Hematology Lab - 10 Hemostasis

HEMATOLOGY LABORATORY • HEMOSTASIS OUTLINE • Introduction • Primary Hemostasis o Steps in Primary Hemostasis o Standard Reference Values o Laboratory Assessment of o Scope of Practice Primary Hemostasis o History o Clinical Significance o Anticoagulants • Secondary Hemostasis o Basic Terminology in o Coagulation Factors Hematology o Coagulation Factors o The Blood Pathways o Blood Components o Bleeding Disorders o Hematologic Parameters and o Laboratory Evaluation of Reporting Format Secondar Hemostasis o Difference Between Capillary and Venous Blood o Hemostasis o Megakaryopoiesis o Laboratory Evaluation of Platelets and Hemostasis • • • • • • • • • • • INTRODUCTION STANDARD REFERENCE VALUES • • Volume o Adults = 5 – 6L or 6-8% of the total body weight (6080mL/kg wt.) o Neonates = 250 – 350 mL Specific Gravity o Whole blood (M) 1.055 – 1.064 o Whole blood (F) 1.052 – 1.060 o Plasma 1.025 – 1.029 o Serum 1.024 – 1.028 ANTICOAGULANTS • SCOPE OF PRACTICE • • • • Identification of reactive or neoplastic alterations in cell population Evaluation of Erythrocyte, Leukocyte and Platelet morphology and function, their cell count as one of the bases for diagnosis Bone Marrow examination Evaluation of Hemostatic function HISTORY • • • • • • • • • Hematology dates back to Aristotle and Plato. 2nd century A.D. - Hemophilia was recognized but pathophysiology was not understood 12th century A.D. - Moises Maimonides described 2 male children who died from excessive bleeding after circumcision 1658 - Swammerdam discovered yhe ERYTHROCYTES or red blood cells Athanasius Kircher (1657) – described “worms” in the blood Anton van Leeuwenhoek (1674) – gave account on RBCs Giulio Bizzozero (late 1800s) – described platelets as “petites plaques” 1803 - Clinical description of families with hemophilia was first published by Hoff 1842 - Platelets were described 1846 - Polymorphonuclear (PMN) was distinguished from other leukocytes by Wharton Jones 1879 - Ehrlich completely classified the leukocytes 1905 - The theory on blood coagulation of Paul Morawitz was accepted 1913 - The whole blood coagulation time by Lee & White was 1st performed 1920 - Hematology was considered a separate science from clinical pathollogys James Homer Wright (1902) – introduced a new visual blood examination through the microscope 1930 - Prothrombin Time was introduced by Quick 1940 - Platelet count, bleeding time determination, and other tests were introduced Joseph and Wallace Coultier (1953) – patented the first electronic cell counter 1964 - Cascade and Waterfall"theory of coagulation was introduced At present, the following are the trends: o Automation o Point Of Care Testing (POCT) o The Advantages of Automation: ▪ More time efficient/ less time-consuming ▪ More accurate and precise results ▪ Less fatigability on the part of the laboratory worker ▪ High throughput • • • EDTA (Ethylene diaminetetraacidic acid) - Chelates calcium from the blood/ converts calcium in a non-ionized form o 1-2 mg/ml of blood shd be used o used in routine hematological tests, like CBC o contained in lavender top evacuation tube o Citrate - same Heparin - natural anticoagulant, but the amount secreted by the liver is not enough to prevent coagulation of blood in vitro. o Its action is Antithrombin & Antithromboplastin o In the presence of heparin the physiologic anticoagulant, antithrombin III acts 2000x faster. Citrate- same action as EDTA, used for hemostatic and coagulation testing o ratio: ▪ coagulation tests -1 part citrate + 9 parts blood. citrate is 0.109 Molar in concentration ▪ ESR(Westergren method) • 1 part citrate +4 parts blood. citrate is 3.8% in concentration BASIC TERMINOLOGY IN HEMATOLOGY COMMON PREFIXES AND SUFFIXES • • • • • • Dyserythropoiesis – abnormal development of RBC Anisocytosis – unequal size of RBC Thrombophlebitis – inflammation of vein due to blood clot Polychromatophilia – affinity for many colors Aplasia – absence of development, cell production Hypogammaglobulinemia – decrease of gamma globulins in the blood • • • • Polycythemia –many blood cell RBC Schistocytosis – abnormal increase of fragmented cells Megaloblastic anemia – giant immature cells in the blood Thrombolysis – breakdown of clots WHITE BLOOD CELLS • • • THE BLOOD ANATOMY AND PHYSIOLOGY OF THE BLOOD • • • • • • Transport – minerals, oxygen, carbon dioxide, nutrients, waste products, hormones Homeostasis – have buffer systems to maintain acidbase equilibrium; normal body temperature Immunity – fights against bacterial invasion carried out by WBC and Immunoglobulins Hemostasis – balance of blood; carried out by platelets and clotting factor **Bicarbonate/Carbonic acid – Buffer system of blood **Albumin – main regulator of osmotic pressure, present in the cell for water balance PHYSICAL CHARACTERISTICS OF THE BLOOD • • • Viscosity – 3.5-4.5 more viscous than water pH – 7.35-7.45 (slightly alkaline) maintained by buffer system Osmolality – 281 -297 milliosmoles/kg H2O • • Leukocytes Protection from infection and injury Can be counted by using: Microscope, Hemocytometer and a Thoma Pipette Granulocytes o Neutrophil - 60-70% o Eosinophil - 2-4% o Basophil - 0-1% Agranulocytes o Monocyte - 3-8% o Lymphocyte - 20-25% PLATELETS • • • Thrombocytes 2 to 4 μm in diameter Round, oval, anucleated