• To understand blood coagulation tests it is helpful to have a basic understanding of the role of the different blood clotting factors and the coagulation process.

• The coagulation process is a complex series of enzymatic reactions involving the proteolytic activation of circulating coagulation factors and activity of co-factors (V, VIII), leading to the production of thrombin which converts soluble plasma fibrinogen into fibrin.

• The fibrin enmeshes the platelet plug, forming a stable thrombus which prevents further blood loss from the damaged vessel.

• The are two interrelated clotting pathways: Intrinsic and the extrinsic pathway.

• The terms intrinsic and extrinsic have traditionally been used in describing the clotting process in the classical blood coagulation theory.

• It is now known that the intrinsic pathway (involving factor XII and kallikrein) is of importance in the in vitro process (laboratory tests), but is less significant in the in vivo (in the body) clotting process.

• Defects in these factors can be detected by means of two clotting-time tests.

PT APTT

Objectives

1. Describe the intrinsic and extrinsic clotting systems.

2. Describe why bleeding time is prolonged in cases of hemophilia and vitamin K deficiency.

3. Demonstrate the tests for prothrombin time and for activated partial thromboplastin time (APTT).

4. Identify the normal values for each, and explain how these tests are used to diagnose bleeding disorders.

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

1. The first event is vasoconstriction, which decreases the flow of the blood in the damaged vessel.

2. The next event is the formation of a platelet plug.

This response occurs in two steps:a. In the first step, platelets adhere to the exposed collagen

(connective tissue protein) of the damaged vessel and then release adenosine diphosphate (ADP), serotoinn and thromboxaine A2.

b. The second step occurs when the ADP, by making the adherent platelets sticky, causes other platelets to cling at this site, forming a platelet clump.

3.The third event is the sequential activation of clotting factors in the plasma, resulting in the formation of an insoluble fibrous protein, fibrin, around the platelet clump. This produces a blood clot. The formation of fibrin from its precursor, fibrinogen, requires the presence of the enzyme thrombin.

• The insoluble fibrin is formed instantly whenever the enzyme thrombin is present, and thus the formation of thrombin must be a carefully regulated event in the body.

• The formation of thrombin from its precursor, prothrombin, requires the sequential activation of a number of other clotting factors.

• When the sequence of events leading to the formation of thrombin is initiated by the release of tissue thromboplastin from damaged tissue cells, fibrin is rapidly formed (10–15 seconds).

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.

• Have a normal prothrombin time but an abnormal APTT

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): o These oral anticoagulants that antagonize the effects of

vitamin K. o Monitored by PT times. o These anticoagulants are used to treat patients with deep-vein

thrombosis (DVT), pulmonary embolism (PE), atrial fibrillation (AF), and mechanical prosthetic heart valves.

• Heparin

Heparin

• It works by activating anti-thrombin III, which blocks thrombin from clotting blood.

• Heparin Therapy is Monitored by PTT times.

• 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

• Bleeding time is a screening test for detecting disorders involving platelet function and for vascular (i.e., capillary) defects that interfere with the clotting process.

• Bleeding time measures the duration of bleeding after a standardized skin incision has been made.

• Normal Values: 1–9 minutes

Clotting time or Coagulation time

• The time required for blood to clot in a glass tube.

• The normal range for the test described below is 5 to 15 min. but each laboratory should determine its own normal values.

Reagent & equipment 1. Water bath, 37C.2. Glass test tube. 3. Stopwatch.4. Plastic syringe and 20-gauge needle.

• Specimen: fresh whole blood , 4 ml .

Procedure1. Label 3 glass test tube with patient name and number them, #1,

#2, and #3.2. Add 1 ml of blood in each tube. The last 1 mL of blood may be

discarded. 3. Start the stopwatch as soon as the blood is placed in tube #3.4. Place the three test tubes in a 37°C water bath.5. At exactly 5 min., title test tube #1 gently to a 45° angle.

Repeat this procedure every 30 seconds, until the test tube can be completely inverted without spilling the contents (that is, until the blood is completely clotted).

6. Record the time it took the blood in test tube #1 to clot.

7. 30 sec. after the blood in test tube #1 is clotted. Proceed with tube #2, and repeat the preceding procedure, tilting the test tube every 30 seconds, until a clot is formed. Record the results. Repeat this procedure for test tube #3.

Clotting time - capillary method

Material

1. Sterile disposable pricking needle or lancet. 2. Stop watch 3. Dry glass capillary tube (narrow diameter 1 top 2 mm,

minimum 10 cm long.) 4. Cotton Swab of absorbent cotton. 5. Spirit wetted, cotton swab. 6. 70 % v/v ethyl alcohol

Clotting time of whole blood

Clotting Time - Slide Method

• The surface of the glass tube initiates the clotting process. This test is sensitive to the factors involved in the intrinsic pathway

• The expected range for clotting time is 4-10 min.4-10 min.