UNIT NINE HAEMOSTASIS & BLOOD COAGULATION: 1 UNIT NINE HAEMOSTASIS & BLOOD COAGULATION By: Bamlaku Enawgaw university of Gondar, GCMHS, MLS email@example.com +251 913 15 02 39 Objectives: Objectives At the end of this chapter, you will be able to: Normal and abnormal hemostasis Discuss how the components of normal hemostasis interact Explain the intrinsic and extrinsic pathways Normal control of the clotting process and the fibrinolytic system State the principles of the different tests of the bleeding disorders HEMOSTASIS : 3 HEMOSTASIS Haemostasis or Hemostasis ( Greek : aimóstasis , from aíma "blood" + stásis "stagnation") is a complex process which continually ensures: prevention of spontaneous blood loss and stops hemorrhage caused by damage of vascular system Most times this includes the changing of blood from a fluid to a solid state and then to fluid state. Function of Haemostasis : 4 Function of Haemostasis Arrests bleeding Keeps blood in fluid state Repair and reestablish the blood flow through the injured vessels Remove haemostatic plug If any of the above functions is exaggerated or impaired it will cause either thrombosis or hemorrhage respectively; so hemostasis is a balance between thrombosis and hemorrhage Mechanism of Hemostasis: 5 Mechanism of Hemostasis Haemostasis involves a series of delicately balanced physical and biochemical changes following an injury to a blood vessel . As the most immediate response, the blood vessel constrict Then platelets adhere and aggregate at the site of the injury and form a plug Mechanism of Hemostasis: 6 Mechanism of Hemostasis These activated platelets secretes substances that initiate the coagulation factors which interact serially, forming a fibrin network or clot in which white cells, red cells and platelets are trapped and form a solid plug of blood ( coagulation) which seals off the injury vessel completely. Finally slow lysis of the clot, fibrinolysis, begins and the site of the injury is repaired. Components of normal hemostasis: 7 Components of normal hemostasis vessels Tissue Tissular coagulation factor Platelets Thrombocytic Platelet Coagulation factors Coagulation (activator & inhibitors) Humoral ( plasma factors) Fibrinolysis (activater & inhibitors) Phases: Phases Three –phase process Primary hemostasis Coagulation (secondary hemostasis) Fibrinolysis Phases cont’d: Phases cont’d I. Primary hemostasis Involves Blood vessels (vascular vasoconstriction phase and release of tissue or exogenous factors) Thrombocytes (platelate or endothelial –thrombocyte phase, platelate aggregation and release of platelate factors). After 3 to 5 minutes , blood flow is arrested with the formation of a platelate plug . Phases cont’d: Phases cont’d II. Coagulation Involves Plasma coagulation factors (plasma phase) Platelate factor 3 provides for definitive hemostasis Takes 5 to 10 minutes by formation of fibrin Reinforces the platelate plug. Phases cont’d: 11 Phases cont’d III Fibrinolysis Essential final step in any hemostasis mechanism, Enabling in 48 to 72 hours, and a Return to normal by destroying fibrin and healing the injured vessel. I. Primary hemostasis: I. Primary hemostasis Is a result of a three –way interaction between: Vascular wall Platelets plasma coagulation factors Triggered by Small injuries to blood vessels The plasma coagulation factors desquamation (damaging of epithelial cells in pinpricks) Primary hemostasis: 13 Primary hemostasis Involves: Vasoconstriction Platelet adhesion Platelet aggregation Platelet secretion A. Vasoconstriction : A. Vasoconstriction The Blood vessel wall First line of defense for normal hemostasis Lined with endothelial cells which synthesize von Willebrand factor (vWF) vWF is secreted into the circulation or onto the collagen-containing sub-endothelium. Following endothelial cell damage and sub-endothelial exposure, platelets bind to vWF and collagen to initiate hemostasis. These form a tight selective membrane that keeps blood cells and plasma inside the vessel The vascular system cont.d: The vascular system cont.d Endothelial cells also produce a fibrinolytic activator protective function to prevent blockage of blood vessels by clots Nerve and muscular tissue in the supporting sub endothelial (under the endothelium) tissue allow Constriction of the vessel when injured (muscle cells contract) The vascular system cont…d: The vascular system cont…d The vascular system cont….d: The vascular system cont….d The blood vessels allowing the passage of gases, nutrients, selected cells to enter or leave the system In the normal state, the endothelial cells produce a substance called prostacyclin & other substances Inhibit platelet function (cause disaggregation of platelets). The vascular system cont.d: The vascular system cont.d Vasoconstriction: Constriction of small vessels such as arterioles, venules, capillaries slows or stops blood flow Activated and controlled by neural mechanisms and other substances released from platelets Serotonin ---- constricts blood vessels Thromboxane A2 --- Is a powerful vasoconstrictor and platelet aggregating agent The vascular system cont.d : The vascular system cont.d Slowed bleeding more effective platelet contact activation adhesion of platelets to the exposed subendothelial tissue wound sealed and vascular lumen narrows and closes and blood flow to the injured site minimized Note: this effect is temporary lasting up to 20 minutes needs to be supplemented by platelets and blood coagulation factors B. Platelets : B. Platelets Platelets are Enucleated, Cytoplasmic fragments of the megakarocyte mother cell 4 stages