Hema 2

• Platelets


  Fragments that arise from the megakaryocyte, the largest cell found in the bone marrow
• Platelets


  • Small, colorless, moderately refractile bodies, that appear as azure granules with scanty light blue cytoplasm when stained
  • Essential for maintaining the balance of bleeding and clotting within the body (Hemostasis)
• Materials


  • Phlebotomy Kit
  • Anticoagulated Blood(EDTA)
  • Hema Quick-Giemsa stain
  • Glass slide
  • Applicator stick
  • Microscope
  • Personal Protective Equipment
• Platelets


  • Around 2-3 um(diameter), that adhere, aggregate and secrete the contents of their granules
• Platelet estimate


  1. Prepare a blood smear

  2. Dry the smear

  3. Stain the prepared smear

  4. View the smear under the microscope

  5. Count the number of platelets in ten field under 100x magnification

  6. Average the platelet count

  7. Multiply the average by 15,000

  8. Report the platelet count
• Reese-Ecker Method

  Diluting fluid composition: Brilliant cresyl blue, 40% formalin, and sodium Citrate
• Reese-Ecker Method


  1. Draw blood up to 0.5 mark of the RBC pipette

  2. Dilute blood with Reese Ecker diluting fluid up to 101 marks

  3. Shake the pipette for 1-5 minutes

  4. Discard 5-6 drops and charge the counting chamber

  5. Place the counting chamber on a petri dish with a wet filter paper to allow the platelet to settle

  6. Count the platelets in all 25 tertiary squares of central secondary square (1mm2)
• Ammonium Oxalate technique


  Draw blood up to the 1.0 mark of the RBC pipette and ammonium oxalate up to the 101 marks
• Direct Methods


  • Nygard's
  • Guys and Leake's
  • Brenker-Cronkite
• Indirect Methods


  • Dameshek
  • Fonio's
  • Olef's-The best indirect method
• Sources of Error


  • Platelet clumps
  • Imperfect sources of blood
  • Blood in EDTA kept 20 degrees Celsius
• Platelets are stained light blue in the Reese-Ecker Method
• Computation for Reese-Ecker Method: No. of cells/mm3=Total no. of cells counted / (area (1) x depth (1/10) x dilution (1/200)) or cells counted x 2,000
• Computation for Ammonium Oxalate technique: No. of cells/mm3=Total no. of cells counted / (area (1) x depth (1/10) x dilution (1/100)) or cells counted x 1,000
• Thrombocytosis is a condition that may lead to an increased platelet count
• Thrombocytopenia is a condition that may lead to a decreased platelet count
• Mixing studies


  A special laboratory test that is performed if the Prothrombin Time (PT) and Activated Partial Thromboplastin Time (aPTT) is prolonged
• Intrinsic pathway


  1. XIIa

  2. XIa

  3. VIIIa:IXa

  4. Xa:V

  5. IIa

  6. Ia
• Extrinsic pathway


  VIIa:TF
• Common pathway


  1. Xa:V

  2. IIa

  3. Ia
• Mixing study is performed for the prolonged clotting time only
• Patient plasma to NPP ratio

  • 1:1 mixing study (equal parts patient plasma and NPP) is most commonly used
  • 4:1 mixing study (4 parts patient plasma and 1 part NPP)
• Incubation time


  • Immediate: clotting time is evaluated immediately after mixing patient plasma and NPP
  • Incubated: mixed patient plasma and NPP is incubated at 37 °C for 1-2 hours
• Normal Plasma Pool (NPP)


  • Can be purchased from a vendor
  • If making NPP locally, it is recommended to use platelet poor plasma from a minimum of 20 - 30 donors with coagulation factor levels of approximately 100%
• Immediate correction of clotting time

  Means a factor deficiency pattern

  Principle: at least 50% activity of a given factor is sufficient to produce a normal clotting time
• Not corrected in the immediate (PT) or immediate or incubated mixing study (aPTT)

  Interpretation: Inhibitor pattern

  Principle: the inhibitor binds all available factor, from both the patient and NPP and the clotting time remains prolonged
• Rosner index


  Used in conjunction with a 1:1 mixing study, originally described for use in lupus anticoagulant testing

  Rosner index > 15 suggests presence of an inhibitor
• Chang percentage


  Used in conjunction with a 4:1 mixing study (optimal sensitivity)

  Chang percentage result of < 50% suggests the presence of an inhibitor
• Problems that might be encountered

  • Multiple factor deficiencies
  • High hematocrit or underfilled collection
  • Anticoagulant effect

  Heparin: affects aPTT mixing studies

  Direct thrombin inhibitor (DTI) may affect aPTT > PT mixing studies

  Direct Xa inhibitors may affect PT > aPTT mixing studies
• Mixing studies


  A special laboratory test that is performed if the Prothrombin Time (PT) and Activated Partial Thromboplastin Time (aPTT) is prolonged
• Intrinsic pathway


  1. XIIa

  2. XIa

  3. VIIIa:IXa

  4. Xa:V

  5. IIa

  6. Ia
• Extrinsic pathway


  VIIa:TF
• Common pathway


  1. Xa:V

  2. IIa

  3. Ia
• Mixing study is performed for the prolonged clotting time only
• Patient plasma to NPP ratio

  • 1:1 mixing study (equal parts patient plasma and NPP) is most commonly used
  • 4:1 mixing study (4 parts patient plasma and 1 part NPP)
• Incubation time


  • Immediate: clotting time is evaluated immediately after mixing patient plasma and NPP
  • Incubated: mixed patient plasma and NPP is incubated at 37 °C for 1-2 hours
• Normal Plasma Pool (NPP)


  • Can be purchased from a vendor
  • If making NPP locally, it is recommended to use platelet poor plasma from a minimum of 20 - 30 donors with coagulation factor levels of approximately 100%
• Immediate correction of clotting time

  Means a factor deficiency pattern

  Principle: at least 50% activity of a given factor is sufficient to produce a normal clotting time
• Not corrected in the immediate (PT) or immediate or incubated mixing study (aPTT)

  Interpretation: Inhibitor pattern

  Principle: the inhibitor binds all available factor, from both the patient and NPP and the clotting time remains prolonged
• Rosner index


  Used in conjunction with a 1:1 mixing study, originally described for use in lupus anticoagulant testing

  Rosner index > 15 suggests presence of an inhibitor