Intro to Hemostasis PDF

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Summary

- Hemostasis, the process in which the body stops bleeding and maintains blood in the fluid state within the vascular system. - Fibrinolysis, involves the proteolytic digestion of fibrin by the enzyme plasmin. - Coagulation, the process of stopping blood flow from a wound. Requires coagulation facto...

Description

MLSC-3121U, Clinical Hematology II -

Hemostasis, the process in which the body stops bleeding and maintains blood in the fluid state within the vascular system. Fibrinolysis, involves the proteolytic digestion of fibrin by the enzyme plasmin. Coagulation, the process of stopping blood flow from a wound. Requires coagulation factors, fibrin and platelets. Coagulopathy, a disease affecting the blood-clotting process.

Platelets in Hemostasis -

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Platelets provide a negatively charged phospholipid surface where reactions can occur. They release substances that mediate responses to cell damage and bleeding. The surface membrane glycoproteins allow them to attach to other platelets. Alpha Granules, contain coagulation factors in platelets. Dense Granules, contain ADP for energy in platelets. Lysozyme, contained by lysosomes in platelets. Dense Tubular System, location of thromboxane synthesis. Open Circular Canalicular System, allow entry and exit of platelet factors from the liver into the platelet. Platelets are activated by the clotting enzyme thrombin. The stimuli causes adhesion, shape change and aggregation in the platelets on the opening in the blood vessel. Once stuck together, the platelets will release their alpha dense granules to secrete their coagulation factors into the injured area to promote fibrin clot formation. Adhesion, von Willebrand factor stimulates platelets to stick to the fibrin via their platelet receptors. Aggregation, results in the formation of a platelet plug when platelets come together via fibrinogen and glycoproteins. Primary Hemostasis, defined by platelet adhesion to exposed collagen within the endothelium of the vessel wall. It forms the platelet plug and does not involve fibrinogen. Involves platelet aggregation. Secondary Hemostasis, involves the enzymatic activation of coagulation proteins to produce fibrin from fibrinogen. It is the fibrin forming system and uses many coagulation factors.

Platelet Plug Formation -

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Adhesion, when the vessel wall is damaged, the blood is exposed to the sub endothelium which is high in vWF and Collagen. The platelet receptors interact with collagen and vWF and stick to the sub endothelium. vWF is the plasma protein that links platelets to the sub endothelium, Gp1b is the platelet membrane receptor and collagen fibres make up the sub endothelium. Adhesion causes dense granule release and aggregation follows. Shape Change, once adhered, the platelets will change shape in the plug. They develop pseudopods to enhance adhesion with other aggregating platelets. This is caused by

MLSC-3121U, Clinical Hematology II

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agonists like collagen, thrombin or ADP and the amount of shape change varies depending on the strength of the agonist. Primary Reversible Aggregation, agonist causes release of ADP from dense granules. The platelets will aggregate and can let go afterwards to reverse the aggregation. Secondary Irreversible Aggregation, stronger than primary aggregation in that a strong agonist that stimulates complete dense and alpha granule release to cause an irreversible aggregate. Plug Formation, platelet factors stimulate the conversion of fibrinogen into fibrin to form the clot. Consolidation, the clot/platelet plug contracts into a dense thrombus. Stabilization, mediated by activation of coagulation cascade with fibrin formed between and on aggregated platelets. Fibrin stabilizes the clot when FXIII to FXIIIa cross link fibrin to make sure it maintains stability.

Platelet Function Tests -

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Measures adhesion and aggregation. Challenging due to reliability, accuracy and simplicity of methods. Historically, people intentionally caused clot formation by making a cut into the patient and timing their bleeding for coagulation testing. Optical Aggregometry, using spectrophotometry based on turbidity of the sample decreasing as clotting occurs. Luminaggregometry, uses fluorescence and optical density to measure ATP release during aggregation. Whole Blood Aggregometry, uses electrical impedance to measure decreases in current from platelet aggregation at electrodes. Mucosal bleeding is a common manifestation of platelet disorders.

Coagulation Factors -

Formed in the liver. Liver disease commonly results in under production of coagulation factors and bleeding abnormalities. Substrate, fibrinogen (Factor I) Cofactors, labile factor (Factor V) Enzymes, serine proteases: IIa, VIIa, IXa, Xa, Xia, XIIa, prekallikrein, Transaminase (Factor XIIIa)

Coagulation Cascade -

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The process requires plasma proteins (factors) and phospholipids and calcium. Intrinsic Pathway, happens in the bloodstream and starts with the contact factors and factor XII. Tested by the APTT test initiated in vitro by activation on negatively charged surfaces such as glass. Extrinsic Pathway, involves tissue factor and calcium is activated in this pathway. Starts by the activation of factor VII to factor VIIa which interacts with the intrinsic pathway.

