Hemostasis & Blood Coagulation: First Teaching Hospital Zhengzhou Univ

Hemostasis & Blood Coagulation First Teaching Hospital ZhengZhou Univ.

Normal Hemostasis • A well regulated process • Maintains blood in a fluid, clot free state in normal vessels • Induces the rapid formation of a localized hemostatic plug at the site of vascular injury

Normal sequence of Hemostasis (4 steps)

1. Arteriolar vasoconstriction (transient) 2. Exposure of subendothelial ECM when there is endothelial injury 3. Tissue factor released at the site of injury (by endothelial cells) 4. Formation of permanent plug

The Main Players in Hemostasis

• Endothelial cells • Platelets • Coagulation cascade

Endothelial Cells have Prothrombotic Effect • Via von Willebrand factor (vWF) and tissue factor • factors that depress fibrinolysis • factors needed for the clot are not destroyed before clot forms

Platelets • Express glycoprotein receptors on membranes • Have three types of granules – Alpha granules • Fibrinogen, fibronectin, factor V and VIII, β −thromboglobulin

– Dense bodies or delta granules • ATP/ADP, ionized calcium, histamine, serotonin, epinephrine

– Lysosomal granules

Platelets continued

• When stimulated by contact with subendothelial tissue, platelets undergo three general reactions: 1. Adhesion and shape change mediated by vWF and glycoprotein Ib 2. Secretion (release reaction) – calcium required in coagulation cascade – ADP as mediator of platelet aggregation – Surface expression of phospholipid complex • Binding site for calcium ions and coagulation factors

Platelets continued

• 3. Aggregation – ADP and TXA2 (vasoconstrictor thromboxane A2) are the stimuli for the formation of the primary hemostatic plug • Aspirin inhibits synthesis of TXA2

– Fused mass of platelets • Created by coagulation cascade that produces thrombin • Thrombin also converts fibrinogen to fibrin cementing platelets in place

Coagulation Cascade • A series of conversions of inactive proenzymes to activated enzymes, – culminating in the formation of thrombin

• Thrombin then coverts the soluble plasma protein fibrinogen to insoluble fibrous protein fibrin

Two pathways of coagulation cascade

• Intrinsic – Surface contact • Extrinsic – Tissue injury

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Control of cascade to prevent clotting elsewhere • Antithrombins

• Plasminogen– activated by heparin like plasmin system molecules on endothelial cells • Clinical administration of heparin minimizes thrombosis

• Proteins C and S – Vitamin K dependent – Inactivate cofactors Va and VIIIa

– Breaks down fibrin and inhibits its polymerization – Products of split fibrin are anticoagulants

Laboratory Tests

Capillary Fragility Test (capillary resistance test)  the

capillary fragility test is a nonspecific method for evaluating bleeding tendencies.

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A positive-pressure test, used to measure the capillaries ability to remain intact under increased intracapillary pressure, is controlled by a blood pressure cuff around the patient's upper arm.

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method: Select and mark a space (diameter is 5-cm) on the patient's forearm. Ideally, the site should be free of petechiae; otherwise, record the number of petechiae present on the site before starting the test. Fasten the cuff around the arm, and raise the pressure to a point midway between the systolic and diastolic blood pressures (approximately 100 mm Hg). Maintain this pressure for 5 minutes; then release the cuff. Count the number of petechiae that appear in the space. Record test results.

Precaution • Do not repeat this test on the same arm within 1 week. • This test is contraindicated in patients with disseminated intra vascular coagulation (DIC) or other bleeding disorders, and in those with significant petechiae already present.

Reference values • A few petechiae may normally be present before the test. Less than 10 petechiae on the forearm 5 minutes after the test is considered normal, or negative; more than 10 petechiae is considered a positive result.