of development: Megakaryoblast, Promegakarocyte, Megakaryocytic, Thrombocyte Each megakaryocyte produces 2000-4000 platelets Platelets cont’d: Platelets cont’d Young platelets are larger & less dense than older platelets. also metabolically active and more effective in hemostasis Platelet turn over rate equals 35,000 4,300 per L each day Size 2-20 fL; 2-4 m in diameter, colorless, with Wright’s stain they stain blue with pink granules Platelets cont’d : Platelets cont’d Platelets cont’d : Platelets cont’d Life span ~9 days (7-10 days) Production regulated by thrombopoietin (plus others) Shaped like a porous disc & resemble a microscopic sponge Normally 2/3 of the platelets released from the bone marrow stay in the circulation; the remaining sequestered in a splenic pool that is freely exchangeable with circulating platelets Platelets: Platelets Function of platelets Maintain the functional integrity of the endothelial surface Initially arrest bleeding by forming temporary hemostatic platelet plug Provide phospholipids (Platelet factor 3) acts as a catalytic surface for initiation of the coagulation process When there is an injury platelets undergo the following actions: Adhesion Release reaction Aggregation B. Platelet Adhesion : B. Platelet Adhesion It is the binding of platelet to non platelet surface: sub endothelial collagen involves changes from a disc shape to a slightly broader, plate like form to increase surface area a number of plasma proteins are required for normal platelet adhesion. Thrombin Fibronectin vWF vWF is the largest component of factor VIII and secreted by platelets and by vascular endothelial cells. Adhesion cont’d : Adhesion cont’d Collagen – vWF –Platelet vWF bridge physical distance between platelate and sub endothelial collagen Increase bond that seal platelet to the vessel wall reversible C. Platelet secretion: C. Platelet secretion It is release of contents of the granules of platelet Primarily ADP stimulates aggregation Cathecolamine (especially epinephrine) and serotonin enhance vasoconstriction Platelets contain 3 types of secretary granules: Lysosome containing acid hydrolyses α-granules containing platelate specific proteins (Plt factor 4, β- thromboglobulin, as well as other proteins such as Platelet derived growth factor and coagulation proteins found in plasma (fibrinogen & von Willebrand’s factor) -granules containing ATP, ADP, Calcium & serotonin Release reaction cont’d : Release reaction cont’d Slide 29: 29 D. Platelet aggregation Platelet-Platelet interactions Platelets are bind to each other (platelets fill open space to form a plug.) Triggered by: PLT factor 3, adenosine diphosphate (ADP), Thrombin and Thromboxane Need fibrinogen to bridge platelet to platelet distance and encourage platelet plug. Irreversible once platelet aggregate they do not disaggregate. Aggregation : Aggregation Platelates shed membranes rich in phospholipid (appearance of PLT factor 3 on the PLT membrane) this happens during PLT plug formation serves as a catalytic site for the coagulation proteins Aggregation is also a response to helps initiation of the coagulation mechanism Aggregation cont’d: Aggregation cont’d platelet aggregation SECONDARY HAEMOSTASIS (COAGULATION): 32 SECONDARY HAEMOSTASIS (COAGULATION) coagulation : 33 coagulation In the coagulation or plasma phase , blood changes from the fluid state to the gelled state , a result of the transformation of a soluble protein , fibrinogen , into an insoluble protein , fibrin . This forms the network around which the clot will be formed. This change in state corresponds to a cascade of enzyme activity whose first step have the function of amplifying the entire process of fibrin formation. This cascade requires a large number of protein factors , most of which are present as pro enzymes and which are transformed by partial proteolysis to active forms. Procoagulants are the enzymes ( zymogens) and Co - factors Enzymes( Serine Proteases): 34 Enzymes( Serine Proteases) Are proteolytic enzymes Hydrolyze peptide bonds Synthesised as inactive zymogen Activated zymogen –cleaved at sites by another proteases Activation is localized ( at sites of injury) DIC-fatal condition Plasma coagulation factors Nomenclature : 35 Plasma coagulation factors Nomenclature Plasma coagulation factors have various names but an internationally standardized nomenclature system is using Roman numeral designations. A lower case “ a” indicates the active factor (e.g. factor IXa) Roman numerals indicate inactive forms as they exist in the plasma except factors III & IV. They reflect order of discovery but not the sequence of reaction in the coagulation system Slide 36: 36 Factor I (Fibrinogen) Large, stable globulin protein Precursor of fibrin, which forms clot With thrombin, fibrinogen molecule, leaving a fibrin monomer. The monomers aggregate together to form the final polymerized fibrin clot product Slide 37: 37 Factor II (Prothrombin) Is a stable protein In the presence of ionized calcium, prothrombin is converted to thrombin by the enzymatic action of thromboplastin from both extrinsic and intrinsic sources. Slide 38: 38 Factor IIa (Thrombin) THROMBIN is considered the most important protease of the coagulation pathway 70% consumption during clotting Is the activated form of prothrombin This proteolytic enzyme, which interacts with fibrinogen, is also a potent platelet-aggregating substance Activates co factors V and VIII , F XI and F XIII Cont..: 39 Cont.. Tissue thromboplastin (factor III) Tissue thromboplastin is the term given to any non-plasma substance containing lipoprotein complex