI-1: Production and Circulation: o Produced by vascular endothelial cells and hepatocytes. o Circulates in plasma bound to vitronectin with an Principle of the TPA Assay: average concentration of 10 ng/mL. TPA Measurement: o Enzyme Immunoassay: Estimates plasma TPA antigen concentration. TPA Activity Measurement: o Reagent plasminogen is added to patient plasma. o TPA activates plasminogen to plasmin, and plasmin activity is measured with a chromogenic substrate. o Color intensity is proportional to TPA Activity. o Optional: Soluble fibrin can be included to enhance o Diurnal variation affects PAI-1 levels. o Plasminogen (inactivated) → (to be functional) → convert → Plasmin → needs TPA/UPA Storage in Platelets: High concentrations of an inactive PAI-1 form is stored in platelets. Function: Inactivates free TPA by covalent binding, playing a regulatory role in fibrinolysis. Prepared by: Rosa Mikaela Dumago HEMA2 LEC- Finals  EMILIO AGUINALDO COLLEGE Gov. D. Mangubat Ave., Brgy. Burol Main, City of Dasmariñas, Cavite 4114, Philippines Tel. Nos. (046) 416-4339/41 www.eac.edu.ph SCHOOL OF MEDICAL TECHNOLOGY HEMATOLOGY 2 LECTURE Clinical Significance of PAI-1: immobilized with anti-PAI-1 and detected using anti-urokinase Elevated PAI-1: Associated with an increased risk of antibodies. venous thrombosis, cardiovascular disease, and is a potential marker of aging. o Chromogenic Substrate Assay: Commonly PAI-1 Deficiency: used in indirect measurement method. o Rare and usually associated with hemorrhage only in ▪ Example: Spectrolyse PAI-1 Activity cases of complete deficiency due to homozygous or Assay compound heterozygous SERPINE1 mutations. ▪ Patient PPP is mixed with a fixed o Complete deficiency is more common in specific amount of reagent TPA, and residual populations, such as the Old Order Amish of Indiana, TPA is assayed in a plasminogen system. due to a founder variant. ▪ Result: Color intensity in inversely Diagnosis of Complete Deficiency: Confirmed by serum proportional to plasma PAI-1 activity. PAI-1 assay, platelet-derived PAI-1 should be absent in true deficiency cases. Clinical Significance of PAI-1: Specimen Collection o Collected from patients at rest into acidified citrate tubes to prevent in vitro platelet PAI-1 release. o Centrifugation is performed immediately to prepare platelet-poor plasma (PPP). Assay Methods: o Immunologic and Chromogenic Assays: ▪ Immunoassay: Uses urokinase to bind PAI-1, forming a urokinase-PAI-1 complex, Prepared by: Rosa Mikaela Dumago HEMA2 LEC- Finals  EMILIO AGUINALDO COLLEGE Gov. D. Mangubat Ave., Brgy. Burol Main, City of Dasmariñas, Cavite 4114, Philippines Tel. Nos. (046) 416-4339/41 www.eac.edu.ph SCHOOL OF MEDICAL TECHNOLOGY HEMATOLOGY 2 LECTURE Antithrombotic Therapies and Its Arterial Thrombosis Management: Laboratory Assessment Medications: UFH, LMWH, Fondaparinux, Coumadin, Hematology 2 Lecture and antiplatelet drugs like aspirin, clopidogrel, prasugrel, and ticagrelor. Thrombosis and Anticoagulation Aspirin Prophylaxis: Low-dose aspirin (20 mg/day) for therapeutic effect. o Used intravenously for VTE treatment, AMI, stent reocclusion prevention, and cardiac Switching and Reversing Anticoagulation: surgeries with cardiopulmonary bypass. o Initial dosage: 5000-10,000 units bolus, Switching from Intravenous to Oral Anticoagulants: followed by continuous infusion adjusted to o Crossover from UFH to Coumadin requires 5 days patient weight. to reach full Coumadin anticoagulation. o DTIs (e.g., argatroban, bivalirudin) can double PT during transition; chromogenic factor X assay is Monitoring Heparin Therapy: preferred for monitoring. PTT Monitoring: o Baseline PTT obtained before UFH initiation. Coumadin Overdose Reversal: o Follow-up PTT 4-6 hours after UFH bolus to Vitamin K (oral or intravenous) to counteract overdose. ensure it falls within the therapeutic range. Severe bleeding may require plasma, recombinant factor VII, o Daily PTT and platelet count monitoring are or prothrombin complex concentrates. essential for dose adjustments and HIT Reversal effects monitored by clinical observation and detection. PT/INR assays. oPTT Therapeutic Range: Established by pairing PTT with anti-Xa results (0.3 – 0.7 units/mL) Unfractionated Heparin (UFH) Therapy in a set of patient samples. and Monitoring