PMTP_LAB_M6

• Blood smear


  • Should be made within 2 hours after collection
• Pseudothrombocytopenia


  Adherence of platelet on the surface of WBC (Neutrophils)
• Correction for pseudothrombocytopenia


  1. Recollection blood specimen using 3.2% Sodium Citrate

  2. Results of platelet count obtained from the light blue top should be MULTIPLIED by 1.1
• Pseudothrombocytopenia


  Platelet form large clumps as large as WBC
• Pseudoleukocytosis


  Platelet form large clumps as large as WBC
• Correction for pseudothrombocytopenia and pseudoleukocytosis

  1. Recollect using 3.2% Sodium Citrate

  2. Result of platelet count and WBC count obtain from the light blue top should be multiplied by 1.1
• Free blood


  Best for evaluation of blood cell morphology
• Source of specimen


  • EDTA blood
  • Anticoagulant-free blood
• EDTA


  • Advantages: Multiple blood smear can be made, Prepared at a later time, Prevents platelets clumping or clotted specimen
  • Disadvantages: EDTA-induces platelet clumping
• Anticoagulant-free blood


  • Advantages: Made at the patient's side, Some artifacts may be prevented
  • Disadvantages: Platelets clumping, Few films can be made
• Methods of blood film preparation

  • Two glass slides method (Manual Wedge Technique)
  • Coverslip technique
  • Automated methods
• Important considerations for blood film preparation

  • Angle between two slides
  • Size of the drop of blood
  • Speed of the spreader
  • Hematocrit of the patient
• Characteristics of an ideal blood smear

  • Gradual transition from thick to thin area
  • 2/3 to 3/4 the length of the film slide
  • Finger-shaped
  • Visible lateral edges
  • Without irregularities, holes, or streaks
  • Feathery edge and has rainbow appearance
  • Whole drop of blood is picked up and spread
• Blood smear distribution


  • Uneven distribution of WBCs
  • Feather edge and side edges: MORE segmented neutrophils, monocytes, and eosinophils
  • Center of the film: MORE small lymphocytes
• Coverslip techniques


  • Glass slide-Cover slip Method (Beacom's Method)
  • Two-coverslip Method (Ehrlich's Method)
• Automated methods


  • Miniprep- semi- automated, portable instrument
  • Centrifugal (Spinner Type)- uses 0.2 mL of anticoagulant blood
  • Coulter LH- slide makers and slide stainers
  • Sysmex SP- 10- slide makers and slide stainers
• Purpose of blood film staining

  • For evaluation of cellular morphology
• Important solutions used for blood film staining

  • Fixative: Methanol
  • Stain: Wright or Wright- Giemsa Stain
  • Buffer: 0.05M Sodium Phosphate (pH+ 6.4)
• pH range for blood smear staining

  6.4 - 6.8
• Romanowsky-type stain


  Any stain that contain methylene blue and halogenated fluorescein dye (common is Eosin B or Eosin Y)
• Methylene Blue


  Basic Stain - colors the nucleus and some cytoplasm structure a blue or purple (basophilic stained structure)
• Eosin


  Acidic stain - colors the cytoplasmic structures an orange-red color (acidophilic stained structures)
• Manual staining technique


  1. Flood the slide with Wright stain

  2. Allow stain to remain on slide for 1-3 mins

  3. Buffer is added

  4. 5-10 mins to stain batch of slides
• Automated staining technique


  1. 1 min process

  2. Uses modified Wright or Wright- Giemsa Stain filtered into a Coplin jar or staining dish

  3. Aged distilled water is used as buffer
• Characteristics of a well-stained blood smear

  • Macroscopic: pink to purple
  • Microscopic: RBCs - orange to salmon pink, WBCs nuclei - blue to purple, Neutrophil cytoplasm - pink to tan (w/ violet to lilac granules), Eosinophil granules - Bright orange, red to orange, Basophil's cytoplasm granules - dark blue to black
• Problems encountered in blood smear staining


  • RBCs: gray (or blue) - Usual causes: stain/buffer too basic more than 6.8 (MOST COMMON)
  • WBCs: too dark - Usual causes: Inadequate staining
  • Eosinophil: gray - Usual causes: Heparinized blood was used
  • RBC's: too pale or red - Usual causes: Stain/buffer too acidic less than 6.4 (MOST COMMON)
  • WBCs: barely visible - Usual causes: Under buffer, Over rinse
• Parasites that may appear in the blood smear: Malaria
• Storage of blood smear slides

  • 7 DAYS before proper disposal