0.2

• Evaluation of Primary Hemostasis

  1. Bleeding time

  2. Capillary Fragility/Resistance Test

  3. Tourniquet/Rumple-Leede/Hees test/Positive pressure test

  4. Suction CuP/Petechiometer/Negative pressure technique
• Quantitative Evaluation of Platelet (PLT Count)

  1. Indirect Method: Fonio’s Method

  2. Direct Method: Light Microscopy: Rees & Ecker (Toncantin’s) and Guy & Leaks, Phase Contrasts: Brecher-Conkrite, Unopette, Tocantins, Nygard’s, Walker & Sweeney’s, Van Allen method, Automation, Plt count estimation
• Quantitative Evaluation of Platelet (PLT Function Tests)

  Platelet Adhesion: Duke’s method, Ivy’s method, Modified Ivy’s/Modified Simplate, Adelson-Crosby Method, Cody lalitch, Mac Farlane for bleeding time, Aspirin Tolerance test, Glassbead Retention test, Aspirin Tolerance test, Platelet Aggregation: turbidity, Transmittance of Light, Clot retraction test: Hirschboeck Method, MacFarlane for Clot retraction, Platelet Factor Assay (PF3 and PF4), Contents (internal and External)
• Vascular Integrity


  Rumple-Leede/Tourniquet/Hees test/Positive pressure test: Apply pressure cuff on the upper forearm and inflate to pressure midway systolic and diastolic (for 5 minutes), Remove cuff and observe for the presence of Petechiae (<3mm) on the volar surface of arm and dorsal area of hands and fingers, 15 min after release of pressure cuff, choose an area of 1 inch diameter and count the number of petechiae and grade: Grade, No of petechiae 1+ (normal) 0-10, 2+ (equivocal) 10-20, 3+ 30-50, 4+ >50, Sunction CuP/Petechiometer/Negative Pressure: Uses modified Da Silva Melle instrument, CuP applied at 200 mmHg for 1 min at the outer surface of arm, Observe for Macroscopic petechiae, NV: <4 petechiae
• Difficulties in counting platelets
• Normal platelet count values
• Indirect Method: Fonio’s Method

  Not reliable, Platelets are counted in relation to 1,000 RBCs in blood smear, Formula: Plt/mm3= No of plts counted x RBC Count / 1000, Alternative: Plt/mm3= average no. of plts/OIF x 20000
• Direct Method: Light Microscopy

  Rees and Ecker’s (Tocantin’s), Guy and Leake’s, Diluting fluid components: Brilliant cresyl blue, Sodium citrate, Distilled water, Crystal violet, Sodium citrate, Distilled water, Formalin 40%, Count Platelet in 4 large squares (as in WBC count), Count Platelet in all 25 intermediate squares in the central large squares (as in RBC count), Shortcut formula: Plt count
• Materials for Platelet Count


  • Brilliant cresyl blue
  • Sodium citrate
  • Distilled water
  • Crystal violet
  • Formalin 40%
• Platelet Count in 4 large squares

  Count Platelet in 4 large squares (as in WBC count)
• Platelet Count in all 25 intermediate squares


  Count Platelet in all 25 intermediate squares in the central large squares (as in RBC count)
• Shortcut formula for Platelet Count

  1. Plt count (/mm3)= Plt count x 10x 200 / 4

  2. Plt count (/mm3)= Plt count x 100 x 200 x 1
• Shake pipet for 3-5 times before loading
• Charge counting chamber and allow it to stand for at least 3 minutes
• Phase Contrast Microscopy for Platelet Count


  Procedure is same as Rees Eckers except platelets are counted w/ the use of Phase Contrast Microscope
• Methods for Platelet Count Estimation

  • Brecher-Conkrite (REFERENCE METHOD)
  • Unopette, Tocantins, Nygard’s
  • Walker & Sweeney’s, Van Allen method
• Uses 1% ammonium oxalate as diluting fluid
• Automation Machines for Platelet Count