Mechanism of UFH Chromogenic Anti-Xa Heparin Assay: Action: o Less interference and more stable than PTT; Structure and Binding: reflects only antithrombin-Xa activity. o UFH is a sulfated glycosaminoglycan mixture from o Used when PTT is compromised by factors like porcine mucosa, with molecular weights ranging lupus anticoagulant, fibrinogen levels, or factor from 3000 to 30,000 Daltons. VIII elevation. o Approximately one-third of UFH molecules have a high-affinity pentasaccharide sequence that binds Heparin Resistance and Monitoring antithrombin. Challenges: Anticoagulant Action: Heparin Resistance: Due to high fibrinogen/factor VIII o UFH enhances antithrombin activity, catalytically or depleted antithrombin. inhibiting factors IIa (thrombin), IXa, Xa, XIa, and Alternative Monitoring: Anti-Xa assay or antithrombin XIIa. concentrate administration. o Thrombin-antithrombin bridging drives thrombin Specimen Handling: Platelet Factor 4 (PF4) in whole inhibition faster than Factor Xa inhibition. blood neutralizes heparin if not promptly processed, requiring centrifugation within 1 hour of collection. Activated Clotting Time (ACT): ACT: Point-of-care test for high UFH levels (1-2 units/mL), used in extracorporeal procedures. Target ACT range for PCI: 200 – 240 seconds. Target ACT range for CABG: 480 – 600 seconds. Reversing Heparin: Protamine Sulfate: Neutralizes UFH at a ratio of 1 mg per 100 units of UFH. Administered after CPB discontinuation or UFH overdose; monitored by ACT. Risks: Protamine can cause delayed HIT, necessitating routine platelet count checks. Prepared by: Rosa Mikaela Dumago HEMA2 LEC- Finals  EMILIO AGUINALDO COLLEGE Gov. D. Mangubat Ave., Brgy. Burol Main, City of Dasmariñas, Cavite 4114, Philippines Tel. Nos. (046) 416-4339/41 www.eac.edu.ph SCHOOL OF MEDICAL TECHNOLOGY HEMATOLOGY 2 LECTURE Low-Molecular-Weight Heparin (LMWH) Calibration: and the Anti-Factor Xa Assay: o Laboratory uses LMWH calibrators to prepare a calibration curve, potentially creating a hybrid LMWH Production and Mechanism: curve if the assay monitors both UFH and o Source: Derived from UFH by fractionation, LMWH. resulting in shorter polysaccharide chains (mean molecular weight of 4500 – 5000 Daltons). LMWH Reversal and HIT Considerations: Mechanism: o Retains the same antithrombin-binding Protamine Sulfate: Partially reverses LMWH-associated pentasaccharide sequence as UFH, primarily bleeding; however, its effect is incomplete. inhibiting factor Xa rather than thrombin. HIT Risk: o Thrombin-Antithrombin Bridging: Reduced due o LMWH reduces HIT risk by 90% compared to to shorter chains, but factor Xa inhibition remains UFH in patients without prior UFH exposure. potent. o Contraindicated in patients with existing HIT antibodies, as LMWH may cross-react. LMWH Therapy: Administration: Fondaparinux and the Anti-Factor Xa o Delivered subcutaneously once or twice daily with Assay premeasured doses (e.g., 30 mg every 12 hours or Fondaparinux Overview: 40 mg daily). o Commonly used for VTE prophylaxis in orthopedic Structure: or general surgeries and trauma. o Synthetic pentasaccharide modeled on the active o Preferred in pregnancy for VTE risk due to the sequence in UFH and LMWH. teratogenic effects of Coumadin. o Consists of a single, defined molecular structure o Employed in “bridging” for patients transitioning rather than a mixture, providing high off Coumadin pre-surgery due to its shorter half- antithrombin affinity. life. Mechanism of Action: o Inhibits only Factor Xa via antithrombin, with no Advantages Over UFH: effect on thrombin or other serine proteases. o Increases antithrombin activity 400-fold. Predictable dose response and longer half-life (3-5 hours vs. 60-90 minutes for UFH). Clinical Use of Fondaparinux: Increased bioavailability, less frequent dosing, home administration, and lower HIT risk. Administration: o Given once daily by subcutaneous injection Monitoring LMWH Therapy: (2.5 to 7.5 mg), with a half-life of 17-21 hours. Indications: When to Measure: o Approved for VTE prevention after orthopedic o Required in patients with renal insufficiency and abdominal surgery and for treating acute (creatinine clearance 4 mg/dL), low or high body weight, children, and o Contraindicated in patients with creatinine during pregnancy. clearance