from tissues. These tissues can be from the brain, lung, vascular endothelium, liver, placenta, or kidneys; Are capable of converting prothrombin to thrombin Slide 40: 40 Ionized calcium (formerly factor IV) Necessary for the activation of thromboplastin, and for conversion of prothrombin to thrombin. Ionized calcium is the physiologically active form of calcium in the human body and only small amounts are needed for blood coagulation. Cont..: 41 Cont.. Factor V (Proaccelerin) Extremely labile globulin protein. Deteriorates rapidly Consumed in the clotting process and is Essential to the thromboplastin formation Slide 42: 42 Factor VII (proconvertin) It is not destroyed or consumed in clotting and is found in both plasma and serum Activates tissue thromboplastin and the acceleration of the production of thrombin from prothrombin. Cont..: 43 Cont.. Factor VIII (Antihemophilic Factor) This factor is consumed during the clotting process and is not found in serum. Factor VIII is extremely labile, with a 50% loss within 12 hours at 4 o C in vitro and a similar 50% loss in vivo within 8-12 hours after transfusion. Consists of the von Willebrand factor (vWF). Slide 44: 44 Factor IX (Plasma Thromboplastin component) Stable protein factor that is neither consumed during clotting nor destroyed by aging at 4oC for 2 weeks. It is an essential component of the intrinsic thromboplastin generating system, where it influences the amount rather than the rate of thromboplastin formation. Cont..: 45 Cont.. Factor X (Stuart Factor) Relatively stable factor that is not consumed during clotting. Together with factor V, factor X in the presence of calcium ion forms the final common pathway Factor XI (Plasma Thromboplastin Antecedent) Partially consumed during the clotting process. Essential to the intrinsic thromboplastin-generating mechanism Cont..: 46 Cont.. Factor XII (Hageman factor) Stable factor that is not consumed during the coagulation process. Adsorption of factor XII and kininogen (with bound prekallikrein and factor XI) to negatively charged surfaces such as glass or subendothelium (Collagen) exposed by blood vessel injury intitiates the intrinsic coagulation pathway. Surface absorption alters and partially activates factor XII to factor XIIa by exposing an active enzyme (protease) site. Kallikrien (activated Fletcher factor) cleaves partially activated factor XIIa molecules adsorbed onto the subendothelium to produce a more kinetically effective form of XIIa. Cont..: 47 Cont.. Factor XIII (Fibrin-Stabilizing Factor) In the presence of ionized calcium produces a stabilized fibrin clot The clotting mechanism responsible for the formation of fibrin involves a cascade of reactions in which inactive enzymes (zymogens) are activated, and the activated enzymes in turn activate other inactive enzymes. Cont..: 48 Cont.. Can be gouped as 1. Fibrinogen group Thrombin sensitive I, V, VIII,XIII 2. Prothrombin group Vitamin-K dependent II,VII,IX and X 3. Contact group XI, XII, PK, HMWK 4. Co factors: : 49 4. Co factors: Each bind serine protease -gain stability -increase reactivity - HMWK- K,XI - VIII-IX - V -X - Tissue factor (III)-VI - non circulating constituent protein of the sub endothelium - Tissue thromboplastin= mixture of tissue factor + phospholipid : 50 5. Additional components: Ca 2+, v W F, Phospholipid Platelate Factor 3 or endothelial cell membrane (phospholipid) Not in fluid phase Phospholipid is an assembly molecule COAGULATION PATHWAYS: 51 COAGULATION PATHWAYS Contact activation pathway (intrinsic) : 52 Contact activation pathway (intrinsic) Can be brought about in vitro by exposing the blood to electro-negatively charged surfaces such as glass & collagen fibers Activation in vivo occurs when blood is exposed to collagen fibers underlying the endothelium in the vessels Cont..: 53 Cont.. Contact factors do not have an in vivo procoagulant function. But responds to the negatively charged surfaces: non siliconized glass Serine protease bind negatively charged phospholipid surface through positively charged Ca 2+ Ca involved in most reaction Ca bridge platelate factor with phospholipid, and platelate factor to platelate factor binding In vivo activated by Valve prostheses Artificial implants in surgery Exposure to foreign substances: sub endothelial collagen Cont..: 54 Cont.. Although intrinsic pathway is more complex & slower, it accounts for the majority of the coagulation activity in vivo Factor XII, Prekallikrein, HMW kininogen are referred to as the contact proteins, because their activation occurs on contact with an abnormal surface or (glass or kaolin) Contact activation pathway (intrinsic): 55 Contact activation pathway (intrinsic) The contact activation pathway begins with formation of the primary complex on collagen by high-molecular-weight kininogen (HMWK), prekallikrein , and FXII (Hageman factor). Prekallikrein is converted to kallikrein and FXII becomes FXIIa. FXIIa converts FXI into FXIa. Factor XIa activates FIX, which with its co-factor FVIIIa form the tenase complex, which activates FX to FXa. Slide 56: 56 Tissue factor pathway (extrinsic): 57 Tissue factor pathway (extrinsic) Following damage to the blood vessel, FVII leaves the circulation and comes into contact with tissue factor (TF) expressed on tissue-factor-bearing cells (stromal fibroblasts and leukocytes), forming an activated complex (TF-FVIIa). TF-FVIIa activates FX. FXa and its co-factor FVa form the prothrombinase complex, which activates prothrombin to thrombin. Thrombin then activates fibrinogen. Finally leads to formation of fibrin network. Slide 58: 58 