Bleeding time Screening test for detecting disorders of functional platelet and for Von Willebrand disease, and is directly affected by platelet count and the ability of platelets to form a platelet plug Prolonged in thrombocytopenia, Von Willebrand disease, and following ingestion of aspirin, aspirin-containing compounds, anti-inflammatory drugs, anticoagulants, some antibiotics and certain other drugs. Methods 01 Duke method 02 Ivy method 03 Template method DUKE METHOD Site: earlobe/ fingertip Device: feather lancet Incision size: lancet – size dependent Normal value: 1-3 minutes DUKE METHOD 1. Cleanse the ear lobe of the patient with alcohol and air dry. 2. Puncture the ear lobe with the right hand while the left hand is holding the glass slide. 3. Turn on the stopwatch. 4. Hold the filter paper under the ear and let blood drop from the ear lobe to the filter. Do not apply pressure to the ear. 5. When blood no longer drops from the ear, turn the stopwatch off and record the time. The length of time from the puncture until the blood no longer flows is the bleeding time. IVY METHOD Site: volar surface of the forearm approximately 5 cm below the antecubital crease Device: feather lancet with blood pressure cuff inflated to 40 mmHg Incision size: parallel to antecubital, 3-5 mm (depends on the size of the lancet) Normal value: 2.5 – 7 minutes IVY METHOD 1. Place a blood pressure cuff around the arm above the elbow and inflate to 40mmHg. Maintain this pressure throughout the procedure. 2. Clean the skin of the fleshy portion of the forearm below the antecubital fossa with alcohol and allow to airdry. 3. Tense the skin and proposed test site by gasping the posterior portion of the arm between the thumb and forefinger of the left hand. 4. Pierce the stretched skin with disposable lancet all the way down; turn the stopwatch on. 5. Absorb the drops of blood at 30-second interval on the filter paper until the blood ceases. Turn off the stopwatch and record time. 6. Label the center of the filter paper with name, date and bleeding time. TEMPLATE METHOD Site: volar surface of the forearm approximately 5 cm below the antecubital crease Device: disposable template device with blood pressure cuff inflated to 40 mmHg Incision size: Adult (white) – 5.0 mm long, 1 mm deep Pediatrics (pink) – 3.5 mm long, 1 mm deep Normal value: 2-8 minutes Simplate Contains a springloaded blade within a plastic case Guillotine-like cut in the skin Surgicutt Utilizes a slicing action using a surgical blade Spring loaded; blade automatically retracts into the housing after it has made a standardized cut TEMPLATE METHOD 1. Place the patient’s arm on a steady support with the volar surface exposed. Lightly shave the area if body hair will interfere with the test. 2. Place the sphygmomanometer cuff on the upper arm. Inflate the cuff to 40mmHg. For newborns: Venostasis pressure should be maintained in accordance with the weight of the newborn. The following pressures are recommended for use in conjunction with the newborn weight: *The time between inflation of cuff 20 mmHg for infants - 1,000g or less and incision should be 30-60 seconds. 25 mmHg for infants - 1,000g - 2000g Hold at this exact pressure for the 30 mmHg for infants - 2,000g or greater duration of the test. 3.Cleanse the area with an antiseptic swab and allow to air dry. 4. Remove the template device from the blister pack, being careful not to contaminate or touch the blade-slot surface. 5. Remove the safety clip. Once removed, do not push the trigger. 6. Hold the device securely between the thumb and the middle finger. 7.Gently rest the device on the patient’s forearm and apply minimal pressure on the skin. TEMPLATE METHOD 8.Gently push the trigger, starting the stopwatch simultaneously. 9. Remove the device from the patient’s forearm immediately after triggering. 10. Every 30 seconds, wick the blood with blotting paper. Bring the blotting paper close to the incision, but do not touch the paper directly to the incision so as not to disturb the formation of the platelet plug. 11. Wick the blood every 30 seconds until blood no longer stains the paper. Stop the timer. Bleeding time is determined to the nearest 30 seconds. 