MLSC-3121U, Clinical Hematology II

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Tested by the PT test to determine how long it takes for the plasma to create a clot when thromboplastin calcium is added. Common Pathway, have processes similar to of both the intrinsic and extrinsic pathways and is where the other two pathways meet. Measured by both the PT and APTT test.

Evaluation of Hemostasis -

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Family history is very useful in determining family history of genetic bleeding disorders. Lab testing done through Plt counts, template bleeding time, PT, APTT and Thrombin time. Platelet Related Disorder, patients exhibit petechiae and mucous membrane bleeding. Coagulation Defects, deep spreading hematomas, bleeding into the joints with evident hematuria. Specimens one are collected for analysis in blue top NaCitrate tubes in a 9:1 NaCitrate to blood ratio. NaCitrate binds Ca ions to prevent coagulation. EDTA also binds Ca, but it interferes with other factors and has some anticoagulant properties. Platelet Poor Plasma, required for clot based coagulation testing. Removes platelets from the plasma through centrifugation until the platelet count is less than 10 x 10^9/L. Spun at 1500g for 15 minutes. Platelets may release membrane phospholipid phosphatidylserine which will neutralize lupus anticoagulants. They also secrete factors V and VIII and vWF to falsely shorten the tests and platelet factor 4 which is a heparin agonist. Platelet poor plasma should be inspected for hemolysis, lipemia and icterus. Hemolysis indicates potential platelet or coagulation pathway activation when they release tissue juice. Lipemia and icterus may affect the end point results in optical instruments. PT Testing should have sample stored at RT for 24 hours. APPT testing samples should be stored at RT for 4 hours (if on heparin, centrifuge for 1 minute and test within 4 hours).

Prothrombin Time -

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Measures the extrinsic pathway that starts with factor XII. Done at 37 degrees to mimic body temperature. Thromboplastin calcium mixture is the reagent that acts as the tissue juice to stimulate clot formation in the plasma. The time between adding the reagent and the clot is recorded to evaluate clotting time. Internal Normalized Ratio (INR), the PT test is used to monitor patients on oral blood thinners but it becomes hard to evaluate these patients off PT alone. The INR adjusts for thromboplastin sensitivity using an index. Each thromboplastin has a unique ISI used in the INR equation to standardize results for comparison. Human brain thromboplastin is the most sensitive with an ISI of 1. INR = (PTPatient/PTNormal Range)ISI. INR above 4.0 is a critical value and should be phoned as such because the patient could start bleeding out. Thrombin Time, similar to the PT procedure.

Activated Patrial Thromboplastin Time

MLSC-3121U, Clinical Hematology II

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Measures the intrinsic system. Uses prewarmed partial thromboplastin reagent and 0.0025 CaCl2 which is considered as a start reagent. Similar procedure to the PT test just with different incubation times and reagents.

Testing Methodologies Mechanical Endpoint Detection -

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Electrochemical Detection, uses electrochemical detection of clots. Uses two probes in a solution that normally conduct a current only when they’re both in solution. When a clot forms at the probe, the electrical contact between the two probes remains, even when the moving probe is out of the solution. Steel Ball Detection, the movement of a steel ball is plasma is monitored by magnetic sensors. As clotting starts the plasma gets thicker and start to slow down the rocking of the ball. Once he ball stops moving, the time is recorded by the machine for a result.

Photo-Optical Endpoint Detection -

Turbidimetric Detection, plasma becomes more turbid as a clot starts to for. Machine uses turbidimetry to measure the change in optical density of the plasma which it proportional to clot formation.

Nephelometric Endpoint Detection -

Uses a light emitting diode at a high wavelength to detect light scattering as Ag-Ab complexes are formed. Detects the amount of agglutination of particles by reading the increasing amount of light scattered by reading the increasing amount of light scattered at a 90-degree angle as agglutinates are formed. The IL ACL series does this.

Chromogenic Endpoint Detection -

Uses a colour producing chromophore to measure it at a predetermined wavelength. Free pNA has a yellow colour with an intensity proportional to the amount of free pNA. Can use direct or indirect measurements.

Immunological Endpoint Detection -

Latex microparticles are coated with a specific antibody directed against the analyte. As the particles agglutinate, more light is absorbed. The increase in light absorbance is proportional to particle size and antigen level in the sample....