diagram Common pathway: 59 Common pathway Activated factor X, in association with cofactor on phospholipid surface and calcium, converts prothrombin to thrombin Thrombin converts fibrinogen to fibrin Slide 60: 60 Fibrinolysis : 61 Fibrinolysis Fibrinolysis is the process wherein a fibrin clot , the product of coagulation , is broken down. Its main enzyme plasmin cuts the fibrin mesh at various places, leading to the production of circulating fragments that are cleared by other proteases or by the kidney and liver . Necessary for tissue repair to proceed and for normal circulation to resume For hemostasis to be effective, normal balance must exist between clot formation and removal Slide 62: 62 Control of coagulation : Control of coagulation Equally important are mechanisms that prevent inappropriate activation of the cascade. Natural or innate inhibitors and anticoagulants circulate in the plasma, limiting the initiation and extent of fibrin formation. There are several protective mechanisms against thrombosis; the most important are: Removal of activated clotting factors by blood flow past the clot or by the liver (Hepatocytes) Inactivation of clotting factors by circulating inhibitors. Slide 64: The natural anticoagulant system in vivo includes: anti-thrombin III , whose activity is facilitated by heparin, is synthesized by hepatocytes, megakaryocytes, and vascular endothelium. It inhibits activated factors II, IX, X, XI, and XII; Heparin: produced endogenously by mast cells, inhibits thrombin or fibrinogen. Also affected are thrombin induced platelet aggregation and release. Protein C, which requires Protein S as a co-factor synthesized by the liver (both are vit K dependent) Consumption of platelets and clotting factors by the clotting process Degradation of the clot by the fibrinolytic enzyme, plasmin PHYSIOLOGICAL COAGULATION INHIBITORS: PHYSIOLOGICAL COAGULATION INHIBITORS The extent of the clot should be confined to the immediate surrounding area of the vascular lesion. Thus, the inhibitory systems control thrombin production. 1. Antithrombin III Is a serine protease inhibitor A glycoprotein of hepatic origin Most powerful physiological coagulation inhibitors Markedly inhibiting thrombin (FIIa) To a lesser degree on factors Xa, IXa, XIa, XIIa and kallicrein Requiers Heparin for effective anticoagulant activity Cont..: Cont.. 2. Protein C-Protein S Protein C : vitamin K-dependent - synthesized in the liver - zymogen which is expressed after activation, as a serine protease (activated Protein C) activated by thrombin in the presence of Ca 2+ and a cofactor located on the surface of endothelial cells, thrombomodulin. Protein Ca (activated Protein C) inactivates the major proteins, factors Va and VIIIa. It requires as a co factor a phospholipids surface, Ca 2+ and is greatly enhanced by a plasma protein, Protein S Cont..: Cont.. Protein S is - Vitamin K- dependent factor -synthesized in the liver present in two forms in the plasma, a circulating form, and a form bound to the fourth component of complement (C4b) binding protein only circulating Protein S is active as a cofactor of activated Protein C Protein Ca activity is regulated by inhibitor ( PCI) Cont..: Cont.. 3 . Heparin cofactor II (HC II) a glycoprotein synthesized in the liver unlike AT III, HC II is a very specific inhibitor and only neutralizes thrombin efficiently this action is accelerated by heparin and dermatan - sulphate Cont..: Cont.. 4) Tissue factor pathway inhibitor (TFPI) - is emerging as the most important regulatory mechanism in vivo coagulation A) synthesised by endothelial cells and circulates in plasma bound to low density lipoproteins B). also present in platelets and bound to heparan sulphate at the endothelial surface TFPI halts continued direct generation of factor Xa. Thus,continued Xa formation early becomes dependent on ongoing activation factor X by the IXa-VIIIa phospholipid complex C). TFPI inhibits coagulation by binding to factor Xa and TF:VIIa complex and inhibiting their proteolytic activity PHYSIOLOGICAL INHIBITOR OF THE FIBRYNOLYTIC SYSTEM: PHYSIOLOGICAL INHIBITOR OF THE FIBRYNOLYTIC SYSTEM As in coagulation, fibrynolysis is confined to the clot surface and controlled by inhibitors Their target is : - either the activation system The strongest inhibitor is the Plasminogen Activator Inhibitor (PAI). PAI inhibits t-PA and u-PA or plasmin whose principal inhibitor is alpha 2- antiplasmin Bleeding and Coagulation disorders : Bleeding and Coagulation disorders Vascular defects Platelet defects Coagulation factors Vascular defects Inability to contract after injury Causes include: Ascorbic acid (vit C) deficiency Inflammation Certain toxins Aging and Congenital defects e.g. hereditary hemorrhagic telangiectasia In these conditions, bleeding in to the skin produces ecchymoses called “vascular purpura” Slide 75: Platelet defects Quantitative and qualitative defect Thrombocytopenia Decreased production Hypoplasia Marrow replacement by tumor or malignant cells Immune damage from toxins, drugs, bacterial & viral infections Idiopathic (ITP) Ineffective maturation e.g. in megaloblastic anemia Increased destruction or utilization Autoimmune antibodies DIC (disseminated intravascular defect) Slide 76: Disorders of PLT function (qualitative defect) Acquired or inherited Acquired Platelet disorders Many drugs e.g. asprin, other non-steroidal anti-inflammatory drugs By inhibiting platelet prostaglandin synthesis (TXA2) Other disorders causing thrombocytosis Hereditory Platelet disorders Thrombasthenia defect in primary platelet aggregation Coagulation