and granular HEMATOLOGIC PARAMETERS AND REPORTING FORMAT Normal Value RBC Count Female RBC Count Male WBC Count Platelet Count Conventional Unit 4.5 - 5.5 x 106 uL or cm3 5.0-6.5 x 106 uL 4.5-11 x 103 uL or cm3 150-450 x 103 uL or cm3 14-18 g/dL 40-54% 86-90 uL3 Hemoglobin (Male) Hematocrit (PCV) Mean Corpuscular Value (MCV) Mean Corpuscular 27-33 uug Hemoglobin (MCH) Mean Corpuscular 33-36 g/dL Hemoglobin Concentration (MHCH) SI Unit 4.5-5.5 x 1012 L 5.0-6.5 x 1012 L 4.5-11 x 109 L 150-450 x 109 L 140-180 g/L 0.4-0.54 L/L 86-90 fL 27-33 pg 33-36% DIFFERENCES BETWEEN CAPILLARY & VENOUS BLOOD • BLOOD COMPONENTS RED BLOOD CELLS • • • • • • • • • Erythrocytes Anucleate biconcave cells filled with Hemoglobin Pink to red that measure 6 to 8 μm in diameter with a zone of pallor covering 1/3 of their center Hemoglobin – reddish protein, transports oxygen and carbon dioxide Hematocrit – ratio of the volume of RBC’s to the volume of whole blood; Also called as PCV. MCV (Mean Cell Volume) – RBC diameter on a WrightStain blood film. MCHC (Mean Cell Hemoglobin Concentration) – RBC Staining intensity or degree of pallor MCH (Mean Cell Hemoglobin) – Mass of Hemoglobin and closely reflects the MCHC RDW (Red Cell Distribution Width) – RBC Distribution Width; Degree of Variation in RBC Volume • • • • Capllary blood contains more glucose than venous blood(10-20 mg/dL higher) Capillary blood contains more leukocytes than venous blood(up to 1000cells/ ul) Capillary blood has a lower erythrocyte count and hemoglobin value than venous blood(5%) Capillary blood has alower platelet count than venous blood(platelets adhere to injured tissues) The red blood cells in capillary blood are more fragile than those of venous blood ( lower pH of capillary blood) HEMOSTASIS • Primary Hemostasis o First response of blood vessel to injury by vasoconstriction o Platelets will adhere to the exposed subendothelium (vWF) which leads to platelet activation due to GP1b/IX/V complex o Platelet activation will cause shape change from resting phase to activated phase leading to formation GPIIb/IIIa complex (only adhere/occur after shape change) which allows platelet aggregation/formation of platelet plug ▪ • • During shape change, there is a fusion of platelet granules with the open canalicular system (OCS) to serve as an avenue for the release of platelet granular contents ▪ Shape change can lead to release reaction/platelet secretion o Activated platelet aggregates o End result is formation of temporary platelet plug due to platelet aggregation by action of GPIIb/IIIa complex Secondary Hemostasis o Aka coagulation ▪ Not synonymous with hemostasis ▪ Only refers to secondary hemostasis o Stability of fibrin clot that is brought about by proclotting factors (fibrinogen, prothrombin, etc.) ▪ Also brought about by factor XIII (fibrin-stabilizing factor) o End result is formation of stable fibrin clot Fibrinolysis o Dissolution of stable fibrin clot o Marks the return to normal state • • • • • MEGAKARYOPOEISIS • • • • Megakaryoblast → Promegakaryocyte → Megakaryocyte (Granular & Mature) As cell matures, cell decreases in size, nucleoli decrease, nucleus to cytoplasm ration decreases, nuclear chromatin condenses, cytoplasmic staining become less basophilic Unique due to o Endoreduplication o Cell size increases instead of decreasing size ▪ Only hematopoietic series where cell size increase as the cell mature o Synchronistic maturation (cytoplasm & nucleus mature at the same rate) Mitotic ability is arrested at telophase (phase where daughter cells are separated) o Causes enlargement of megakaryocyte as cells mature and increases in chromosomal material o Enlarged cell size & enlarged nucleus o Nucleus enlargement is slower LABORATORY EVALUATION OF PLATELETS AND HEMOSTASIS • • • Laboratory Evaluation of Platelets Laboratory Evaluation of Coagulation Special Laboratory Evaluation o Fibrinolysis o Anticoagulant Therapy ▪ Blood thinners • EVALUATION OF PLATELETS • • • • Platelet count o Direct Platelet Counting o Indirect Platelet Counting Bleeding Time - screening test for the evaluation of entire primary hemostasis Platelet Aggregometry - evaluates platelet function/ response to platelet agonists (platelet activator) Ristocetin Cofactor Assay - for von Willebrand Disease (vWD) • Anti-PF4 - screening test for Heparin-Induced Thrombocytopenia (HIT) Platelet counts are used to screen for quantitative diseases of platelets Several methods are employed in platelet counting. This is owing to the fact that platelets are difficult to count Reasons for difficulty in platelet counting are o Platelets are small, and easily confused with dirt, dust, and epithelial debris o Platelets tend to aggregate and disintegrate o Platelets tend to adhere to foreign substances including glass pipettes o Platelets are distributed unevenly Direct Platelet Counts o Specimen: EDTA-anticoagulated blood o Breecher-Cronkite: ▪ Reference of manual platelet counting ▪ Diluent: 1% (NH4)2C2O4; 1:100 dilution ▪ Area counted in: 10/25 intermediate squares of central large square of INCC ▪ Microscopy: Phase-contrast ▪ Formula: 𝑷𝒍𝒂𝒕𝒆𝒍𝒆𝒕𝒔/µ𝑳=𝑷𝒍𝒂𝒕𝒆𝒍𝒆𝒕𝒔 𝒄𝒐𝒖𝒏𝒕𝒆𝒅 𝒙 𝟐.