Bleeding Time And Clotting Time (Bt And Ct) PDF

HEMA312 PRELIM LABORATORY TOPIC #1 6. Being careful not touch the puncture site. Blot the filter paper MR. MIKHAIL KRISTOFFER GONONG every 30 seconds, until the bleeding stops....

HEMA312 PRELIM LABORATORY TOPIC #1 6. Being careful not touch the puncture site. Blot the filter paper MR. MIKHAIL KRISTOFFER GONONG every 30 seconds, until the bleeding stops. 7. Record the bleeding time. BLEEDING TIME AND CLOTTING TIME (BT AND CT) IMPORTANT NOTE: BLEEDING TIME - Should be the blood flow more than 15 minutes. Discontinue the - used to measure the duration of bleeding after a measured skin test and report the test as “greater than 15 minutes” incision. - Test for/to assess primary hemostasis REFERENCE RANGE: 2-8 minutes - Test for in vivo platelet adhesion - Affected to congenital platelet disorders: IVY’S METHOD PROCEDURE 1. Obtain a piece of filter paper, stopwatch, and sphygmomanometer ✓ Bernard-Soulier Syndrome 2. Position the patient’s arm with the volar surface exposed. − Platelet adhesion defect 3. Place the sphygmomanometer on the upper arm − GP Ib59 receptor 4. Moisten a piece of cotton with 70% alcohol or Povidone iodine ✓ Von Willbrand Disease solution and thoroughly cleanse the puncture site. − Platelet adhesion defect 5. Apply the cuff and inflate to 40-60 mmHg and hold at this exact − VWF factor pressure for the duration of the test. ✓ Glanzmann’s Thrombasthenia − Platelet aggregation defect IMPORTANT NOTE: − GP IIb3a receptor - The time between inflation and incision should be 30 to 60 ✓ Storage Pool Defects seconds. − Wiskott-Aldrich Syndrome 6. After applying the pressure cuff and preparing the test site. Make − May-Hegglin Syndrome three (3) same punctures with a disposable lancet (1mm wide and - may be measured by one of the three methods: 3mm deep) Caution: Avoid subcutaneous veins. 1. Modified template/simplate – MCU and most accurate 7. Start the stopwatch immediately due to incision size is standardized. (wound size: 5mm x 8. After 30 seconds, wipe the flow of blood with the filter paper. 1mm incision) (Bring the paper close to the incision, but do not touch the paper 2. Ivy (forearm) directly to the incision, so as not to disturb the formation of the 3. Duke (earlobes) for young and infant children platelet plug) - Bleeding time depends on the elasticity of the blood vessel wall 9. Blot each site with filter paper every 30 seconds thereafter, until and on the number and functional capacity of platelets. no blood stains the paper. 10. Stop the timer when only clear fluid is absorbed onto the filter USES: paper. The bleeding time is determined to the nearest 30 seconds. - Performed on patients with a personal or family history of 11. Release the pressure of the sphygmomanometer. bleeding disorders. 12. Record the bleeding time for each of the puncture. (divided by 3) - Useful along with a platelet count for preoperative screening. 13. Get the average of the bleeding time in minutes and seconds. - The test is usually not recommended for patients with a platelet count of less than 75,000/µl. (not recommended for patients with SOURCES OF ERROR: severe thrombocytopenia) ✓ If body hair will interfere, lightly shave the area. ✓ Patient should be advised of a potential to produce a scar. This can PRINCIPLE OF BLEEDING TIME: usually be avoided using a butterfly bandage applied for 24 hours. ✓ The test is performed by using a sterile blood lancet to make a ✓ Aspirin and aspirin containing products may cause a prolonged measured skin puncture in the fingers, earlobe, forearm. bleeding time for up to 2 weeks ✓ The time is started immediately after the puncture. ✓ A standardized cut is necessary for valid results. Too little pressure ✓ Touching a piece of filter paper to the cut every 30 seconds until on the device and the wound will be shallow or nonexistent. Too bleeding ceases and record the time and reported as bleeding much pressure and the wound will be too deep. This is the one time. area where standardization has not been completely controlled. ✓ Low skin temperature produces constriction of the capillary PRE-ANALYTICAL PHASE (PATIENT PREPARATION) vessels, resulting in decreased blood flow. (no aspirin intake 24 hrs ✓ Explain to the patient this test is used to measure the time prior to test) required to form a clot and stop bleeding. ✓ Check the patient’s history for recent use of drugs (anti-platelet PRECAUTION: drugs) of that prolong bleeding time, including sulphonamides, ✓ Be sure to maintain a cuff pressure of 40-60 mmHg throughout thiazide diuretics, antineoplastic, anticoagulants, non-steroidal the test. If bleeding does not diminish after 15 minutes, anti-inflammatory drugs, aspirin and aspirin compounds, and discontinue the test. some non-narcotic analgesics. ✓ Report the test as “greater than 15 minutes” ✓ Aspirin – only drug that affects BT/ prolong BT REFERENCE RANGE: 3 to 6 minutes ANALYTICAL PHASE BORDERLINE: 6 to 11 minutes ✓ DUKE’S METHOD Materials: INTERPRETATION OF RESULTS 1. Sterile Blood Lancet - The bleeding time depends primarily on extra-vascular and 2. Stopwatch vascular factors and, to a lesser degree, on the factors of 3. Sterile Filter Paper coagulation. 