factor defect and inhibitors: Coagulation factor defect and inhibitors Coagulation factor defect or abnormal function Factor deficiencies The most important congenital deficiency is Factor VIII deficiency: Called Hemphilia A Graded as severe, moderate, and mild depending on the coagulant activity of Factor VIII Inherited as sex-linked ressesive manner and occurs exclusively in males Slide 78: von Willebrand’s disease: Due to deficiency of vWF Characterized by defects in platelet adhesion Factor IX deficiency: Called Hemophilia B Sex linked recessive Occurs less frequently and milder in its clinical presentation than factor VIII deficiency (Hemopilia A) Abnormal coagulation factors function : Abnormal coagulation factors function Abnormality in function is seen in vit K deficiency. The binding of Ca++ to factors II, VII, IX, X is required for normal clotting. Without the attached Ca++, these fators will not bind to phospholipids and rate of factor activation will be sharply decreased. To bind Ca++ they need gamacarboxylation with vit K (to make II, VII, IX & X functional) Vit K dependant factors are: Factors II, VII, IX, X, Protein C & protein S Slide 80: Consumption of coagulation factors e.g. DIC acquired coagulation defect secondary to other pathologic processes which results in accelerated consumption of platelets and several coagulation factors, particularly fibrinogen Inhibitors of coagulation e.g Lupus anticoagulant in patients with SLE and other related disorders. Lupus anticoagulant interferes with the phospholipid portion of Factor Xa-V-Ca++-Plt phospholipids complex. Slide 81: Disturbance of the balance between promoters and inhibitors of coagulation due to: Various disorders including bacterial, viral, rickettsial, infections Complication of pregnancy AML Tissue damage (shock, heat strock, burns) Hemolytic transfusion reactions Venome snake bites Laboratory investigation of bleeding and coagulation disorders : 82 Laboratory investigation of bleeding and coagulation disorders Bleeding Time Coagulation time Platelet count Clot retraction time Prothrombin time (PT) Accctivated Partial Thromboplastin Time (APTT) The Thrombin time Fibrinogen quantitative assays Bleeding time: : 83 Bleeding time: Measures the time required for the blood to stop flowing after a standardized capillary puncture. This depends on the number and function of platelets and capillary integrity Assesses platelet adhesion & aggregation, number of platelets and capillary integrity. prolonged in: Shortage of platelets Inadequate function of platelets von Willebrand’s disease Poor retract ability of capillaries (e.g. scurvy-vit C deficiency) Deficiency of plasma factors Different methods:: 84 Different methods: Duke method: Oldest method performed by Puncture (3mm) the ear lobe and record time blotting the blood every 30 seconds until bleeding ceases and record time. Blotting is done without allowing the filter paper to touch the wound Time between the puncture and the cessation of bleeding is the BT NR = 1-3 min (3-6 min boarder line) Drawback: impossible to standardize depth of incision; Cont..: 85 Cont.. B. Ivy method A blood pressure cuff is placed on the patient’s arm above the elbow, inflated & maintained at a constant pressure (40 mmHg) This is to standardize the pressure in the vascular system. Two or three standardized (3 mm) punctures of the forearm are made. The length of time required for bleeding to stop is recorded. Report the average of the two results Cont..: 86 Cont.. NR = 1-7 min; 7-11 min boarder line Bleeding > 15 min, discontinue the procedure and report as >15 min. It is recommended to repeat the procedure on the other arm if bleeding time < 1min and >7 or 15 min Sources of error No bleeding because of too gentle an incision Severe bleeding: superficial veins have probably been cut If filter paper touches the wound, a platelet aggregate might be removed resulting in prolonged bleeding Cont..: 87 Cont.. Decrease bleeding time: Failure to cleanse the area Puncturing a cold blood less area Making superficial puncture Increase bleeding time: Puncturing a red flushed area Too deep puncture Applying pressure to the punctured area Slide 88: 88 Interpretation Conditions associated with prolonged bleeding time include : (i) Thrombocytopenia (ii) Inherited or acquired platelet function defects (iii) Inherited plasma defects (von Willebrand’s disease, factor V deficiency) (iv) Vascular abnormalities (v) Drugs e.g. aspirin and antihistamine 2. Coagulation time (Clotting time) : 89 2. Coagulation time (Clotting time) This test is a qualitative measurement of factors involved in the intrinsic pathway. Deficiency in the factors of intrinsic pathway (I,II,V,VIII,IX,X,XI,XII) will affect the result Principle The test is based on the fact that the whole blood, when added to a foreign surface such as glass, will form a clot. The time required for the formation of clot is a rough indication of the efficiency of the coagulation factors. Cont…..: 90 Cont….. Capillary blood methods Slide method (NR 2-6 min) Capillary tube (NR 2-6 min) Dale & Laidlaw (NR 1-3 min) Venous blood methods Lee & Wite method (NR 5-15 min) Howell method (NR 10-30 min) Silicone tube method (NR 20-60 min) Note: the report should always include method and normal range Slide 91: 91 A. Slide method Puncturing the finger and record the time, placing 3 drops of blood on a glass slide, drawing the point of the needle or lancet through the drops until fibrin threads appear & recording the time B. Capillary tube method Puncturing and record the time, Filling a capillary tube with blood allow 3 