Tests for Hemostasis (Bleeding time and Clotting time) Salman bin Abdul-Aziz University College of Pharmacy Clinical Pharmacy Department Physiology-I (Practical Part) Tests for Hemostasis (Bleeding time and Clotting time) 11/16/2018

Objectives After completing this experiment, you should be able to: 1. Define bleeding. 2. Define hemostasis and describe how bleeding stops from a finger prick. 3. Describe the physiological basis of bleeding disorders. 4. Determine BT and CT by the routine laboratory methods, and give their normal values. 5. Indicate the clinical importance of doing BT and CT. 11/16/2018

Introduction Bleeding is the name commonly used to describe blood loss. It can refer to blood loss inside the body (internal bleeding) or blood loss outside of the body (external bleeding). Bleeding after injury is a common experience for most of people. But bleeding stops automatically within a few minutes. However, suspicion of a disease arises when there is frequent and prolonged bleeding with minor injuries, such as during shaving or cutting of nails In others, there may be spontaneous bleeding (without any apparent trauma) in the skin, gums, or into joints, and muscles, etc. It is in all such cases that various tests are carried out. 11/16/2018

Hemostasis The term hemostasis refers to the process of stoppage of bleeding after blood vessels are punctured, cut, or otherwise damaged. Hemostasis, which is a homeostatic mechanism to prevent loss of blood, is a result of a complex, natural, physiological response. The term is also used for surgical arrest of bleeding. Hemostasis involves the following 4 interrelated steps: 1. Vasoconstriction (contraction of injured blood vessels). 2. Platelet plug formation. 3. Formation of a blood clot. 4. Fibrinolysis (dissolution of the clot). 11/16/2018

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Three mechanisms of hemostasis, and platelets are involved in each: Vascular spasm, Platelet plugs and Chemical clotting. Vascular spasm: when a large vessel such as an artery or vein is severed, the smooth muscle in its wall contracts in response to the damage. Platelets in the area of the rupture release serotonin, which also brings about vasoconstriction. The smaller opening may then be blocked by a blood clot. Platelet plugs: when capillaries rupture, the damage is too small to initiate the formation of a blood clot. The rough surface, however, causes platelets to change shape and become sticky. These platelets stick to the edges of the break and to each other. The platelets form a mechanical barrier or wall to close off the break in the capillary. Chemical clotting: The stimulus for clotting is a rough surface within a vessel, or a break in the vessel, which also creates a rough surface. Chemical clotting has 3 stages. 11/16/2018

Cont'd Stage 1 begins when a vessel is cut or damaged internally. The product of stage 1 is prothrombin activator. This chemical is an enzyme whose substrate is prothrombin. The stages of clotting may be called a cascade, where one leads to the next. Prothrombin activator, the product of stage 1, brings about the stage 2 reaction: converting prothrombin to thrombin. Thrombin, brings about the stage 3 reaction: converting fibrinogen to fibrin. The clot itself is made of fibrin, the product of stage 3. Once the clot has formed and bleeding has stopped, clot retraction and fibrinolysis occur. Clot retraction involves folding of the fibrin threads to pull the edges of the rupture in the vessel wall closer together. 11/16/2018

PHYSIOLOGICAL BASIS OF BLEEDING DISORDERS Excessive and prolonged bleeding with small injuries, or spontaneous bleeding my result from defects of: 1. A Platelets 2. Blood vessel walls 3. Coagulation of blood. 1,2. DEFECTS OF PLATELETS AND VESSEL WALLS Defects of platelets (i) and vessel walls (ii) typically cause spontaneous bleeding from small vessels, or during cuts and bruises and bleeding in the gums. 11/16/2018

Cont'd 3. Defects of clotting are of 2 types: i. Excessive bleeding into tissues (muscles, joints, or viscera) is usually due to injury to relatively large vessels. The delay in the formation of a clot fails to support the normal action of platelets in checking blood loss. Deficiency of clotting factors—inherited or in liver disease (hepatitis, cirrhosis, vitamin K deficiency) are the usual causes. ii. Thrombosis. It is clotting of blood that occurs within unbroken blood vessels. Bleeding Disorders. Bleeding disorders may be inherited or acquired—the acquired defects being more common. Disorders due to platelet and vessel wall defects are more common than coagulation disorders that are due to deficiencies of clotting factors. 11/16/2018

