PBS & WBC DIFFERENTIAL HEMA 1 LABORATORY

• Specimens received for routine testing in the hematology section of the laboratory have been collected in Tripotassium ethylenediaminetetraacetic acid (EDTA)
• Blood films from EDTA tubes that remain at room temperature for more than 5 hours
• blood cell artifacts: (echinocyte red blood cells [RBCs] spherocytes necrobiotic leukocytes vacuolated neutrophils).
• Vacuolization of monocytes normally occurs almost immediately with EDTA but causes no evaluation problems.
• Main advantages of making films from blood in the EDTA tube ● are that multiple slides can be made if necessary and they do not have to be prepared immediately after the blood is drawn.
• EDTA anticoagulant is used when preparing smears for differential white blood cell counts because it prevents clotting and allows for better visualization of the different types of WBCs.
• After spreading, the slide is allowed to air dry completely before being stained with a dye such as Wright's stain.
• EDTA - generally prevents platelets from clumping on the glass slide, which makes the platelet estimate more accurate during film evaluation.
• Platelet satellitosis ● when anticoagulated with EDTA. The platelets surround or adhere to neutrophils, which potentially causes pseudothrombocytopenia when counting is done by automated methods.
• Low platelet counts and falsely increased WBC counts (pseudoleukocytosis) - can result from EDTA induced platelet clumping.
• Pseudo Leukocytosis - occurs when platelet agglutination are similar in size to WBCs and automated analyzers cannot distinguish the two.
• The platelet clumps are counted as WBCs instead of platelets.
• Another source of blood for films - is from finger and heel punctures.
• Problems with finger and heel sticks can be eliminated with the use of EDTA microcollection tubes, such as Microtainer tubes (Becton, Dickinson).
• Wedge film technique- is probably the easiest to master.
• Manual wedge technique - It is the most convenient and most commonly used method for making peripheral blood films.
• Manual wedge technique - This technique requires at least two 3-inch 3 1-inch (75-mm 3 25-mm) clean glass slides.
• Manual wedge technique used the pusher slide, held securely in the dominant hand at about a 30- to 45-degree angle
• Sysmex SP-10 - is an automated slide-making and staining system
• Drying of Films ● all blood films and bone marrow smears should be dried as quickly as possible to avoid drying artifact
• Blowing breath on a slide causes RBCs to become echinocytic (crenated) , develop water artifact (also called drying artifact).
• Pure Wright stain or a Wright-Giemsa stain (Romanowsky stain) - is used for staining peripheral blood films and bone marrow smears.
• Pure Wright stain or a Wright-Giemsa stain (Romanowsky stain)- These are considered polychrome stains because they contain both eosin and methylene blue
• Giemsa stains- also contain methylene blue azure.
• Methanol in the stain - fixes the cells to the slide.
• Oxidized methylene blue and eosin - form a thiazine-eosinate complex, which stains neutral components.
• 0.05 M sodium phosphate (pH 6.4) or aged distilled water (distilled water placed in a glass bottle for at least 24 hours; pH 6.4 to 6.8)- The buffer that is added to the stain should be
• Free methylene blue - is basic and stains acidic (and basophilic) cellular components, such as ribonucleic acid (RNA)
• Free eosin - is acidic and stains basic (and eosinophilic) components, such as hemoglobin and eosinophilic granules.
• Neutrophils - are so named because they have cytoplasmic granules that have a neutral pH and pick up some staining characteristics from both stains
• Water or drying artifact appearances: ● heavily demarcated central pallor ● refractive (shiny) blotches on the RBCs ● echinocytes (crenation
• Blood films can be made from the blood in the EDTA tube made within 2 to 3 hours of drawing the specimen.
• Artifacts from EDTA tube stored >5 hours
• Three types of Blood smears:

  1. The cover glass smear
  2. The wedge smear
  3. The spun smear
• The WBC-RELATED PARAMETERS of a routine CBC include the following:

  1. Total WBC count (WBCS 3 X 10^9/L)
  2. Relative Counts
  3. Absolute Count
  4. WBC morphology
• Absolute Counts- WBC differential count values expressed as the actual number of each type of cell (E.G., Neutrophils 3 x 10^9/L)
• Relative Counts- WBC differential count values expressed as percentages
• Neutrophilia- neutrophil increases called
• Eosinophil- increases called Eosinophilia
• Basophilia- basophil increases called