𝟓 𝒙 𝟏𝟎 𝒙 𝟏𝟎𝟎 𝑷𝒍𝒂𝒕𝒆𝒍𝒆𝒕𝒔/µ𝑳=𝑷𝒍𝒂𝒕𝒆𝒍𝒆𝒕𝒔 𝒄𝒐𝒖𝒏𝒕𝒆𝒅 𝒙 𝟐𝟓𝟎𝟎 o Rees-Ecker: ▪ Diluent: Formalin, Brilliant cresyl blue, Na citrate; ▪ 1:200 Dilution ▪ Area counted in: 25/25 intermediate squares of central large square of INCC ▪ Microscopy: Brightfield ▪ Formula: • 𝑷𝒍𝒂𝒕𝒆𝒍𝒆𝒕𝒔/µ𝑳 = 𝑷𝒍𝒂𝒕𝒆𝒍𝒆𝒕𝒔 𝒄𝒐𝒖𝒏𝒕𝒆𝒅 𝒙 𝟏𝟎 𝒙 𝟐𝟎𝟎 • 𝑷𝒍𝒂𝒕𝒆𝒍𝒆𝒕𝒔/µ𝑳 = 𝑷𝒍𝒂𝒕𝒆𝒍𝒆𝒕𝒔 𝒄𝒐𝒖𝒏𝒕𝒆𝒅 𝒙 𝟐𝟎𝟎𝟎 o Leake-Guy: ▪ Diluent: Formalin, Crystal violet, Na oxalate; ▪ 1:100 Dilution ▪ Area counted in: 5/25 intermediate squares of central large square of INCC ▪ Microscopy: Brightfield ▪ Formula: • 𝑷𝒍𝒂𝒕𝒆𝒍𝒆𝒕𝒔/µ𝑳 = 𝑷𝒍𝒂𝒕𝒆𝒍𝒆𝒕𝒔 𝒄𝒐𝒖𝒏𝒕𝒆𝒅 𝒙 𝟓 𝒙 𝟏𝟎 𝒙 𝟏𝟎𝟎 • 𝑷𝒍𝒂𝒕𝒆𝒍𝒆𝒕𝒔/µ𝑳 = 𝑷𝒍𝒂𝒕𝒆𝒍𝒆𝒕𝒔 𝒄𝒐𝒖𝒏𝒕𝒆𝒅 𝒙 𝟓𝟎𝟎𝟎 Indirect Platelet Counts o Indirect: counts the number of platelets per 1000 RBCs in a peripheral blood smear making use of the following diluents: o Fonio’s: 14% MgSO4 o Dameshek’s: Formalin, Na citrate, BCB, sucrose o Formula: Platelets/L=Platelets counted/1000RBCs xRBC ct (/L) Bleeding Time o Used as a general screening test for o May indicate impaired platelet function but influenced by different factors ▪ Duke: makes an incision on middle or ring finger ▪ Ivy: makes three incisions on the volar surface of the forearm and makes use of a pressurized cuff ▪ Template (modification by Mielke) ▪ Simplate (General Diagnostics) ▪ Aspirin Tolerance Test • • Platelet Aggregometry o Aggregometer: measures aggregation of platelets in suspension of citrated whole blood or PRP in response to an agonist. o Agonists: ▪ Thrombin ▪ ADP ▪ Epinephrine ▪ Collagen ▪ Arachidonic acid ▪ Ristocetin o Responses to aggregometry ▪ NSAIDS: abnormal respose to arachidonic acid & collagen ▪ thienopyridines (clopidogrel, prasugrel): abnormal response ADP ▪ Eiconasoid pathway enzyme deficiencies (cyclooxygenase, TXA synthase, protein kinase C): abnormal response to ADP, epinephrine, collagen ▪ Storage pool deficiencies: ATP release reduction Platelet Adhesiveness Test o Salzman’s Test o Uses Glass Beads Collecting System ▪ • • • • % 𝑎𝑑ℎ𝑒𝑠𝑖𝑣𝑒𝑛𝑒𝑠𝑠 = 𝑃𝐶𝑖 − 𝑃𝐶𝑓 𝑃𝐶𝑖 • 𝑥 100 o Ref: 26-67% o *PCi: initial platelet count o **PCf: final platelet count Capillary Fragility Test o Aka. ‘Tourniquet Test’ o Aka. Rumpel-Leede CFT o Aka. Hess Test o Performed by inflating a pressurized cuff to midway between the systolic and diastolic BP. After two minutes, petechiae is counted below the antecubital fossa o This test now forms part of the case definition for Dengue (WHO, nd) Clot Retraction Time o Micro method: Hirshboek (Castor Oil) ▪ A drop of blood is placed and allowed to float on the surface Castor Oil. The time it takes for the ‘dimpling phenomenon’ to take place is recorded, o Macro methods: ▪ MacFarlane ▪ Stefanini (Stefanini-Dameshek) Ristocetin Cofactor Assay o aka. vWF activity assay o makes use of reagent platelets and patient’s PlateletPoor Plasma for Ristocetin-induced Platelet Aggregation and compares the result with the RIPA results using normal PPP. Anti-PF4 Immunoassay o Screening assay for Heparin-Induced Thrombocytopenia o ELISA o confirmed by 14C Serotonin Release Assay COAGULATION TESTING • • Lee and White: first laboratory test used to evaluate coagulation o Ref: 7-10 mins Activated Clotting Time (ACT): modification of Lee and White o with clot activator (diatomide) o Ref: ▪ 75-120 secs. (w/o heparin) ▪ 140-185 secs (w/ heparin) o Micro methods (Clotting Time) ▪ Slide Method ▪ Dale-Laidlaw (Capillary Tube Method) • Ref: 2-6 mins Prothrombin Time with INR o Prothrombin Time ▪ reagent: Thromboplastin ▪ sensitive to deficiencies of Factors VII, I, and II and moderately sensitive to Factor V and X deficiencies ▪ used in monitoring Warfarin therapy o International Normalized Ratio (INR) ▪ standardized system of reporting in order to eliminate differences in reporting due to differing performances of different reagents ▪ Prolonged in DIC, liver dse, Vit K def. To differentiate between Liver Dse and Vit K deficiency, perform FV and FVII assay. FV FVII Liver Disease Decreased Decreased Vit. K deficiency Normal Decreased Partial Thromboplastin Time o PTT / APTT o reagent: phospholipid (partial thromboplastin) + negatively-charged activator (silica, kaolin, ellagic acid, celite) o Prolonged in deficiencies of intrinsic pathway factors, presence of LA or antibodies to intrinsic pathway factors o Used to monitor heparin anticoagulant therapy o Prolonged in DIC, and to a lesser extent, Vit K def o Ref: 20-45 secs. o Detection of LA and Specific Inhibitors ▪ PTT prolonged, TCT prolonged = Hep ▪ Px Plasma + rgt PPNP (1:1) ▪ PTT (1:1) corrected & clinical bleed = def ▪ PTT (imm) corrected; PTT (inc) not corrected = inhibitor ▪ If with clinical bleed, specific inhibitor ▪ PTT (imm) & (inc) not corrected = suspect LA; perform Phospholipid neutralization Thrombin Clotting Time o Bovine thrombin rgt cleaves fibrinopeptides to form fibrin polymer. o Prolonged in hypofibrinogenemia, afibrinogenemia, dysfibrinogenemia o Prolonged in the presence of FDPs, paraCHON, or heparin o Reptilase Time: same procedure, using reptilase rgt (atroxin ); insensitive to heparin. o Ref: 10-15 secs. o o • • • • • • Stypven Time o Aka. Russel Viper Venom Time o Detects deficiencies in Factors X, V, II, I (common pathway factors) o Ref: 6-10 secs Anti-FXa assay o Measures available FXa in patients receiving intravenous LMW Heparin therapy. o In patients under LMW Heparin therapy, the LMW Heparin binds to ATIII and the complex binds with and inhibits the activity of FXa but not FIIa. o The unbound FXa from the patient sample reacts with the