  • Coulter S-Plus - Impedance Counting and Conductivity
  • Ortho ELT 81ds - Laser Light Scattering
  • Technicon auto-counter - Optical particle scattering
  • Ultra Flo 100 - Electronic Impedance and Flow cytometry (Hydro dynamic focusing)
  • Baker series 810 - Electronic Impedance and Flow cytometry (Hydro dynamic focusing)
• Examine in thin area of the slide where only a few red blood cells slightly overlap using OIF
• Normal blood smear should demonstrate 8-20 plts/field
• Qualitative Evaluation for Platelets

  1. Platelet Adhesion

  2. Bleeding Time
• Bleeding Time is the time it takes for a standard wound to stop bleeding
• Bleeding Time is a screening test for detecting disorders of platelet function and VWD
• Bleeding Time is not affected by the coagulation mechanism
• Bleeding Time is used for differentiation of Factor 8 and VWD
• Factors affecting Bleeding Time


  • Plt count and platelet function
  • Thickness and vascularity of the skin
  • Quality of blood vessels
  • Aspirin and Aspirin containing compounds
• Aspirin and Aspirin containing compounds should be deferred 7 days before the test
• NSAID’s, Ibuprofen, Tolmetin sodium, Naproxen should be deferred 24 hrs before the test
• Duke Method for Bleeding Time

  1. Finger is punctured 3 mm deep

  2. Start timer as soon as the first drop of blood appears

  3. Blot drop of blood with filter paper every 30 sec (don’t touch the wound)

  4. Stop timer as soon bleeding stops

  5. Normal value: 2-4 mins
• Ivy Method for Bleeding Time

  1. Use sphygmomanometer inflate at 40mmHg at patient's upper forearm (above elbow)

  2. Make 2 incisions (2 mm deep & 2mm long)

  3. Blot drop of blood on each site on 2 separate filter papers
• Bleeding time measurement method: Pears

  Blood w/ filter paper every 30 sec (don’t touch the wound), Stop timer as soon bleeding stops, NV: 2-4 mins
• Bleeding time measurement method: Ivy Method


  Use sphygmomanometer inflate at 40mmHg at px’s upper forearm (above elbow), Make 2 incisions (2 mm deep & 2mm long), Blot drop of blood on each site on 2 separate filter paper every 30 secs, Record time of 2 punctures and report average 2 results, NV: 1-7 mins
• Bleeding time measurement method: Modified Ivy/ Template Bleeding time/ Simplate/ Mieke et Al method


  Uses a template containing standardized Slit in place of disposable lancets, sphygmomanometer inflated in forearm, NV: 2-9 mins
• Bleeding time measurement method: Adelson-Crosby method

  Almost the same w/ Dukes but puncture finger immerse in NSS warmed at 37C at water bath to check bleeding, NV: >3 mins
• Bleeding time measurement method: Cody Lalitch


  Same as Adelson method
• Bleeding time measurement method: Mac Farlane


  Same as Adelson method but the site is earlobe
• Glassbed Retention Test


  In Vitro determination of Plt Adherence, Uses EDTA sample, Reported in %, NV: 75-95%
• Causes of decreased Platelet retention

  • Bernard Soulier, VWD, Glazzman, Chediak Higashi, Aspirin, Myeloproliferative, Uremia, Afibrinogenemia, Thrombotic disorder, Hyperlipidemia, Carcinoma, Oral contraceptive, Pregnancy
• Aspirin Tolerance Test


  Assess effects of standard dose of aspirin on Duke’s Bleeding time
• Platelet Aggregation Test


  More important than adhesion, Principle: Turbidimetry and Light Transmittance, Specimen Consideration: Fasting: 8 hours (Avoid Lipemic samples), pH: 6.5-8.5, Temp: 37C at Rt, Process w/in 3 hrs, No aspirin/NSAIDs: 7 days/1 week, Specimen: Citrated PRP (platelet Rich Plasma), Aggregating agents: Ristocetin, ADP, Collagen, Epinephrine, Serotonin, Arachidonic acid, Thrombin, Results: Aggregation: normal, No aggregation: abnormal