Screening Tests for Haemostasis Coagulation Time & Bleeding Time

Bleeding Time & Coagulation TimePresentation by : Dr. Mohammed Abdul Aleem (1st year PG)Upgraded department of PathologyOsmania Medical College, HyderabadObjectivesTo learn the principle, technique and interpretation of screening tests for haemostasis bleeding time (BT) and clotting time (CT)To know the normal range of BT and CT and abnormal values in diseases.

Haemostasis is the process by which the bleeding from an injured site is arrested by formation of haemostatic plug, followed by removal of that plug spontaneously in due course of time. Components of Haemostasis:

1. Integrity of vascular wall2. Plateletsabnormalities in count and function3. Coagulation systemvarious plasma coagulation factors4. Fibrinolytic mechanism5. Inhibitors of coagulationNormally, a balance is maintained in these factors. Anything that interferes with any of these components results in abnormal bleeding. For investigation, following scheme is followed:

A. Clinical evaluation that includes patients history including intake of drugs, family history, details of sites of bleeding, frequency, and character of haemostatic defect.

B. Screening tests for assessment of abnormalities of various components of haemostasis.

C. Specific tests to pinpoint the cause of abnormalityPetechia & Purpura primary hemostasis defectHematoma: A swelling resulting from a large area of hemorrhage in subcutaneous tissue or muscle. (coagulation disorder)Hemarthrosis: This refers to bleeding into a joint - hemophilia.

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Screening tests for Haemostasis:

Disorders of vascular haemostasis: Bleeding time (BT), Hess capillary test (tourniquest test)Disorders of platelets: Platelet count, Bleeding time (BT)Coagulation system: Whole Blood clotting (coagulation) time (CT), Activated Partial Thromboplastin Time with kaolin (APTTK), one-stage Prothrombin time (PT), Thrombin time (TT) Fibrinolytic system: Fibrinogen, Fibrin degradation products(FDP)Inhibitors of coagulation: FDPs6Blood for coagulation studies is collected by venepuncture in Trisodium citrate in the ratio of 1:9Care must be taken that the sample is neither haemolysed nor clotted.

3.8% Trisodium citrate in the ratio of 1:9 , i.e. 4.5 ml of blood is added to a clean collection tube containing 0.5 ml of citrate.7Bleeding Time

The bleeding time test assesses primary hemostasis.Bleeding time is duration of bleeding from a standard puncture wound on the skin which is a measure of the function of the platelets as well as integrity of the vessel wall. One of the most important preliminary indicators for detection of bleeding disorders. Most commonly done preoperative investigation in patients scheduled for surgery.(vascular and platelet components) 8

A, After vascular injury first transient vasoconstriction. B, Platelets bind via glycoprotein Ib (GpIb) receptors to (vWF) on exposed ECM and are activated, undergoing a shape change and granule release. Released (ADP) and thromboxane A2 (TxA2) induce additional platelet aggregation through platelet GpIIb-IIIa receptor binding to fibrinogen, and form the primary hemostatic plug. C, Local activation of the coagulation cascade (involving tissue factor and platelet phospholipids) results in fibrin polymerization, cementing the platelets into a definitivesecondary hemostatic plug. D, Counterregulatory mechanisms, mediated by tissue plasminogen activator (t-PA, a firinolytic product) and thrombomodulin, confine the hemostatic process to the site of injury.9

METHODS FOR BLEEDING TIME

1. Dukes method

2. Ivys method

Principle: A small puncture is made on the skin and the time for which it bleeds is noted. Bleeding stops when platelet plug forms and breach in the vessel wall has sealed.

Dukes Method:

Procedure: Clean the tip of finger with spirit. Puncture deeply so that blood flows drop by drop without squeezing the finger.Start the stop-watch immediately.At every 30 seconds intervals remove the drops of blood on filter paper without touching the skin.Stop the watch when no more blood spot comes on the filter paper and note the time.

Normal Bleeding time: 1- 4 minutesIvys MethodTie the BP apparatus cuff around the arm and inflate it.Clean an area with spirit over the forearm and allow it dry.Make 2 clean punctures avoiding the superficial veins.Start the stop-watch immediately.Remove the drops of blood on filter paper without touching the skin every 15 seconds.Stop the watch when no more blood spot comes on the filter paper and note the time.