4. Gauze pads or cotton balls - The chief factor controlling bleeding from a small cut is the 5. 70% Alcohol or Povidone Iodine Solution constriction of the minute vessels following injury. ✓ IVY’S METHOD - Accuracy in this test is enhanced by blotting the drops of blood Materials: at shorter intervals of time as the drops of blood become 1. Sterile Blood Lancet progressively smaller. 2. Stopwatch - Thrombocytes play an important part in the formation of the 3. Sphygmomanometer/BP cuff hemostatic plug that seal off a wound. In thrombocytopenic 4. 70% Alcohol or Povidone Iodine Solution purpura there is a decrease in platelets resulting in a prolonged bleeding time due to a defective platelet plug. An additional DUKE’S METHOD PROCEDURE factor prolonging the bleeding time in this condition is a defect in 1. Obtain a piece of filter paper and stopwatch. capillary contraction. In can be hereditary and acquired platelet 2. Moisten a piece of cotton with 70% alcohol or Povidone iodine dysfunction. and thoroughly cleanse the ball of the patient’s middle or ring - In hemophilia, the bleeding time is normal. This explained by the finger. (Puncturing the heel is not recommended) fact that there are no vascular or extravascular or extravascular 3. Allow the skin to air-dry. abnormalities. However, the test should not be performed on a 4. Make a puncture wound 2-3 mm deep in the earlobe or finger known hemophiliac, for delayed oozing blood is a real hazard. with a disposable blood lancet. 5. Start the stopwatch immediately. INCREASED BLEEDING TIME VALENZUELA, ATASHA NICOLE DE JESUS  Thrombocytopenia 5. After about two minutes start snapping off small lengths of the Capillary wall abnormalities tube, at intervals of 30 seconds, each time noting whether the Platelet abnormalities (Bernard-Soldier disease, Glanzmann’s fibrin thread is formed between the snapped ends. disease) 6. Repeat breaking at regular time intervals, till fibrin thread appears Drugs (aspirin, warfarin, anti-inflammatory medications, at the broken end of capillary tube. Do not pull away the catted streptokinase, urokinase, dextran, B-lactam antibiotics, moxalactam) pieces. Disseminated intravascular coagulation 7. Record the time interval between pricking finger and first Myeloproliferative disorders appearance of fibrin thread at the broken ends of capillary tube. Von Willebrand’s disease That is clotting time of blood. OTHER METHODS FOR BLEEDING TIME REFERENCE RANGE: 4-9 minutes ✓ Adelson-Crosby – sterile NSS warmed at 37 C ✓ Cody-Lalitch ✓ Bleeding time by Macfarlane – earlobes puncture ✓ Aspirin Tolerance Test – effect of aspirin on standard ADDITIONAL NOTES ✓ Hemophilia – bleeding time is normal except Factor I deficiency. ✓ Primary hemostasis – blood vessel and platelets IMPORTANT NOTES: ✓ Secondary hemostasis – clotting factors/coagulation factors, BLEEDING TIME and platelet phospholipids. ✓ Prolong BT has problem with primary hemostasis DUKE’S METHOD RR: 2-8 minutes ✓ Aspirin – inhibits platelet aggregation, thus it inhibits the IVY’S METHOD RR: 3-6 minutes action of cyclooxygenase which this enzyme is important to BORDERLINE: 6-11 minutes synthesize thromboxane A2 (important to promote CLOTTING TIME vasoconstriction and platelet aggregation. SLIDE OR DROP METHOD RR: 2-4 minutes CAPILLARY TUBE METHOD RR: 4-9 minutes CLOTTING TIME - interval between the moment when bleeding starts and the moment when the fibrin clot thread is first seen. - Bleeding time and clotting time is not the same. - Bleeding time depends on the integrity of platelets and vessel walls, whereas clotting time depends on the availability of coagulation factors. BLOOD COAGULATION - When the blood vessel ruptures, in a few minutes blood losses its fluidity and set into a semisolid mass called clot. - In vivo, blood clots outside the body on cuts and injuries. In vivo, blood clots inside the blood vessels. In coagulation disorders like hemophilia, clotting time is prolonged but bleeding time remains normal. ANALYTICAL PHASE ✓ SLIDE OR DROP METHOD Materials: 1. Blood lancet 2. Glass slide 3. Timer / Stopwatch 4. Cotton 5. 