reagent substrate and delivers a measurable signal that can be detected by the instrument. Fibrinogen Assay o Modification of TCT (Clauss) using higher conc of reagent and dilution of PPP(Px) o Elevated in pregnancy, moderately sever liver dse, chronic inflammatory conditions o Decreased in DIC, severe liver dse Substitution tests o Previously performed on samples with abnormal PT or PTT results. Different types of plasma or serum are added to the patients plasma and retested for ‘correction’ o Note: ▪ Fresh plasma: all factors are present ▪ Aged plasma: Factors V and VIII:C are absent ▪ Serum: Factors I, II, V, VIII:C and XIII are absent ▪ Adsorbed plasma: Factors II, VII, IX, and X are absent • • • Whole Blood Clot Lysis time o Whole blood is incubated at 37ºC for 24 hours and the time it takes for the clot to dissolve is noted Euglobulin Clot Lysis time o The euglobulin fraction of plasma contains plasminogen, fibrinogen, and plasmin. This fraction is precipitated with 1% acetic acid and resuspended in a borate solution. o The euglobulins are then clotted by the addition of thrombin. The clot is incubated and the time of lysis is reported. o Clinically, the euglobulin lysis test is a screening procedure for fibrinolytic activity. Antithrombin III Assay o In the presence of heparin, thrombin is neutralized at a rate that is proportional to the antithrombin (AT III) concentration. o Following defibrination, plasma is assayed in a twostage procedure that utilizes standardized amounts of heparin, fibrinogen, and thrombin. The resulting clotting time is interpreted using a calibration curve. o Clinically, the AT III assay is useful prior to and subsequent to treatment with heparin in cases of disseminated intravascular coagulation (DIC). TESTS OF FIBRINOLYSIS • • D-Dimer Assay o The fundamental principle of the D-dimer assay has remained largely unchanged: recognition of the unique neo-epitope in D-dimer by specific antisera. Ddimer contains a neo-epitope that is formed following the cross-linking of adjacent D domains by factor XIIIa. It is this epitope that is recognized by specifi c antisera used in clinical assays. o The classic microplate ELISA was considered the gold standard for measuring D-dimer. Today, more than 30 different D-dimer assays are commercially available. FSP / FDP assay o Whole blood is added to thrombin (to ensure complete clotting) and soya bean enzyme inhibitors (to prevent any breakdown of fibrin). After incubation, the patient’s serum is diluted and mixed with latex particles that have been coated with anti–fibrin split products. o If fibrin split products are present, agglutination will occur. If these products are present in increased amounts with normal hepatic and renal function, it is assumed that a recent fibrinolytic event has taken place or is taking place. o Clinically, elevated results demonstrate that the activation of plasmin has occurred or is occurring, such as in primary fibrinolysis and DIC with secondary fibrinolysis. ANTITHROMBOTIC THERAPY AND MONITORING • • “Antithrombotic therapy, which includes therapies targeting platelets as well as the coagulation system, is used to prevent and treat thromboembolic disease” (McPherson and Pincus, 2017) Antithrombotic therapies include Antiplatelet agents and anticoagulants. • Vitamin K antagonists o Vit. K antagonists inhibit the action of Vit. K and blocks its regeneration. Most common is Warfarin, taken orally o Vit. K is necessary for the synthesis of FII, FVII, FIX, and FX. o Monitoring of Warfarin therapy is by measurement of PT, although PTT is also typically prolonged. PRIMARY HEMOSTASIS • • • • o Antithrombin Parenteral Anticoagulants o Unfractionated Heparin (UFH) ▪ Binds with ATIII, accelerating its function in inhibition of Thrombin (FIIa) and FXa, as well as other serine proteases. ▪ Also promotes fibrinolysis and impairs platelet function. ▪ Most common method of monitoring UFH therapy is APTT although several methods of testing may be employed including activated clotting time, anti-FXa, and protamine reversal. o Low Molecular Weight Heparin (LMWH) ▪ Short heparin fragments derived from UFH by chemical and enzymatic processes ▪ Able to inactivate FXa but because of its low molecular weight, cannot inactivate FIIa. ▪ Monitoring is via anti-FXa assays o Fondaparinux ▪ Synthetic pentasaccharide (minimum length) needed to bind with ATIII and inhibit FXa (similar to LMWH) ▪ Monitoring is via anti-FXa assays although PT and APTT may also be affected only by a few seconds. Antiplatelet Therapy o Aspirin (Acetylsalicylic Acid) ▪ Inhibits platelet activation (thus inhibits aggregation) by inhibiting COX activity ▪ Inhibition of COX activity results to a decrease in Thromboxane A2 levels ▪ Measured by quantitative assay of Thromboxane B2, a metabolite of Thromboxane A2 ▪ More recent alternatives to quantitative TXB2 testing is platelet aggregometry in response to Arachidonic acid • • • • • Hemostasis – Cessation of bleeding at a vascular injury site via the formation of a thrombus. o Whenever vessel injury occurs, hemostasis take place o Under normal circumstance, there should not be a formation of thrombus and blood should flow in a fluid state unimpeded in blood vessels o System by which blood is kept in a fluid state is also referred to as hemostasis