12. Remove the sphymomanometer cuff and cleanse the incision site with an antiseptic swab. Potential scarring can be reduced by approximating the skin edges with a non-allergenic wound closure strip for 24 hours. Bleeding time devices Device No. of Incisions Length/Depth of Incision (mm) Simplate Pediatric 1 3.0 x 0.5 Perpendicular/Parallel Simplate 1 5.0 x 1.0 Parallel Simplate II 2 5.0 x 1.0 Parallel Surgicutt 1 5.0 x 1.0 Parallel Surgicutt 2 2 5.0 x 1.0 Parallel Surgicutt Newborn 1 2.5 x 0.5 Perpendicular Surgicutt Jr. 1 3.5 x 1.0 Parallel Position of Choice EQUIPMENT Blood pressure cuff Bleeding time device Stopwatch Wattman filter paper Alcohol prep pads Bandage PRINCIPLE A blood pressure cuff is placed on the patient’s arm above the elbow, inflated, and maintained at a constant pressure throughout the procedure. One or two standardized incisions are made on the volar surface of the forearm. The length of time required for bleeding to stop is recorded as the bleeding time. PROCEDURE 1.) Locate the area for the bleeding time. PROCEDURE 1.) Locate the area for the bleeding time. PROCEDURE 2) Cleanse the site with an alcohol and allow to dry. 3) Place a blood pressure cuff on the patient’s arm above the elbow. Increase the pressure to 40 mmHg (or lower for newborns) and hold this exact pressure for the entire procedure. PROCEDURE 4) Prepare the bleeding time device. Position it in the correct direction and appropriate area on the arm, using only that amount of downward pressure so that both ends are touching the skin and the device does not cause an indentation. 5) Activate the trigger and start the stop watch. Remove the device approximately 1 second after making the incision. PROCEDURE 6) Blot the blood from the puncture site on a clean section of circular filter paper every 30 seconds. 7) When the bleeding ceases, stop the watch and release the blood pressure cuff. Record the results. 8) Place a bandage over the puncture site. DISCUSSION The aspirin tolerance test may be useful in helping to distinguish functionally abnormal platelets from normal platelets When performing a bleeding time on infants, use Surgicutt Newborn The incision must be made consistently in the same direction Clotting time Generally measures intrinsic and common pathways and monitor heparin therapy Time it takes for blood to solidify after it has been out of the vessel and upon activation of the coagulation factors Methods 01 Slide Method 02 Capillary Tube Method 03 Lee and White Method Slide method Also called as drop method Site: fingertip Materials used: pricker, clean glass slide Puncture to a depth of 3mm Normal value: 2-4 minutes Capillary tube method Sabraze’s Method, Dale and Laidlaw’s Method Site: fingertip Materials used: pricker, plain glass capilet Puncture to a depth of 3mm Normal value: Sabraze’s Method: 3-7 minutes Dale and Laidlaw’s Method: 2-4 minutes Lee and White method Routinely used Perform venipuncture to obtain blood sample Material used: syringe Main stimulus: clean glass test tubes Normal value: 5-15 minutes Specimen: Freshly drawn venous blood Lee and White method PRINCIPLE The time required for the clot to form is a measure of the overall intrinsic and common pathway Freshest volume of blood sample is the 1ˢᵗ volume of blood wherein coagulation factors are activated whereas the 1ˢᵗ volume of blood taken out from the blood vessel is the last volume of blood wherein the coagulation factors are activated PROCEDURE 1. Label 3 test tubes (#1, #2, #3) and premoisten with NSS. 2. Perform venipuncture and withdraw 3 ml of venous blood. 3. Start the timer as soon as blood enters the hub of the needle. 4. Carefully transfer 1 ml of blood to each of the 3 tubes and incubate in waterbath. 5. Tilt test tube 1 every 30 seconds until blood clots and record time. 6. Do the same with the 2ⁿᵈ tube until blood clots and also do the same with the 3ʳᵈ tube. 7. Report the time required for the blood in the 3ʳᵈ tube to clot as the clotting time. CENTRIFUGATION Centrifuge uses centrifugal force to separate phases of suspensions by different densities Frequently used in processing blood to derive plasma or serum fractions Urine and body fluids may be centrifuged to concentrate particulate matter as sediment to be examined Conditions should specify both the time and the centrifugal force Centrifuge selection Must have the highest possible centrifugal force and not rotational speed RCF in g units Multiples of the gravitational force Key points to remember Tubes, carriers, or shields of equal weight, shape, and size should be placed in opposing positions in the centrifuge head to achieve appropriate balance Tubes must be balanced across the center of rotation, and each bucket must be balanced with respect to its pivotal axis Specimens must be placed with regard for a geometrically symmetrical arrangement, using waterfilled tubes to attain balance EQUIPMENT 1. Tabletop, general laboratory centrifuges 2. Horizontal head, fixed angle, or angle-head 3. High speed centrifuges 4. Portable floor models, undercounter models 5. Microcentrifuges 6. Refrigerated and unrefrigerated types 7. Ultracentrifuge models Aliquot preparation Often created when multiple tests are ordered on a single specimen Prepared by transferring a portion of the specimen into one or more tubes labeled with the same id information as the specimen tube Once transferred into an aliquot tube, serum and plasma are virtually indistinguishable Serum and plasma or plasma obtained from different types of anticoagulant must not be mixed together If testing is to be delayed, most samples can be refrigerated at 4°C for up to 8 hours