70% ethyl alcohol ✓ CAPILLARY TUBE METHOD Materials: 1. Capillary tube, non-heparinized 2. Blood lancet 3. Timer / Stopwatch 4. Cotton 5. 70% ethyl alcohol SLIDE OR DROP METHOD PROCEDURE 1. Disinfect site of puncture with 70% ethyl alcohol. Air dry. 2. Puncture to a depth of 2-3 mm using a blood lancet. 3. Start timer as soon as the first drop of blood appears. 4. Transfer the three drops of blood onto a clean glass slide. Careful not to touch the skin. 5. Pass the tip of the lancet through the first drop of blood every 30 seconds and not for the formation of fibrin strands. Repeat with second drop and then the third drop. 6. Stop the timer immediately as soon as fibrin strands are seen clinging at the tip of the lance on the third drop. REFERENCE RANGE: 2-4 minutes CAPILLARY TUBE METHOD PROCEDURE 1. Apply 70% alcohol to the clean finger with cotton swab. Allow it to dry naturally. 2. Prick the finger with usual aseptic precautions. Immediately start the stopwatch. 3. Dip one end of the capillary into blood drop gently without pressure. 4. Allow to fill the capillary with blood by lowering the end of fitted capillary. (Do not stuck the blood around ¾ of its length undipped) VALENZUELA, ATASHA NICOLE DE JESUS HEMA312 PRELIM LABORATORY TOPIC #2 - Equipment used: Hemacytometer and Thoma pipet (RBC or WBC) MR. MIKHAIL KRISTOFFER GONONG - Uses phase contrast microscope - Diluting fluid: 1% ammonium oxalate (also used for manual WBC MANUAL PLATELET COUNT | DIRECT AND INDIRECT METHOD counts; lyses RBC’s and allows platelets to form pseudopods) - Count 25 RBC squares (area is 1mm2) DIRECT METHOD - If low counts: PRE-ANALYTICAL PHASE ✓ use WBC pipet (1:20 dilution) Platelets ✓ RBC pipet: 1:100 dilution - smallest formed elements in the blood from megakaryocyte cytoplasm; 2-4 um TOCANTIN’S METHOD - round or biconvex shape - Equipment used: Hemacytometer and Thoma pipet (RBC or WBC) - MPV: 8-10 fL - Uses light microscope - fragments of cytoplasm that have broken off platelet precursors. - Diluting fluid: Rees and Ecker - Platelets function in the coagulation of the blood. - Brilliant Cresyl blue (BCB): dye - Neutral formalin: prevents platelet clumping Platelets are difficult to count due to: - Sodium citrate: anticoagulant ✓ Small size – often hard to differentiate from bacteria and debris - Count 25 RBC squares (area is 1mm2) ✓ Adhesiveness – affinity for adhering to glass ✓ Aggregation – tendency to clump together PROCEDURE A. DILUTION OF BLOOD SAMPLE PRINCIPLE 1. The capillary bore of the RBC pipette is rinsed with Rees-Ecker ✓ Whole blood is diluted with Rees-Ecker diluting fluid which makes diluting fluid. All excess fluid should be removed from the pipette platelets readily visible under the bright-field microscope before blood is drawn into the pipette. This step will ensure ✓ The platelets are then counted in the central large square of the platelet will not adhere on the wall of the capillary bore of the counting chamber (hemacytometer) pipette. ✓ Collection of the blood in the desired amount in the EDTA evacuated 2. Draw the blood into the pipette up to 0.5 mark. If excess blood tube by venipuncture was draw, adjust the blood level to the exact 0.5 mark with NSS moistened cotton. Clean the stem of the pipette first before any MATERIALS AND EQUIPMENT adjustment will be made. ✓ EDTA evacuated tube 3. The blood should be diluted immediately with the Rees-Ecker ✓ Microscope with LPO and HPO solution. Make a 1:200 dilution by filling the pipette with the ✓ Rees & Ecker Diluting Fluid solution up to 101 mark. Two pipettes should be use ✓ Hemocytometer and coverslip simultaneously, and each should be used to charge one side of the ✓ RBC pipette with rubber tubing counting chamber. and mouthpiece 4. After the dilution, the pipettes should be shaken immediately for ✓ Tally Counter or Cell Counter at least 60 seconds. This step will prevent platelet clumping and will ensure accurate counting. B. CHARGING OF SOLUTION ONTO THE COUNTING CHAMBER 5. After mixing, discard the first 5 drops from each pipette. ✓ Note: Do not induce or blot the tip of the pipette by any absorbent material to facilitate the process. The drop should drop freely by gravity alone. Each side of the hemocytometer should be charged with a different pipette 6. After charging the chambers, the hemocytometer should be kept in covered