Factors involved: o Platelets - form platelet plug during primary hemostasis, release factors that promote vasoconstriction, crucial in intrinsic pathway of coagulation (secondary hemostasis) o Coagulation factors - pro-clotting factors, proteins involved in secondary hemostasis ▪ Coagulation - end product is formation of stable fibrin clot and eventually thrombus o Fibrinolytic system - lyse fibrin clot and signify a start the return to normalcy o Anticoagulation system - inhibitors of coagulation (Protein C, S - inhibits cofactors) Cellular Components - Platelets, Granulocytes & Monocytes Protein Systems o Coagulation System (Clot Forming) → serves to form thrombin that initiates the proteolysis of fibrinogen, leading to fibrin clot formation o Fibrinolytic Sytem (Clot Lysing) → functions to lyse the clot formed by thrombin o Anticoagulation System (Regulating) → regulates all enzymes of the coagulation and fibrinolytic systems, so that no inappropriate excess of clotting or bleeding occurs. Vasoconstriction - A short-lived reflex reaction of the smooth muscle in the vessel wall produced by the sympathetic branches of the autonomic nervous system. Controlled by vessel smooth muscle, enhanced by chemicals secreted by Platelets. o Blood vessel constricts to impede blood flow at the site of injury Primary hemostasis – Formation of a temporary, unstable patch made of platelets (Platelet plug) o Adhere at site of injury via GP complex Ib/IX/V binding to collagen in exposed subendothelium ▪ GP Ib - most important; if deficient, may lead to Bernard-Soulier syndrome (abnormal platelet adhesion) o • • Collagen contact would initiate platelet activation to undergo confirmational changes - due to contraction of microtubules o Fusion of platelet granular content and open canalicular system o ADP promote activation of nearby platelets o Recruitment of other platelets o Aggregates/platelet plug form due to platelet-toplatelet contact ▪ Due to GP IIb/IIIa complex - allow platelet aggregation ▪ Deficiency of GP IIb/IIIa leads to Glanzmann’s Thrombasthenia (abnormal platelet aggregation) Secondary hemostasis – Formation of stable fibrin clot. (Responsible: Clotting factors) o Aka coagulation o Lead to stable fibrin clot (end product) o Further activation lead to thrombus formation Fibrinolysis – Breakdown of the thrombus (Responsible: Plasmin) o To not impede normal blood flow • • • Upon injury, the VonWillebrand Factor (vWF) that is secreted by the endothelial cells will adhere to the subendothelial collagen. The glycoprotein Ib (GP1b) expressed on the surface of the platelets will adhere to the vWF that is attached to the S.E.C. Platelet activation will then take place, altering the shape of the platelet by forming pseudopods. STEPS IN PRIMARY HEMOSTASIS • • • Platelet Adhesion – Property by which platelets bind to non-platelet surfaces such as non endothelial collagen. o Activation → shape change → release rxn (secretion) o Mediated by GP Ib/IX/V Platelet Secretion – Property by which platelets release their granules. o Can be direct to blood vessels ▪ Serotonin, thromboxane A2 - promote vasoconstriction o Other granules serve to recruit other platelets Platelet Aggregation – Property by which platelets bind to one another. o Mediated by GP IIb/IIIa complex • A reduction or total absence of GP1b will result from the inability of platelets to adhere on the vessel walls. This condition is called Bernard Soulier Syndrome (BSS). o Signs and symptoms of Bernard-Soulier syndrome ▪ Symptoms: Easy bruising, Nosebleeds, Menorrhagia, Gastrointestinal bleeding (occasional) ▪ Physical findings: Increased bruising, Purpura and petechial rash. PLATELET AGGREGATION • • • PLATELET ADHESION • • • Endothelial cells secrete nitric oxide and prostacyclin which functions as vasodilators and keep the platelets from adhering to surface of endothelial cells. Upon injury to the endothelial cells, the production of nitric oxide and prostacyclin will be interrupted, thus allowing platelets to adhere to the endothelial cells. Platelet adhesion happens 1-2minutes after injury. • • • • • After adhering to the S.E.C., platelets will then secrete ADP in order to activate Glycoprotein IIb/IIIa (GP2b/3a). GP2b/3a is a glycoprotein that can bind to both vWF and Fibrinogen. GP2b/3a will then bind to the vWF that is present in the plasma. Can also bind to fibrinogen if its present. The GP2b/3a of other platelets will then bind to the opposite side of the vWF. This process will result to the formation of a temporary platelet plug. An abnormality with GP2b/3a will result in an inability of platelets to form aggregates. This condition is called: Glanzmanns thrombasthenia. Signs and Symptoms are the same with Bernard Soulier Syndrome. PLATELET SECRETION • • • • • • • • • • Release of granules and other contents. Happens simultaneously during platelet adhesion and aggregation, with most secretion occuring during the aggregation phase. Secretions: o Alpha granules o Dense granules Secretions: Procoagulants. Alpha granules: o Fibrinogen o von Willebrand Factor Dense granules: o Serotonin o Adenosine Diphosphate o Calcium o CAPAS (Calcium, ADP, Pyrophosphate, ATP, Serotonin) Fibrinogen and vWF – Serves as bridges for platelet receptors. Serotonin – Vasoconstrictor ADP – Promotes platelet