min Break the capillary tubes at half min intervals until a span of fibrin is seen and recording the time Slide 92: 92 C. Dale & laidlaw Puncturing and record the time, Allow the blood to flow into a capillary tube which contains a lead bead, Immerse the capillary tube in 37oC water, tilt the tube until the lead bead is held firmly by fibrin threads, & record the time D. Howel method Coat a syringe with petroleum, draw blood from a vein and record the time the blood enters the syringe, Transfer the blood to a test tube, tilt tube until coagulation takes place, & record time Slide 93: 93 E. Silicone tube method Coat a syringe with silicone to decrease the contact of blood with glass Draw blood from a vein & noting the time the blood enters the siliconized syringe, transferring the blood to 2 siliconized test tubes, placing the tubes on 37 o C water bath, tilting the tubes until coagulation takes place & recording the time F. Lee & White method In the past, screening test to measure all intrinsic coagulation system & to monitor heparin therapy. But it is time consuming, sensitive to only severe factor deficiencies & insensitive to high doses of heparin, and has poor reproducibility. Slide 94: 94 Procedure Label 3 clean test tubes (important) Draw 4 ml of blood, start the stopwatch as soon as blood enters the syringe Remove the needle & gently transfer about 1 ml of blood to each of the 3 test tubes Place the 3 test tubes in a 37 o C water bath Allow 4-5 min to elapse & gently tilt test tube #1 to a 45 0 angle every 30 sec until the blood is completely clotted Repeat the process with the 2nd and 3rd test tubes & record the time Coagulation time is the time elapsed between the withdrawal of blood and completion of coagulation in test tube #3. Slide 95: 95 Sources of error Coagulation hastened: Dirty test tubes Poor venipuncture can introduce tissue thromboplastin Air bubbles, faulty venipuncture Excessive agitation during transfer Coagulation retarded: Temperature < 35 o C & > 45 o C Diameter of the test tube; Increased volume of blood per tube Cont..: 96 Cont.. At completion of the Lee & White clotting time, it is suggested that 1 test tube remain in the 37 o C water bath to be checked after 2 & 4 hours for clot retraction. Also the same tube may be allowed to remain in the water bath over night & checked the next day for clot lysis NR 5-15 min Standardization of Clotting time can be achieved when: The amount of blood is consistent Process occurs at specified temperature and Clotting occurs in the same physical environment 3. Platelet count : 97 3. Platelet count The platelet count is the number of platelets in 1mm3 of undiluted blood A platelet count is mainly used in the diagnosis and treatment of hemorrhagic diseases Platelets are difficult to count because: Are very small Disintegrate easily Indistinguishable from dirt Clump easily Before diluting blood examine for clot Method: platelet count can be done by : 98 Method : platelet count can be done by Direct method In this method blood is diluted and platelet are counted with either microscope or electronic instrument Breaker – Cronkite method Principle; whole blood is diluted with 1% ammonium oxalate and platelets appear as a tiny glistering body Rees and Ecker’s method Principle: blood diluted with a solution containing brilliant crycyle blue which stains platelets a light bluish color. Note RBCs are not lysed. Slide 99: 99 Procedure The procedure the same as the procedure for RBC count Platelets appear as round, oval or elongated particles which are highly retractile and stain light bluish color and about 1/10 of the size of the red cell using 40x objective Calculation: Platelets in mm3 = N o of plt in 5 P section X DCF X VCF = n x 200 x 50 = n x 10,000 Note: Amonium oxalate should be stored in refrigerator and always be filtered just before use to remove crystals and other debris, which may be mistaken for platelets. NR=150-450 x 10 9 /L (98-337 x 10 9 /L in Ethiopia) Indirect method:: 100 Indirect method: There are two major steps 1. Determination of the ratio of platelet and red cells from stained smear 2. Count total red cell count and calculate total platelet Total platelet count = No of platelets X total red counted on the slide blood cell count No of RBCs counted on the slide E.g. Total red cell count = 5,000,000/mm3 No of platelets on the slide = 50 No of RBCs on slide = 1000 Platelet count = 50 x 5,000,000/mm3 1000 = 250,000/mm3 Slide 101: 101 Thrombocytosis In Polycythemia Vera: over active bone marrow resulting in increased production of WBC, RBC & Plt Idiopathic thrombocythemia (increased platelet number) CML Following splenectomy Sickle cell anemia: hyperactive bone marrow to produce more RBC, but increased WBC & Plts as well Slide 102: 102 Thrombocytopenia Thrombocytopenia purpura (Idiopathic or secondary to other diseases) Aplastic anemia: characterized by pancytopenia Acute leukemia: decrease in Plt and RBC production Pernicious anemia: decreased production due to deficiency of vit B12 Sometimes following chemotherapy and radiation therapy Platelet count may be below or above NR values in certain normal conditions and activities e.g. below normal: first few days of life and before menstruation above normal: at high altitude and after severe exercise Slide 103: 103 Platelet estimates (for QC purpose) In normal blood smear there are 8-20 Plts per field in the thin area (4-8 Plts/100 RBC; 30 Plts for every 500 RBC) One method of estimating platelets is to determine the average number of platelets per field, using 10 different fields, taking the average and multiply