Normal Bleeding time : 3 5 MinutesAdvantages of the methodi. This is the method of choice.ii. It is a standardised method.iii. Bleeding time is more accurate

13Clinical Application of Bleeding Time:

The bleeding time is prolonged in following conditions:

i. Thrombocytopenia ITP, Infections, Heparin Induced (HIT)ii. Disorders of platelet functions - Bernard-Soulier syndrome, Glanzmans Thrombostheniaiii. Acute leukaemiasiv. Aplastic anaemiasv. Liver diseasevi. von Willebrands diseasevii. DICviii. Abnormality in the wall of blood vessels Allergic PurpuraHereditary Hemorrhagic Telangiectasiasix. Administration of drugs: e.g. aspirin, other NSAIDS.Thrombocytopenia: ITP, Infections: malaria, dengue, Heparin inducedDecreased production : Aplastic anemias, 14

Coagulation TimeClotting Time is also known as whole blood clotting time and is a measure of the plasma clotting factors. It is a screening test for coagulation disorders.

Various other tests for coagulation disorders include:Prothrombin time (PT)Partial thromboplastin time with kaolin (PTTK) or Activated partial thromboplastin time with kaolin (APTTK),Measurement of fibrinogen.

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The extrinsic pathway is initiated when F VII combines with tissue factor after inj.Intrinsic : F XII is activated in vitro ( e.g. by glass) requires high molecular weight kininogen. F IXa combines with F VIII, phospholipid, and calcium to activate F X to F Xa.Thrombin splits off fibrinogen to form fibrin monomers, which polymerize. F XIII stabilizes fibrin polymers by introducing covalent bonds.

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Inhibitors of CoagulationTissue factor pathway inhibitor (neutralizes VII-tissue factor complex), Antithrombin III (most important physiological inhibitor; synthesized in liver;) inhibits mainly thrombin and F Xa), Protein C (activated by thrombin in the presence of thrombomodulin; causes proteolysis of activated forms of F V and F VIII)Protein S acts as a cofactor in this reactionMETHODS FOR CLOTTING TIME Capillary tube method

Lee and White method

Slide Method

1. Capillary Tube MethodProcedure: Clean the tip of a finger with spirit.Puncture it deeply with a disposable needle.Start the stopwatch.Fill two capillary tubes (4 inch size tube, fill upto 3 inch continuous) with free flowing blood from the puncture after wiping the first drop of blood.After 2 minutes and then every 30 seconds interval, break the capillary tube at 1 cm distance to see whether a thin fibrin strand is formed between the two broken ends. Stop the watch and calculate the time from average of the two capillary tubes 22Capillary Tube method:

Advantages:It can be performed when venous blood cannot be obtained.

Disadvantages:

i. Method is insensitive.ii. Method is unreliable.

Normal clotting time 4-7 minutes.

2. Lee and White Method:Procedure:After cleaning the forearm, make a venepuncture and draw 5 ml of blood in a siliconised glass syringe. Start the stopwatch.Transfer 1 ml of blood each into 3 glass tubes which are kept at 37C in a water bath.Third tube is gently tilted every 30 seconds until the blood in it clots. Second tube is then tilted in same manner. Lastly, the first tube is tilted in the same manner. The clotting time is taken when the tubes can be tilted without spilling of their contents.Calculate the clotting time by average of 3 tubes.Lee and White Method:

Advantages:

i. More accurate and standard method.ii. Test can be run with control.

Disadvantages:

There can be contamination of syringe or tubes.

Normal clotting time 6-9 minutes.3. Slide Method:Place several drops of blood on a clean slide from a deep finger puncture. At 30 second intervals, draw a needle through one of the drops.As soon as the needle picks up fibrin threads and drags them along, coagulation has taken place.The time interval between the placing of the drops on the slide and the formation of fibrin threads is the coagulation time.

Normal time : 2- 8 Minutes.Clinical Applications of Clotting Time

Clotting time is prolonged in following conditions:

Deficiency of coagulation factors Haemophilia A (F VIII), Von Willebrands Disease, Haemophilia B (F IX Christmas Disease)Afibrinogenaemia.Disseminated intravascular coagulation (DIC).Liver Disease or Renal DiseaseVitamin K DeficiencyAdministration of drugs such as anticoagulants - HeparinComplete blood count and blood smearActivated partial thromboplastin time, TT, PT,

Other methods for Platelets: Clot retraction methodClot lysis

Test for fibrinogen/fibrin degradation productsTest for D-dimerCoagulation factor assays

27Thank you