Hemostasis Is a complex process which causes the bleeding process to stop. It refers to the process of keeping blood within a damaged blood vessel. Dependent upon: Vessel Wall Integrity. Adequate Numbers of Platelets. Proper Functioning Platelets. Adequate Levels of Clotting Factors. Proper Function of Fibrinolytic Pathway.

Hemostasis is maintained in the body via three mechanisms Vascular spasm: Damaged blood vessels constrict. Platelet plug formation: Platelets adhere to damaged endothelium to form platelet plug (primary hemostasis) and then degranulate. Blood coagulation: Clots form upon the conversion of fibrinogen to fibrin, and its addition to the platelet plug (secondary hemostasis).

Clotting Mechanism IntrinsicExtrinsic Prothrombin Thrombin Fibrinogen Fibrin (II) - K depended (I) (accelerator) V X Collagen VII XII XI IX ( accelerator ) VIII

Causes Bleeding Disorders 1.Vessel defects. Vitamin C deficiency. Bacterial infection & Viral infection. Acquired. 2.Platelet disorders  Thrombocytopenia causes Drug induced. Bone marrow failure. Hypersplenism. Other causes.

 Thrombocytopathy causes Uremia. Inherited disorders. Myeloproliferative disorders. Drudge induced (Aspirin) 3.Clotting factor deficiencies.  Inherited: Hemophilia A. Hemophilia B.  Acquired: Anticoagulant therapy Liver diseases DIC

Hemophilia  Hemophilia A (Classic Hemophilia) 80-85% of all Hemophiliacs. Deficiency of Factor VIII. Lab Results - Prolonged PTT.  Hemophilia B (Christmas Disease) 10-15% of all Hemophiliacs Deficiency of Factor IX Lab Test - Prolonged PTT

Anticoagulants An anticoagulant is a substance that prevents coagulation; that is, it stops blood from clotting, this prevents deep vein thrombosis, pulmonar embolism, myocardial infarction and stroke. Coumadins (Vitamin K antagonists) Heparin

Coumadins These oral anticoagulants that antagonize the effects of vitamin K. Monitored by PT times These anticoagulants are used to treat patients with deep-vein thrombosis (DVT), pulmonary embolism (PE), atrial fibrillation (AF), and mechanical prosthetic heart valves.

Heparin It works by activating anti-thrombin III, which blocks thrombin from clotting blood. Heparin Therapy is Monitored by PTT times.  Liver Disease Liver Disease can Result in Reduced Production of Coagulation Factors (I,II,V,VII,IX,X).

Disseminated intravascular coagulation ) DIC) Is a pathological activation of coagulation mechanisms that happens in response to a variety of diseases. DIC leads to the formation of small blood clots inside the blood vessels throughout the body. The small clots also disrupt normal blood flow to organs (such as the kidneys), which may malfunction as a result. As the small clots consume coagulation proteins and platelets, normal coagulation is disrupted and abnormal bleeding occurs from the skin the gastrointestinal tract, the respiratory tract and surgical wounds.

The PT and APTT are usually very prolonged and the fibrinogen level markedly reduced. High levels of fibrin degradation products, including D-dimer, are found owing to the intense fibrinolytic activity stimulated by the presence of fibrin in the circulation.  Definitive diagnosis depends on the result of DIC: Thrombocytopenia) prolonged bleeding time) Prolongation of prothrombin time and activated partial thromboplastin time A low fibrinogen concentration Increased levels of fibrin degradation products

Bleeding time Provides assessment of platelet count and function.  Normal value 2-8 min

Platelet –Poor plasma (PPP)  <10 x 10 9 /L.  Specimen has been centrifuged for x g. Why is PPP essential?  Contains platelet factor 4 (heparin neutralizer).  Contains phospholipid (affects lupus anticoagulant and factor assay testing).  Contains proteases. Platelet-Rich plasma (PRP)  x 10 9 /L.  Specimen has been centrifuged for x g. Terms

Prothrombin Time (PT) The prothrombin time: is therefore the time required for the plasma to clot after an excess of thromboplastin and an optimal concentration of calcium have been added. Measures the function of the Extrinsic Pathway. Sensitive to Factors II, IV, V, VII, X. The PT evaluates patients suspected of having an inherited or acquired deficiency in these pathways. Normal value (10-15 Sec)

When is it ordered? Used to monitor oral anticoagulant therapy (Warfarin / Coumadin). When a patient who is not taking anti-coagulant drugs has signs or symptoms of a bleeding disorder. When a patient is to undergo an invasive medical procedure, such as surgery, to ensure normal clotting ability.

