M2 LAB EVAL PRIMARY

• QUANTITATIVE PLATELET TESTS


  Assess the number of platelets

  
  

  •NV: 150-400x109/L

  •Thrombocytopenia: decreased in platelets

  •Thrombocytosis: increased in platelets
• METHODS IN QUANTITATIVE PLATELET TESTS


  Direct Method

  Indirect Method

  Automated Method
• Direct Method


  Brecker Cronkite Method

  Tocantin's Method
• Brecker Cronkite Method


  - Reference method

  - Uses phase contrast microscope

  -Diluting fluid: 1% ammonium oxalate
• Tocantin's Method


  - Uses hematocytometers and thoma pipette

  - Uses simple light microscope

  - Reese and Ecker as diluting fluid

  • Brilliant cresyl blue

  • Neutral formalin

  • Sodium citrate

  - Count 25 RBC square

  - If low count: use WBC pipette (1:20 dilution)
• Indirect Method


  - Used for platelet estimation Allows visualization of platelet morphology

  - Fonio's Method and Dameshek Method

  - Fonio's method: 1 OIO should have 3-10 platelets per 100 RBC
• Automated Method


  - Optical and electrical methods

  - Optical degree of light scattering
• QUALITITATIVE PLATELET TESTS


  Assess the function of platelets

  
  

  - Platelet Adhesion in vitro

  - Abnormal Platelet Retention Test

  - Bleeding Time

  - Clot Retraction Test

  - Platelet Factor III Assay

  - Platelet Factor IV and Beta thromboglobulin Assay

  - Platelet Aggregation Test

  - Platelet Aggregation Patterns
• Capillary Resistance Test


  - Pressure cuff is used in 100mg/Hg for 5 min, count petechiae after 15-30 minutes

  - Correlated with degree of thrombocytopenia
• 1+


  0-10Few petechiae on the anterior part of forearm
• 2+


  10-20Many petechiae on the anterior part of the forearm
• 3+


  20-50Multiple petechiae over the anterior and the back of the hand
• 4+


  >50 Confluent petechiae over the anterior and the back of the hand
• Platelet Retention Test


  - Known as glass bead retention dish

  - EDTA/Heparin

  - Blood is passed thru a tube containing beads then platelets adheres to beads
• 1st tube:


  Control
• 2nd tube:


  Platelet count
• 3rd tube:


  Platelet count; Lower count 75-95% of platelet are lost
• DECREASE PLATELET RETENTION

  ‣ Bernard Soulier

  ‣ Glanzmann Thrombasthenia

  ‣ vWD

  ‣ Chediak-Higashi

  ‣ Myeloproliferative disorders

  ‣ Uremia

  ‣ Aspirin Administration
• INCREASED PLATELET RETENTION


  ‣ Thrombotic disorder

  ‣ Hyperlipidemia

  ‣ Carcinoma

  ‣ Oral Contraceptives

  ‣ Pregnancy
• Bleeding Time


  - Time required for blood to stop bleeding

  - Affected if there are congenital platelet problems

  
  

  • Duke's test

  • Modified Ivy's test

  • Modified Simplate Test
• Duke's test


  - For infants, children

  - Done in earlobes

  - NV: 2-8 min
• Modified Ivy's test


  Done 5cm below the antecubital fossa with pressure cuff (40-60mmhg)
• Modified Simplate Test


  Done by making a wound incision of 5mm length and 1mm depth
• Clot Retraction Test


  - Serum is expressed from the clot

  - Assess function of thrombosthenin

  - Asses entire platelet function and coagulation

  -Abnormal CRT: thrombocytopenia, low and abnormal fibrinogens, paraproteins, Glanzmann’s thrombasthenia

  • Hirshboek Method

  • McFarlane Method
• Hirshboek Method


  - Uses castor oil

  - After 15-45 min check for dimpling phenomenon
• McFarlane Method


  - Checked after 1 hour

  - Uses wire loop to check for clot then water bath

  - Blood volume should decrease up to 50%

  - NV: 44-67%
• Platelet Factor III Assay


  - Platelet phospholipids provides surface to which clotting factors bind

  - Needs patient PPP and PRP

  - Control: Pooled PPP, sample, activators, 0.025M CaCl2

  - Activators: Directly stimulates platelets to produce PF3 Kaolin Epinephrine
• Platelet Factor IV and Beta thromboglobulin Assay


  - Specimen must be refrigerated prior to testing (4C)

  - Part of alpha granules

  - Used as indicator for platelet activation in Myocardial infarction, diabetes, venous thrombosis Measured through RIA
• Platelet Aggregation Test


  Uses aggregometer

  Specimen: PRP
• Platelet Aggregation Test Specimen consideration

  • Fasting at least 8 hrs to avoid lipemic sample

  • Must not be placed in ref to avoid aggregation

  • Non hemolyzed to avoid release of ADP

  • No aspirin within 7 days

  • Specimen must be in water bath at pH 6.5 to 8.5
• Aggregating agents


  ADP

  Collagen

  Epinephrine

  Thrombin

  Ristocetin

  Arachidonic acid