the result by 20,000 (comment as NR, decreased or increased) Never report an estimate; this is just for QC purpose Slide 104: 104 Sources of error Capillary blood: (platelets adhere & squeezing disintegration) Dirt or debris on chamber may be counted as platelets; Ammonium oxalate should be refrigerated and must be discarded if there is evidence of bacterial contamination and must be filtered Presence of clumps: test should be repeated. Dirty tubes, pipets, and counting chamber: Plts will stick to the dirt and falsely lower Plt count Results should be double checked by examination of Plts on a Wright stained blood smear. If the count does not agree with the estimate, it should be repeated 4. Clot retraction Time : 105 4. Clot retraction Time When blood coagulation is complete, the clot normally undergoes contraction, where serum is expressed from the clot, and the clot becomes denser (firm). Thrombosthenin, released by platelets is responsible for clot retraction. Clot retraction time measures the ability of the blood clot to retract. The time is affected by quantitative and qualitative defects in platelets. Cont…: 106 Cont… When the red cell count is high, degree of retraction decreased because of large volume of RBC in the clot, and vise versa. There are different methods. Some inspect the clot after 1, 2, 4 and 24 hours NR 2-4 hrs; Poor 4-24 hrs; >24 hrs reported as none 5. Prothrombin time (PT): 107 5. Prothrombin time (PT) Purpose of test: To evaluate the extrinsic pathway of coagulation (factors I, II, V, VII & X) To monitor warfarin anticoagulant therapy (coumarin) Principle : When tissue thromboplastin and calcium ions are added to plasma, extrinsic clotting factors are activated, resulting in the generation of thrombin and the formation of fibrin clot. Thus the test indicates functions of the factors II, V, VII and X A tissue extract serves as a source of thromboplastin. Slide 108: 108 Reagents Thromboplastin from a fresh human ( or rabbit) brain removed at post mortem or commercially available 0.025 M calcium chloride - Dissolve 2.7 g of calcium chloride in distilled water and make up to one liter Specimen Collect blood into a tube containing 3.8 % sodium citrate in the ratio of 9:1. Collect a control blood sample in the same way Slide 109: 109 Technique Centrifuge both the test and the control blood samples at 1000 g for 15 minutes to obtain platelet poor plasma (PPP) Add 0.1 ml thromboplastin and 0.1 calcium chloride in two glass tubes and place them at 37 C water bath Add 0.1 ml of normal control plasma to each tube and incubate for 1 minute and start the stop watch Tilt the tube repeatedly until a clot forms. Noting the time and take the average time Normal value 11 – 16 seconds Slide 110: 110 Diagnostic significance of PT To diagnose deficiencies in the coagulation factors of the extrinsic system It measures the functional activity of factors VII, X, and V, and factor II or I. Useful for checking the synthesis performance of the liver in hepatic disease Especially useful for initiation and monitoring of oral anticoagulant therapy to adjust the dose. Therefore, extreme care is needed. e.g. Warfarin is a vitamin K antagonist and interferes with the production of vit K dependent factors (factor II, VII, IX, and X) and Protein C & protein S. Protein C and S are natural anticoagulants. Slide 111: 111 PT Prolonged in: Deficiency of one or more coagulation factors in the extrinsic pathway: i.e., factors VII, X, V, and II or I Vit K deficiency Certain liver diseases Circulating anticoagulants Anticoagulant therapy (e.g. Coumarin) DIC (disseminated intravascular coagulation) Slide 112: 112 Quality control of PT Control and patient plasma should be run in duplicate and the two results averaged to obtain the final value. Duplicates should agree within 2 sec. Report both patient and control value; for patients on anticoagulant therapy, the control value is twice the normal. If the PT of the control plasma doesn’t lie within the specified values provided by the manufacturers, it indicates failure in equipment, reagent or techniques used and the test must be repeated Cont..: 113 Cont.. Most common sources of error The 9:1 ratio of blood to sodium citrate should be precise Failure to follow directions in the manufacturers instruction (package insert) strictly while preparing patient plasma, control plasma, reconstituting reagents Use of dirty or wet test tubes, pipets etc to perform the test Test must be performed within 4 hrs of specimen collection (within 2 hrs is best) Mistakes in pipeting Hemolysis Timing, incubation temperature, contact activation influence the test Slide 114: 114 INR values preferable to the PT because different thromboplastin reagents have different sensitivities to warfarin induced changes in levels of clotting factors The INR corrects most of reagent differences, expressed as ISI ISI is the international sensitivity index of the thromboplastin reagent; it is a correction factor assigned by the manufacturer International Normalized Ratio (INR) Cont..: 115 Cont.. ISI values reported by manufacturers vary depending on the instrument used to perform the PT The PT, utilized to adjust the dose of oral anticoagulation, should be reported according to the INR and not the PT ratio or PT in seconds The INR is essentaially a “corrected” PT INR = PT patient ISI PT normal 6. Activated partial thromboplastin time ( APTT) : 116 6. Activated partial thromboplastin time ( APTT) This test demonstrate deficiencies within the intrinsic pathway (factors I, II, V, VIII, IX, X, XI & XII). Also used to monitor patients treated with heparin. Specimen The citrated plasma is centrifuged at 1500 – 2000 g for 15 minutes to remove platelets. Reagents 1. Kaolin Suspend 0.5 g kaolin in 100 ml saline 2. Phospholipids (brain extract or partial thromboplastin) 3. 