Clinical significance: 

Abnormality of blood vessel Vascular purpura

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Abnormality of platelet Quantity: Thrombocytopenia (<20×109/L), Leukemia, etc. Quality: von Willebrand disease, myeloproliferative disease, disproteiniemia, cardiopulmonary bypass, Drug inhibition , Uremia

2. Bleeding time, BT Bleeding time is used to measure the duration of bleeding after a measured skin incision. Bleeding time may be measured by one of three methods: template, Ivy, or Duke. The template method is the most commonly used and the most accurate because the incision size is standardized. Bleeding time depends on the elasticity of the blood vessel wall and on the number and functional capacity of platelets.

method : • Template method: Wrap the pressure cuff around the upper arm and inflate the cuff to 40 mm Hg. Apply the appropriate template lengthwise to the forearm. Use the lancet to make two incisions, 1 mm deep and 9 mm long. Start the stopwatch. Without touching the cuts, gently blot the drops of blood with filter paper every 30 seconds, until the bleeding stops in both cuts. Average the time of the two cuts, and record the result.

Reference values • The normal range of bleeding time is from 3 to 7 minutes in the template method • The abnormal range of bleeding time is longer than 9 minutes in the template method

Clinical significance: Abnormality of blood vessel: hereditary hemorrhagic telangiectasia, etc Abnormality of platelet: ITP, etc Decrease of coagulation factors: DIC, etc Anti-coagulation therapy: Aspirin, etc.

3. Platelets count Platelets, or thrombocytes, are the smallest formed elements in blood. They promote coagulation and the formation of a hemostatic plug in vascular injury. Platelet count is one of the most important screening tests of platelet function. Accurate counts are vital

Normal value: 100~300 × 109/L

Clinical significance: Physiological fluctuation: meal & exercise↑ menstruation↓

Pathological reduction(<100 × 109/L ): Disturbance of pemopoiesis: AA, metastatic cancer of bone marrow

Hyperactivity of destruction: ITP, hypersplenism, SLE, radiation

Hyperactivity of platelets consumption: TTP, DIC, huge blood vessel tumor.

increase(>400 × 109/L): An increased platelet count can result from hemorrhage, infectious disorders, malignant disease, iron deficiency anemia, recent surgery, pregnancy, splenectomy, or inflammatory disorders such as collagen vascular disease. In such cases, the platelet count returns to normal after the patient recovers from the primary disorder. However, the count remains elevated in primary thrombocythemia, myelofibrosis with myeloid metaplasia, polycythemia vera, and chronic myelogenous leukemia.

4.Clot retraction test • The clot retraction test measures the amount of time taken for the clot to retract from the sides of a glass container by gently rimming the top of the clot and is dependent upon normal platelet count and function. • Inspect at ½, 1,2,4 and 24 hours for signs of retraction (separation of clot with expression of serum)

Mechanism and method

Complete retraction

Partial Inadequate nonretraction retraction retraction

Normal value: ½~1h retraction begins and completed within 24hrs.

Clinical significance Directly related to platelets count. Impaired in thrombocytopenia. Normal in hemophilia.

Inadequate retraction Abnormality of platelet Deficiency of coagulation factors Polycythemia vera

5. Coagulation time, CT Mechanism and method

Intrinsic pathway

Normal value: 4~12 min

Clinical significance (Prolongation) Marked reduction of Factor VIII, IX, XI; Marked reduction of prothrombin: serious liver damage, etc. Reduction of fibrinogen:congenital deficiency of fibrinogen, etc. Administration of heparin,dicumarol, and other anticoagulation drugs. Hyperactivity of fibrinolysis with a large amount of FDP

SCREENING TESTS OF COAGULATION Prothrombin Time - PT The extrinsic pathway of coagulation

The (Activated) Partial Thromboplastin Time - APTT The intrinsic pathway of coagulation

The Thrombin Clotting Time - TCT The final common pathway of coagulation

7. Prothrombin time, PT Prothrombin time (PT) measures the time required for a fibrin clot to form in a citrated plasma sample after addition of calcium ions and tissue thromboplastin (factor III). Normal value: 12~14 s

Clinical significance (prolongation) • Prolonged PT may indicate deficiencies in fibrinogen; prothrombin; factors V, VII, or X (specific assays can pinpoint such deficiencies); or vitamin K as well as hepatic disease. It may also result from DIC or ongoing oral anticoagulant therapy. Prolonged PT that exceeds two and one-half times the control value is commonly associated with abnormal bleeding .