Activation and Aggregation. Calcium – Needed for secondary hemostasis. ROLE IN HEMOSTASIS Promotes aggregation SUBSTANCE SOURCE FUNCTION/S High Molecular Weight Kininogen (HMWK) Alpha granules Contact activation of intrinsic coagulation pathway Fibrinogen Alpha granules Factor V Alpha granules Factor VIII: vWF Alpha granules Converted to fibrin for clot formation Cofactor in fibrin clot formation Assists platelet adhesion to subendothelium to Provide coagulation surface SOURCE ADP Dense granule FUNCTION/S Promote platelet aggregation, promote platelet activation Calcium Dense Promote platelet granule aggregation, required in coagulation Platelet Factor 4 Alpha Promote platelet granules aggregation Thrombospondin Alpha Promote platelet granules aggregation Promotes Serotonin Dense Promotes vasoconstriction granules vasoconstriction at site of injury Thromboxane Membrane Promotes A2 (TxA2) Phospholipids vasoconstriction at site of injury Promotes Platelet derived Alpha Promotes smooth vascular repair growth factor granules muscle cell (PDGF) growth for vessel repair Beta Alpha Chemotactic for thromboglobulin granules fibroblasts to help in vessel repair Other systems Plasminogen Alpha Precursor to affected granules Plasmin which induces clot lysis Alpha 2Alpha Major inhibitor of antiplasmin granules Plasmin: inhibits clot lysis INHIBITORS OF PLATELET ACTIVITY • • • ACTIVATORS F PLATELET ACTIVITY ROLE IN HEMOSTASIS Promotes coagulation SUBSTANCE Prostacyclin and Nitric oxide – Vasodilators and platelet receptor blockers. CD 39 – Enzyme produced by endothelial cells that metabolizes ADP and ATP in platelets thereby impeding its function. o ADP - activators of platelets o ATP - metabolic functions of platelet Aspirin and NSAIDS - Prevent formation of TxA2 by inhibiting function of cyclooxygenase (Cox) o Artificial/synthetic inhibitors o Aspirin - promote bleeding LABORATORY ASSESMENT OF PRIMARY HEMOSTASIS • • For Vascular Integrity: o Bleeding time o Capillary fragility test ▪ Rumpel-Leede test/Tourniquet test/Hess test/Positive pressure technique ▪ Petechiometer method/Suction cup method/Negative pressure method Quantitative Evaluation of Platelets: o Direct method (Reese and Ecker aka Tocantin’s method) o Indirect method (Fonio’s method) o Unopette system o Automated counting machine ▪ Tool used to differentiate whether the bleeding is due to fragile veins or problems in blood. • Qualitative Evaluation of Platelets: o Tests for platelet adhesion ▪ Bleeding time • Duke’s method • Ivy and Modified Ivy’s method • Adelson-Crosby method and Macfarlane methodfrrrr • Coply-Lalitch method o Tests for platelet aggregation/aggregometry ▪ Turbidimetry ▪ Nephelometry • Agents such as ADP, collagen, epinephrine, snake venom, thrombin, and ristocetin are used to aggregate platelets. ▪ Platelet aggregation - The principle of the test is that platelet-rich plasma is treated with a known aggregating agent. If aggregated, cloudiness or turbidity can be measured using a spectrophotometer. • Increased turbidity o Clot retraction ▪ Reflects the number and quality of platelets, fibrinogen concentration, fibrinolytic activity, and packed red cell volume. ▪ Overall evaluation of entire hemostatic processes • • • Macfarlane method o Same as Duke but with the addition of immersion of punctured earlobecc in sterile Physiologic saline solution (NSS) warmed at 37℃ waterbath to check bleeding. Bleeding time is no longer recommended due to variables involved o Thickness, elasticity of skin o Platelet count (adhesion & aggregation of platelets) o Conditions causing abnormal platelet aggregation Bleeding time is general screening test for hemostatic disorders CAPILLARY FRAGILITY TEST • • The capillary fragility test measures platelet’s ability to maintain capillary integrity. The degree of thrombocytopenia will correlate with the test, as with the bleeding time. In normal patients, none to very few petechiae are formed during this test. A positive test will be found in thrombocytopenia, decreased fibrinogen and in vascular purpura. Petechiae - small red spots BLEEDING TIME • • • • • • Incision made on skin of patient making use of lancet Used to monitory aspirin therapy Duke’s method o Most practical method; Only needs lancet, cotton, filter paper, and a timer. o Patient is pricked using a lancet on the fingertip or earlobe. ▪ Earlobe - not recommended due to less venous access despite less nerves ▪ Fingertip - adults ▪ Plantar surface of heel - newborns o Time is them measured from the time of pricking up to the time when bleeding stops. ▪ Blot every 15 seconds o 2-5 mins Ivy’s method o Better then Duke’s method; Constant pressure is applied (Use of pressure cuff) o Puncture volar surface of forearm with blood Pressure cuff set at 40mmHg using a lancet. ▪ Three incisions are made (triangle form) ▪ Average time is taken o Time is them measured from the time of pricking up to the time when bleeding stops. o Longer due to use of pressure; 2mins to 7mins Modified Ivy’s method o Same as Ivy’s method with the exclusion of pressure cuff application. o Aka Modified simplate method. o 3 to 5 mins. Adelson-Crosby method o Same as Duke but with the addition of immersion of punctured finger in sterile Physiologic saline solution (NSS) warmed at 37℃ waterbath to check bleeding. o