Coagulation cascade 11/16/2018

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Cont'd Hemophilia: There are several forms of hemophilia; all are genetic and are characterized by the inability of the blood to clot properly. Hemophilia A is the most common form and involves a deficiency of clotting Factor 8. Without factor 8, the first stage of chemical clotting cannot be completed, and prothrombin activator is not formed. Without treatment, a hemophiliac experiences prolonged bleeding after even minor injuries and extensive internal bleeding, especially in joints subjected to the stresses of weight-bearing. 11/16/2018

TESTS FOR HEMOSTASIS 1. Bleeding time. 2. Clotting time. Bleeding Time (BT) is the time interval between the skin puncture and spontaneous, unassisted (i.e. without pressure) stoppage of bleeding. The BT test is an in vitro test of platelet function. Clotting time (CT) is the time interval between the entry of blood into the glass capillary tube, or a syringe, and formation of fibrin threads. 11/16/2018

Bleeding time “Duke” Bleeding time (finger-tip; ear-lobe): Materials: Equipment for sterile finger-prick. Clean filter papers. Stopwatch. Procedures 1. Get a deep finger-prick under aseptic conditions to get free- flowing blood. Start the stop watch and note the time. 11/16/2018

Cont'd 2. Absorb/remove the blood drops every 30 seconds by touching the puncture site with the filter paper along its edges, without pressing or squeezing the wound. Number the blood spots 1 onwards. 3. Note the time when bleeding stops, i.e. when there is no trace of blood spot on the filter paper. Encircle this spot and number it as well. This is the end point. (Do not keep the filter paper on the table and then press your wound on it). 4. Count the number of blood spots and express your result in minutes and seconds. Normal bleeding time = 1–5 minutes. 11/16/2018

Cont'd PRECAUTIONS 1. The skin site chosen for BT should be scrubbed well with alcohol to increase the blood flow. 2. The skin should be dry and the puncture should be 3–4 mm deep to give free-flowing blood. 3. Do not press the filter paper on the puncture site. 4. If bleeding continues for more than 10–12 minutes, stop the test and press a sterile gauze on the wound. Inform me. 11/16/2018

Observations of bleeding time using filter paper: Normal bleeding time = 1 to 5 min 11/16/2018

CLOTTING TIME (CT) I. CAPILLARY BLOOD CLOTTING TIME (WRIGHT’S CAPILLARY GLASS TUBE METHOD) Procedure: 1. Absorb the first 2 drops of blood on a separate filter paper and allow a large drop to form. Now dip one end of the capillary tube in the blood; the blood rises into the tube by capillary action. This can be enhanced by keeping its open end at a lower level. 2. Note the time when blood starts to enter the tube. This is the zero time. 11/16/2018

Cont'd 3. Hold the capillary tube between the palms of your hands to keep the blood near body temperature (in winter, you may blow on it). 4. Gently break off 1 cm bits of glass tube from one end, at intervals of 30 seconds, and look for the formation of fibrin threads between the broken ends. The end-point is reached when fibrin threads span a gap of 5 mm between the broken ends (“rope formation”). Note the time. Normal clotting time = 3–6 minutes. 11/16/2018

Normal clotting time = 2–4 minutes. II. Drop Method Procedure: Place a large drop of blood from a skin puncture on a clean and dry glass slide. Draw a pin through the drop every 30 seconds, and note the time when fibrin threads adhere to the pin and move with it out of the blood drop. The time elapse between placing the blood drop on the slide and the formation of fibrin threads is the clotting time. Normal clotting time = 2–4 minutes. This method is less accurate than the above method. 11/16/2018

Note BT and CT are important in the following situations: i. History of frequent and persistent bleeding from minor injuries, or spontaneous bleeding into tissues. ii. Before every minor and major surgery (tooth extraction, etc.). iii. Before taking biopsy, especially from bone marrow, liver, kidney, etc. iv. Before and during anticoagulant therapy. v. Family history of bleeding disorders. 11/16/2018

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