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VASCULAR FUNCTION Bleeding Time -Measures the time taken for bleeding to stop from a standardized wound. -Preferred method: Ivy method- standardized cuts in forearm 3mm long, 3mm deep. (Henry) -1-6 mins: normal bleeding time (Guyton) Capilllary fragility test -petechiometer creates a negative pressure through a suction cup placed on the volar surface of forearm. (Henry) -negative pressure: maintained for 2min then released. Area under the cup is examined for petechiae and considered positive if 1 or more are present. -alternative method: tourniquet test- sphygmomanometer cuff is placed on the arm. Inflated to a pressure halfway between systolic & diastolic, maintained for 5mins, then released. >5 = + (3cm diameter area) (Henry)

B. PLATELET FUNCTION Platelet count -Rees-Ecker method or Brecker-Cronkite method (manual) -Whole blood is diluted with a 1% ammonium oxalate solution. The isotonic balance of the diluent is such that all erythrocytes are lysed while the leukocytes, platelets, and reticulocytes remain intact. -The standard dilution for platelet counts is 1:100. This dilution is prepared using the leukocyte/platelet Unopette system. The dilution is mixed well and incubated to permit lysis of the erythrocytes. Following the incubation period, the dilution is mounted on a hemacytometer. The cells are allowed to settle and then are counted in a specific area of the hemacytometer chamber under the microscope. The number of platelets is calculated per L (x109/L) of blood. (Becton-Dickinson) C. CLOTTING TIME -In order for blood to clot, the enzyme thrombin must be generated from the plasma precursor prothrombin. Thrombin then converts soluble fibrinogen into insoluble fibrin. Generation of thrombin involves the sequential activation of a number of other plasma clotting factor, this process is also being assisted by Ca++ and by factors released by platelets and damaged tissues . The time taken for blood to clot mainly reflects the time required for the generation of thrombin in this manner. If the plasma concentration of prothrombin or of some of the other factors is low (or if the factor is absent, or functionally inactive), clotting time will be prolonged. The expected range for clotting time is 4-10 mins.

Activated Partial Thromboplastin Time Screening abnormalities in all coagulation factors of intrinsic pathway

-Measures all clotting factors except 7 & 13 -Uses an activator substance incubated with plasma activating the contact group of factors; followed by addition of phospholipids substitute for platelets & calcium (32-46 secs clot formation). Thrombin Time -time taken for conversion of fibrinogen to fibrin -thrombin is added to citrated plasma & time taken for a clot to form is recorded. -12-18 secs normal range Prothrombin Time -evaluation of deficiencies in the common & extrinsic pathway; effectiveness of oral anticoagulant therapy

-addition of pre-warmed platelet-poor plasma to pre-warmed thromboplastin-calcium reagent activates coagulation cascade by forming thromboplastin complex. D. FIBRINOGEN ASSAY -Fibrinogen defects may be quantitative (hypo- or hyper-fibrinogenaemia) or qualitative (dysfibrinogenaemia). Inherited dysfibrinogenaemia is rare with only 250-300 patients reported worldwide but an acquired defect of fibrinogen function is more common, especially in liver disease when the fibrinogen molecule is excessively glycosylated impairing its activity. Elevated levels of fibrin degradation products (FDPs) also impair the action of fibrinogen. -Fibrinogen levels are a useful as part of the investigation of a bleeding tendency or an unexplained prolongation of the APTT or PT. -The reference range for fibrinogen is generally between 1.5-4.0g/L. Turbidimetric method -fibrinogen is precipitated from plama by ammonium sulfate & resultant turbidity is measured. Fibrin clot method - Thrombin test is performed first - Fibrin clot is then separated & washed with saline to remove proteins. - Fibrinogen is determined as protein using a biuret reagent. - Normal value: 200-400mg

Fibrinogen Assays
Assays Clauss A functional assay based upon the time for fibrin clot formation

PT-derived Fibrinogen A derived fibrinogen based upon the prothrombin time Assays Immunological Gravimetric An immunological method which measures fibrinogen antigen rather than functional fibrinogen A method based upon clot weight

E. FIBRINOLYSIS Latex agglutination test -test for fibrin and/or fibrinogen degradation products -The latex agglutination test is a laboratory method to check for certain antibodies or antigens in a variety of bodily fluids including saliva, urine, cerebrospinal fluid, or blood.

-How performed: Blood is drawn from a vein, usually from the inside of the elbow or the back of the

hand. The site is cleaned with germ-killing medicine (antiseptic). The health care provider wraps an elastic band around the upper arm to apply pressure to the area and make the vein swell with blood. Next, the health care provider gently inserts a needle into the vein. The blood collects into an airtight vial or tube attached to the needle. The elastic band is removed from your arm. Once the blood has been collected, the needle is removed, and the puncture site is covered to stop any bleeding. - The sample is sent to a lab, where it is mixed with latex beads coated with a specific antibody or antigen. If the suspected substance is present, the latex beads will clump together (agglutinate). D-Dimer Test -newer assay specific for fibrinolysis involving plasmins degradation of fibrin. -principle of this test is similar with latex except that the antiserum is a highly specific monoclonal antibody to dimers. -normal: .5g/ml -useful in diagnosing DIC ELISA -uses antibody or antigen bound enzymes to measure antigen concentration (or antibodies) -specificity is enhanced by monoclonal antibodies -sensitive in detecting plasma concentration in nanoram/ml range -plasma is reacted with specific antibody immobilized on a solid surface -measures a variety of inhibitors, coagulation, activation products & other coagulation proteins.