Same as dukes (2-5mins) • • Rumpel-Leede test/Tourniquet test/Hess test/Positive pressure technique o Uses blood pressure cuff (maintained at pressure midway between systolic & diastolic pressure of px o 80 mmHg o Reference Range: 1 – 10 petechiae (0-10) o 8 minutes Petechiometer method/Suction cup method/Negative pressure method o 200 mmHg o Reference Range: <4 petechiae o 1 minute CLINICAL SIGNIFICANCE • • • Due to o Prolonged bleeding time o Increased capillary fragility - increased petechiae Vascular disorders o Purpuras - also called blood spots or skin hemorrhages; refers to purple-colored spots that are most recognizable on the skin. Quantitative platelet disorders o Thrombocytopenia - Decreased number of platelets in the circulation. o Thrombocytosis - Increased number of platelets in the circulation. ▪ Have same manifestations w/ thrombocytopenia • Qualitative platelet disorders o Bernard-Soulier Disease - inherited disease wherein there is a lack of GP1b; abnormal adhesion o Glanzmann’s thrombasthenia - inherited disease wherein there is an abnormality in the production og GP2b/3a; abnormal aggregation o Others (Granules deficiencies eg. Chediak-Higashi Syndrome, Grey platelet syndrome) ▪ Grey platelet - observable in PBS • Lacks platelet granules ▪ PBS • Check for adequacy - 4-20 plts/OIF o <4 - inadequate • Check for plt morphology o Size - ¼ - 1/3 RBC size • Check for plt granules CLASSIFICATIONS OF COAGULATION FACTORS • • SECONDARY HEMOSTASIS • • • Involves activation of plasma proteins (serine proteases) in the coagulation system to form a fibrin meshwork. Coagulation - A process whereby, on vessel injury, plasma proteins, tissue factors, and calcium interact on the surface of platelets to form a stable fibrin clot. Platelets also interact with fibrin to form a stable platelet-fibrin clot (end product). o Stable - product/action of CF XIII Coagulation factors are numbered in roman numerals, not hindu-arabic. COAGULATION FACTORS Roman Preferred Descriptive I Fibrinogen II Thrombin III Tissue factor IV Calcium V Proaccelerin VI Omitted VII Proconvertin COAGULATION FACTORS PATHWAYS Synonyms Biochemistry Ia-Fibrin IIa-Thrombin Thrombloplastin Glycoprotein Labile factor According to hemostatic function: o Zymogens ▪ Inactivated form of the coagulation factor. ▪ 2, 5, 7, 9-12, Prekallikrein, 13 o Cofactors ▪ Substances needed by the enzyme to proceed with the reaction. ▪ 3, 5a, 8a, HMWK o Enzymes/Serine Protease ▪ Activated form of the coagulation factor. ▪ Enzymes that cleave peptide bonds in proteins. ▪ 1a, 9-11a, 13a, Kallikrein, Thrombin According to Physical properties o Prothrombin group/Vit. K dependeng group ▪ Requires Vitamin K for its production in the liver. ▪ Synthesized by the liver in the presence of Vit. K ▪ 2, 7, 9, 10 ▪ Also requires presence of Calcium for activation o Fibrinogen group/Labile group ▪ Factors consumed during the clotting process. ▪ Calcium dependent ▪ 1, 5, 8, 13 ▪ 5 & 8 - lost in non-fresh sample (>8hrs) o Contact group ▪ First factors that are activated during the coagulation process. (Intrinsic pathway) ▪ Not consumed; Involved in intrinsic pathway ▪ 11, 12, PK, HMWK • • Lipoprotein Metal ion Glycoprotein Stable factor, Serum Glycoprotein prothrombin conversion accelerator VIII Antihemophilic Antihemophilic (VIII:c) Factor (AHF) Globulin (AHG), Antihemolytic factor A IX Plasma Antihemophilic factor Thromboplastin B/Christmas factor Component (PTC) X Stuart-Prower Stuart factor Factor Prower factor XI Plasma Antihemophilic factor Beta/Gamma Thromboplastin C globulin Antecedent (PTA) XII Hageman Contact factor Sialoglycoprotein Factor Glass factor XIII Fibrin Laki-Lorand Factor Beta/Gamma Stabilization Fibrinase Globulin Factor Pk Prekalikrein Fletcher factor Plasma protein HMWK High Molecular Fitzgerald/William/ Weight Flaujeac factor Kininogen (HMWK) • Factor VI - omitted due to being activated form of CF V • Intrinsic pathway o Activated by contact with S.E.C. Extrinsic pathway o Activated by contact with Tissue thromboplastin Common pathway o A cascade that results in the activation of prothrombin that is needed to convert fibrinogen to fibrin. EXTRINSIC PATHWAY • • Composed of Factor VII. Activated by the release of Tissue factor from the injured vessel into the plasma. • • • • Tissue factor – Also known as tissue thromboplastin; Secreted by tissues. Tissue factor is capable of binding to Factor VII, converting it to Factor VIIa. Factor VIIa will then activate Factor X to Factor Xa. Tissue factor is secreted by the ff organs: lungs, liver, kidney, brain, placenta, spleen. BLEEDING DISORDERS (C.F. DEFICIENCIES) Coagulation Factor Deficiency Factor I INTRINSIC PATHWAY • • • • • Composed of Factors VIII, IX, XI, XII, HMWK and Prekallikrein. Activated when the S.E.C. comes into contact with the coagulation factors. Factors XI, XII, Prekallikrein, and HMWK interact on the surface to activate Factor IX to Factor XIa Factor Xia will then react with Factor VIII, PF3, and Calcium to activate Factor X to Factor Xa. Thromboplastin is derived from phospholipoproteins and organelle membranes from disrupted tissue cells. These membrane lipoproteins, termed tissue factors, are normally extrinsic to the circulation. Clinical Significance Afibrinogenemia (absence) Hypofibrinogenemia Dysfibrinogenemia (dysfunction) Factor II Prothrombin deficiency Hypoprothrombinemia Factor V Owren’s Disease Parahemophilia Factor VII Hypoproconvertinemia Factor VIII Hemophilia A Classic Hemophilia Royal’s disease Factor IX Christmas factor disease HemoPhilia B Factor X Stuart-Prower disease Factor XI HemoPhilia C Rosenthal Diseas Factor XIII Associated with Poor wound healing • A deficiency of even 1 coagulation factor will result in bleeding tendencies. LABORATORY EVALUATION OF SECONDARY HEMOSTASIS COAGULATION TIME • • • • • Negatively charged surfaces are capable of activating Factor XII. o In vivo – Collagen, Elastin o In vitro – Glass, Kaolin COMMON PATHWAY • • • • Starts with the activation of Factor X to Factor Xa by either intrinsic or extrinsic pathway. Factor Xa, with the help of Calcium, Factor V, and PF3, will activate Prothrombin to Thrombin. Thrombin will then activate Fibrinogen to become Fibrin. (Fibrin monomer) Upon the action of Factor XIII, Fibrin monomers will be strengthened and produce a stable fibrin clot. • Aka whole blood clotting time Tests the composite action of all plasma factors acting simultaneously. Clotting time is a measure of the ability of the blood to clot and is not influenced by the platelet functions other than PF3 (platelet phospholipid). It also measures only the time required for the formation of the traces of thrombin sufficient to produce a visible clot. Micro Methods o Sample: Fresh whole blood from prick o Slide or Drop Method 1. Puncture the ring/middle finger 2. Start timer then transfer blood to slide every 30 seconds. 3. Observe for fibrin string. o Capillary or Dale and Laidlaw’s Method 1. Collect non-heparinized blood 2. Start timer and fill 3 capillets 3. After 30 secs, break the capillet 4. Observe for fibrin string. o There may be a tendency of milking site of puncture ▪ Milking site of puncture initiates extrinsic pathway of coagulation which introducing extra tissue fluid activating extrinsic pathway • • Macro Methods o Superior for there is less contamination of the plasma with tissue fluids when blood is drawn from a vein. o Sample: Whole Blood o Lee-White Method or Whole Blood Clotting Time ▪ Use three test tubes 1. Wash the test tube using sterile NSS 2. Collect 3 ml of blood and transfer it to the test tube 3. Every 30 secs, tilt the tube and observe for clot. Normal Value: 5-10 minutes OTHER TESTS • • PLASMA RECALCIFICATION TIME • • • • • More sensitive method than the coagulation time of whole blood because there is an activator added. May reveal abnormality which is not detectable by the determination of the clotting time of venous blood. The activated recalcification time makes use of 0.25 M CaCl2 as reagent o Factor II, VII, IX, X depend on calcium for coagulation cascade for clot formation Sample: Platelet-poor Plasma (heavy spin) Normal Value: Less than 50 seconds PARTIAL THROMBOPLASTIN TIME (PTT) • • • • Test for the intrinsic and common pathways of coagulation. Calcium ions and phospholipids that substitute for platelet phospholipids are added to blood plasma. Detects deficiencies in Factors XII, XI, IX, VIII:c, X, V, II, I o Increased PTT Reagents: phospholipids and calcium ion ACTIVATED PARTIAL THROMBOPLASTIN TIME (APTT) • • • • • Similar to PTT test but with the addition of activators to hasten the production of a visible result. o silica or ellagic acid, kaolin, and phospholipids Test for the deficiencies of factors in the intrinsic system. For monitoring unfractionated heparin therapy o LMWH - tested for anti-Xa Normal Range: 25-35 seconds Detects deficiencies in Factor VIII:c, IX, XI, XII. X, V, II, I PROTHROMBIN TIME • • • • • • Measures the extrinsic and common pathway of coagulation. It is used to monitor oral anticoagulant therapy (Warfarin) Used to detect VII deficiency. o Also prolonged in deficiency in X, V, II, I Principle: o Tissue thromboplastin and calcium added to plasma react to fibrinogen to form a clot. The thromboplastin added to the plasma takes the place of the tissue juice formation of extrinsic thromboplastin. The prothrombin time is therefore prolonged if there is a deficiency of Factors V, VII or X or a very severe deficiency of Factor I and II. International Normalized Ratio (INR) is reported instead o INR = (PTpx / mean PTnormal)ISI o ISI = International Standardized Index Normal Range: 10-14 seconds • • • Thrombin Time (TT) o Measures the time of the conversion of fibrinogen to fibrin clot. o Reagent: thrombin ▪ Cleaves fibrinogen to form fibrin Reptilase Time o Evaluation of a prolonged thrombin time (TT): It is mainly used to confirm or exclude the presence of heparin in the specimen or specimen type. o Uses Reptilase from the snake venom of Bothrops atrox.(same action as thrombin) o TT - affected by heparin therapy o Reptilase time - not increased in presence of heparin o Normal Range: 18-20secs Stypven Time (Dilute Rusell’s viper venom Time) o Used to distinguish deficiencies of Factor X and those of Factor VII. It is also used to detect deficiencies in prothrombin, fibrinogen and factors V and X. It differs from prothrombin time in that deficiencies in factor VII are not detected. o Assess common pathway of coagulation o Prolonged in deficiencies of X, V, II, I o Reagent: Rusell viper venom Duckert’s Test o Used to detect deficiency in Factor XIII.