container for about 10 to 15minutes with wet gauze or cotton pads beneath the hemocytometer to prevent evaporation, this step will allow the platelets to settle aid in accurate counting later. C. COUNTING OF PLATELETS 7. Carefully place the hemocytometer on the microscope stage so as not to disturb the platelets. Count platelets in 5 RBC squares in the center of the counting chamber. Both sides of the counting chamber should be counted and the results averaged. Using the high power magnification count all the platelets, they appear as small, roundish, unevenly shaped structures. NOTE: o If 5 RBC squares are counted area should be 0.20mm² o If 25 RBC squares (central large square) are counted area should be 1 mm² IMPORTANT NOTE: ✓ Platelets should be counted on each side of the hemacytometer, and the difference between the totals should be less than 10% ✓ The accuracy of the manual platelet count should be verified METHODS FOR DIRECT PLATELET COUNT by performing a platelet estimate on a Wright-stained 1. Brecher-Cronkite, Tocantins/Rees-Ecker peripheral blood film made from the same specimen. 2. Guy and Leake’s 3. Nygard’s 4. Walker and Sweeney’s 5. Van allen’s 6. Unopette (calibrated) BRENCHER-CRONKITE METHOD - Reference method for manual platelet counts  COMPUTATION INDIRECT METHOD - Parameters: PRE-ANALYTICAL PHASE ✓ theaverage number of platelets per square millimeter ✓ the dilution factor (which is 200, just like the RBC PRINCIPLE count) ✓ A well-made smear is stained with Wright’s or Wright’s-Giemsa to ✓ the volume of the diluted blood counted, which is the visualize cellular elements of blood including the platelet area times depth (1mm² x 0.1 mm=0.1µl) ✓ Is done in the portion of the smear where the red cells start to overlap ✓ 10 OIO consecutive fields are examined for platelet and reported as NORMAL VALUE: 150,000-400,000 platelets/uL or 150-400 x 103 platelets/uL cells per microliter of blood FORMULA MATERIALS AND EQUIPMENT ✓ EDTA evacuated tube ✓ Microscope ✓ Glass slides (including spreader slide) ✓ Wright’s stain ✓ Tally counter ✓ Cedarwood oil PROCEDURE 1. Make a well-made blood smear and stain with Wright’s or Wright’s-Giemsa stain. See the product insert for the specific INTERPRETATION OF RESULTS instructions. Manufacturer has sometimes have modifications. BELOW NORMAL: Thrombocytopenia 2. Focus the slide using low-power magnification. Examine the smear - Aplastic anemia for areas that is suitable for counting. Care must be taken to stay - Pernicious anemia in an appropriate area of the slide. The RBC’s should barely be - Acute leukemia touching one another in the area of the estimate. - Idiopathic thrombocytopenia purpura 3. Using the high magnification, check the area of smear for suitability for counting the platelets. ABOVE NORMAL: Thrombocytosis 4. Shift the objective to the 1000x magnification. Examine the strip - Polycythemia vera of the film moving from one field to next systematically (See - Hemolytic anemia Figure 1.1) - Chronic myeloproliferative disorders 5. Count and record the platelets seen in then consecutive oil - Post-splenectomy immersion fields. SOURCES OF ERROR ✓ Light adjustment is critical. If the condenser is not lowered it will fade out the platelets. ✓ Bacteria and debris can be misinterpreted as platelets, this type of artifact is generally much more retractile than platelets. ✓ Clumping of platelets sometime occurs, and if so the specimen must be recollected. EDTA is the anticoagulant of choice for preventing platelet clumping. ✓ General hemacytometer errors, i.e. overloading chamber, counting wrong borders, etc. ✓ The phenomenon of “platelet satellitosis” may occur when EDTA anticoagulant is used. This refers to the adherence of platelets around neutrophils, producing a ring or satellite effect. Using sodium citrate as the anticoagulant should correct this problem. Because of the dilution in the citrate evacuated tubes, it is necessary to multiply the obtained platelet count by 1.1 for accuracy COMPUTATION - A platelet count should always be checked on the smear to make sure it looks accurate. To estimate a platelet count from a blood smear you regard each platelet seen per oil power field as being equivalent to 20,000 platelets/mm³ or uL - By determining the average number of platelets seen per oil field multiplying this number by 20,000 you can estimate the platelet count. For example, if you see 10 platelets per oil field your estimated could be: 10 x 20,000 or 200,000/mm³. - Usually 7-20 platelets per OIF represent a normal platelet count. FORMULA