6. Activated partial thromboplastin time, APTT The APTT is used to evaluate all the clotting factors of the intrinsic pathway - except platelets - by measuring the time required for formation of a fibrin clot after the addition of calcium and phospholipid emulsion to a plasma sample. An activator, such as kaolin, is used to shorten clotting time. Purpose To aid preoperative screening for bleeding tendencies. To screen for congenital coagulation deficiencies of the clotting factors To monitor heparin therapy Normal value: Normally, a fibrin clot forms 30 to 45 seconds after addition of reagents. >10 s of normal control. .

Clinical significance (prolongation) • Prolonged APTT may indicate a deficiency of certain plasma clotting factors, the presence of heparin, or the presence of fibrin split products, fibrinolysins, or circulating anticoagulants that are antibodies to specific clotting factors

8. Thrombin time, TT It measures how quickly a clot forms when a amount of standard thrombin is added to a platelet poor plasma sample from the patient and to a normal plasma control sample . After thrombin is added, the clotting time for each sample is compared and recorded. This test allows a quick but imprecise estimation of plasma fibrinogen levels. Normal value: 16~18s Abnormality: 3s longer than normal control.

Clinical significance – Late stage of DIC and fibrinolysis – Increase of heparin-likes substances – serious liver damage, allergic shock, pancreatic disease. – Abnormal increase of globulin: Multiple myeloma.

9. Determination of anti-thrombin III It is of considerable value in evaluating the patient with DIC. As the natural inhibitor of thrombin, antithrombin III is rapidly depleted whenever thrombin generation increases. The assay for the level of antithrombin III uses as a standard a dilution curve of normal plasma, so that a normal individual is expected to have an antithrombin III level of close to 100 percent. Normal value: activity 80~120%

Clinical significance – Activity increase: Lead to bleeding.

later stage of DIC, fibrinolysis –

Activity decrease: Lead to thrombin formation.

10. Plasma protamine paracoagulation test, 3P Normal value: negative

Clinical significance (positive) DIC, Thrombosis, Anti-thrombosis therapy.

11. Determination of FDP After a fibrin clot forms in response to vascular injury, the clot is eventually degraded by plasmin, a fibrin-dissolving enzyme. The resulting fragments are known as fibrinogen degradation products. In this test, FDP are detected in the diluted serum that is left in a blood sample after clotting. FDP = X, Y, D, E fragments Normal value: Serum contains less than 10 mg/L of FDP

Clinical significance (increase) FDP levels increase in primary fibrinolytic states, due to increased levels of circulating profibrinolysin; in secondary states, due to DIC and in subsequent fibrinolysis , deepvein thrombosis, acute promyelocytic Leukemia, malignant tumor

12.Determination of D-dimer • The test is specific for secondary fibrinolysis because it confirms the presence of fibrin split products.

• Normal value: normal D-dimer test results are negative or < 250 ug/ml Clinical significance: Increased D-dimer values may indicate DIC, pulmonary embolism, arterial or venous thrombosis, neoplastic disease, pregnancy (late and postpartum), surgery occurring up to 2 days before testing, subarachnoid hemorrhage (spinal fluid only), or secondary fibrinolysis

13.Quantitative test of fibrinogen Fibrinogen (factor I) originates in the liver and is converted to fibrin during clotting. Because fibrin is necessary for clot formation, fibrinogen deficiency can produce mild-to-severe bleeding disorders. This test is used to determine the amount of fibrinogen present in a blood sample. Note that fibrinogen deficiency may also be indicated by prolonged activated partial thromboplastin time, prothrombin time, and thrombin time.

• Normal value: Fibrinogen levels normally range from 175 to 350 mg/dl • Clinical significance: • Depressed fibrinogen levels may indicate disseminated intravascular coagulation; congenital afibrinogenemia; hypofibrinogenemia or dysfibrinogenemia; fibrinolysis; severe hepatic disease.

THE LABORATORY EXAMINATION OF DIC Item of tests SCREEN TEST

Diagnostic standard

Platelet count

<100×109/L

Prothrombin time control)

>3s (compared w/ N.

Fibrinogen

<200mg/100ml blood

AFFIRMATIVE TEST 3P test

Positive

(FDP/DD)

(Increase)

Thrombin time

>3s (compared w/ N. control)

Fibrinogen

Significant reduction.

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