The procedure uses a tissue thromboplastin reagent with CaCl (Calcium Chloride) to provide a one step procedure for evaluating plasma clotting. The normal values for the prothrombin time range from 10.0 to 13.0 seconds.  An elevated prothrombin time may indicate the presence of 1.Vitamin K deficiency, 2.DIC, 3.liver disease, Principle

 a deficiency in one or more of the following factors: Factor I (Fibrinogen) Factor II (Prothrombin) Factor V (Labile Factor) Factor VII (Stable Factor) Factor X (Stuart-Prower Factor) Factor XIII (Fibrin Stabilizing Factor)  In addition, inhibitors can cause prolonged PT.

Calcium ions and Thromboplastin from brain tissue (Rabbit). Thromboplastin (Tissue Factor) protein-lipid complex found in tissues outside blood vessels. PT Reagent

PT Reagent Calibration Reagents are calibrated against standard PT reagent established by the WHO. ISI = International Sensitivity Index. ISI is assigned by the manufacturer for each lot of reagent using reference material from WHO. The lower the ISI the more sensitive the Reagent  ISI of 1.8 to 2.4 = Low sensitivity  ISI of 1.4 to 1.8 = Average sensitivity  ISI 1.0 to 1.4 = High Sensitivity

PT: INR Values INR = International Normalized Ratio. MNP = Mean Normal Plasma. INR = (PT / MNP) ISI An INR of 1.0 means that the patient PT is normal. An INR greater than 1.0 means the clotting time is elevated.

Performing a PT test Citrate tube; 3.2% citrate tubes. Pre-warm PT reagent and sample to 37 o C Add 100  L sample to tube Add 200  L of PT reagent to tube Start timer Record time to clot in seconds  If the results from run 1 and run 2 are within + 1 second from each other, average the two results and report with appropriate units.  If results are not within required limits, a third run should be performed and average the two that match within acceptable limits. Calculate INR

Coagulation Cascade APTT test measures  the intrinsic pathway factors.  & common pathway factors.  The PTT is also used to monitor heparin therapy. Intrinsic Pathway:  Factors VIII, IX, XI, and XII.  Activated on surface of exposed endothelium.  Complexes form on platelet phospholipids. Common Pathway:  Factors X, V, prothrombin and fibrinogen

When is it ordered? When a patient presents with unexplained bleeding or bruising, It may be ordered as part of a pre-surgical evaluation for bleeding tendencies, When a patient is on intravenous (IV) or injection heparin therapy, the APTT is ordered at regular intervals to monitor the degree of anticoagulation.

Sample Citrate tube; 3.2% citrate tubes. Ratio of anticoagulant to blood should be 1:9 After centrifugation, the Plasma contains all the intrinsic coagulation factors except calcium (removed during anticoagulation) and the platelets (removed during centrifugation).

Reagents Phospholipids and a surface activator Calcium Chloride reagent added to start the reaction. APTT reagent mimics the surface of a platelet.

Procedure Prewarm sufficient volumes of CaCl 2 (0.025 M) to 37 o C Label 2 test tube & 2 control tubes Add 0.1 ml of sample or control to the appropriate tube Add 0.1 ml of APTT reagent to each tube Incubate at 37 o C for 5 minutes Add 0.1 ml of prewarmed CaCl 2 & start stop watch immediately Record the time at which clot is detected

Expected APTT Values Normal Range: 25 to 43 seconds Slightly Elevated: 45 to 65 seconds Extremely Elevated > 70 seconds

What does the test result mean? APTT prolongations are caused by either factor deficiencies (especially of factors VIII, IX, XI, and/or XII), Inhibitors (most commonly, lupus anticoagulants{ immunoglobulin that binds to phospholipids and proteins associated with the cell membrane}) Therapeutic anticoagulants such as heparin. DIC Liver disease