0.025 M calcium chloride Dissolve 2.77 g in one liter distilled water Slide 117: 117 Test principle of APTT: The plasma after centrifugation contains all intrinsic coagulation factors except Ca and platelets. Calcium and a substitute for Platelet factor III (partial thromboplastin-Cephalin) and an activator such as kaolin, to ensure maximal activation, are added to the plasma. The time required for the plasma to clot is the APTT. The activator is added to ensure maximal activation. Slide 118: 118 Mix 0.1 ml of well mixed kaolin suspension and 0.1 ml of phospholipids in a test tube. Keep it at 37 C for 15 seconds to warm Add 0.1 ml of plasma, previously warmed to 37 C Incubate for exactly two minutes At two minutes, add 0.1 ml of 0.025 M calcium chloride solution and start a stop watch. Leave the tube for 30 seconds, and then remove and check for clotting by tilting the tube Record the time taken to clot Normal range 36 – 50 seconds Technique Slide 119: 119 The activated partial thromboplastin time may be prolonged due to 1. Defects in the intrinsic system: e.g. Factor VIII: c, IX, XI, XII etc. 2. Defects in the common pathway e.g. Factors X, V, II, I 3. Inhibitory action of heparin, specific inhibitors of factors etc. Cont..: 120 Cont.. To control the variable of plasma contact, plasma is exposed to a specified amount of an activator to a standard amount of time. When an activator is not added the test is called PTT and the amount of time required for normal plasma to clot is prolonged Normal plasma should be run each time a new reagent is opened The APTT assay reflects the activity of prekallikrein, HMWK, and factors XII, XI, IX, VIII, X, V, II, and I APTT may be prolonged due to a factor decrease or presence of circulating anticoagulants. 7. Thrombin Time: 121 7. Thrombin Time Determines the rate of thrombin induced cleavage of fibrinogen to fibrin monomers and subsequent polymerization of fibrin polymers NR=<20 sec Prolonged When fibrinogen concentration is <100 mg/dL( Hypofibrinogenemia) Dysfibrynogenemia Afibrinogenemia:DIC,Liver disease Thrombin inhibitors or substances that interfere with fibrin formation (e.g heparin, fibrin degradation products) Thrombin time (TT) : 122 Thrombin time (TT) This test is used to detect deficiency of fibrinogen or inhibition of thrombin. It also measures formation of fibrin. Principle: Thrombin is added to citrated plasma at 370C and the time taken for the mixture to clot is noted. Reagent: Thrombin time kit Specimen : platelet poor plasma Slide 123: 123 Procedure Add 0.2 ml of plasma in to a small glass tube and incubate at 370C for 1-2 minute Add 0.1 ml of thrombin, mix and start stopwatch. Then hold the tube in water bath & tilt the mixture back and forth, looking clot formation. Normal value: 12 – 15 seconds. Prolonged TT is due to: Hypofibrinogenemia Dysfibrinogenemia Disseminated intravascular coagulation (DIC) Liver disease Treatment with heparin Fibrinogen levels: 124 Fibrinogen levels Useful to detect deficiencies of fibrinogen and alterations in the conversion of fibrinogen to fibrin NR= 200-400mg/dL (2 – 4 g/l) May be decreased in liver disease or due to consumption of fibrinogen when there is accelerated intravascular clotting mainly affected by the concentration of Fibrinogen Elevated ;-in infection, Inflammation Traumatic injury 8. Assay of plasma fibrinogen level : 125 8. Assay of plasma fibrinogen level Principle During the process of coagulation, enzyme thrombin converts soluble fibrinogen into insoluble fibrin. The time required for this conversion is proportional to the concentration of fibrinogen in the plasma. By plotting a graph of known concentrations of fibrinogen and their clotting time, the concentration of fibrinogen in the sample can be determined. Reagents Vernol buffer with PH 7.4 Calibration plasma with known level of fibrinogen PPP (platelet poor plasma) of patient and control Thrombin solution suspended in 9g/l NaCl Slide 126: 126 Step I : To prepare a normal activity curve using normal pooled plasma with factor deficient plasma: Prepare serial dilutions of the normal pooled plasma ranging from 1:10 to 1:320 as follows Tube No. 1 2 3 4 5 6 Veronal buffer (ml) 1.8 1.0 1.0 1.0 1.0 1.0 Normal plasma (ml) 0.2 Final dilution 1:10 1: 20 1: 40 1:80 1:160 1:320 Conc. Of the 100 50 25 12.5 6.25 3.12 factor (%) Technique Slide 127: 127 Mix 0.1 ml of 1: 10 dilution of the normal plasma with 0.2 ml of pre- warmed thromboplastin solution. Keep at 37 C for two minutes Add 0.1 ml of 0.025 M calcium chloride solution. Start the stop watch and record the clotting time Determine the clotting time for each dilution of the normal plasma in the same way Plot the dilutions against the clotting time (seconds) on a graph paper. The graph should be a straight line with 5 – 50 seconds. Slide 128: 128 Step II: To determine the test plasma of fibrinogen 1. Dilute the test plasma (patient plasma) 1: 10 with the veronal buffer 2. Determine the clotting time for the dilution in the same way in step I 3. Fibrinogen level can be read from the graph if time is b/n 5 & 50 seconds. If fibrinogen level is low time will be >50second, at this time dilute the sample 1:5 and divide the result by 2 and read from the graph. Also if fibrinogen level is high time will be <5second, at this time dilute the sample 1:20 and multiplay the result by 2. and read the result from the graph.