PBS

• A well-made, well-stained, and carefully examined peripheral blood film can provide valuable information regarding a patient's health
• More can be learned from this test than from many other routinely performed hematologic tests
• White blood cell (WBC) and platelet count estimates can be achieved, relative proportions of the different types of WBCs can be obtained, and the morphology of all three cell lines can be evaluated for abnormalities
• Skilled and talented laboratory professionals are essential to the reporting of reliable test results
• Accurate peripheral film evaluation is quite likely to be needed for some time
• Peripheral film evaluation


  The capstone of a panel of tests called the complete blood count (CBC) or hemogram
• CBC


  Includes enumeration of cellular elements, quantitation of hemoglobin, and statistical analyses that provide a snapshot of cell appearances
• Numerical values should be consistent with the assessment derived by examining the cells microscopically
• Careful examination of data in a systematic way ensures that all relevant results are noted and taken into consideration in the diagnosis
• Specimen collection


  Essentially all specimens received for routine testing in the hematology section of the laboratory have been collected in lavender (purple)-topped tubes containing disodium or tripotassium ethylenediaminetetraacetic acid (EDTA)
• EDTA


  Anticoagulates the blood by chelating the calcium that is essential for coagulation
• High-quality blood films can be made from the blood in the EDTA tube, provided that they are made within 4 hours of drawing the specimen
• Blood films from EDTA tubes that remain at room temperature for more than 5 hours often have unacceptable blood cell artifacts
• Advantages of making films from blood in the EDTA tube

  Multiple slides can be made if necessary, they do not have to be prepared immediately after the blood is drawn, EDTA generally prevents platelets from clumping on the glass slide
• Some patients' blood undergoes an in vitro phenomenon called platelet satellitosis when anticoagulated with EDTA
• Platelet-specific autoantibodies that react best at room temperature are one of the mechanisms known to cause platelet satellitosis
• Platelet satellitosis


  Platelets surround or adhere to neutrophils, which potentially causes pseudothrombocytopenia when counting is done by automated methods
• Spuriously low platelet counts and falsely increased WBC counts (pseudoleukocytosis) can result from EDTA-induced platelet clumping
• Pseudoleukocytosis


  Occurs when platelet agglutinates are similar in size to WBCs and automated analyzers cannot distinguish the two, so the platelet clumps are counted as WBCs instead of platelets
• Problems such as platelet satellitosis and pseudoleukocytosis can be eliminated by recollecting specimens in sodium citrate tubes
• Sodium citrate tubes


  Light blue top, proper ratio is nine parts blood to one part anticoagulant
• WBC and platelet counts from sodium citrate specimens must be corrected for the dilution of blood with the anticoagulant
• Dilution factor

  The reciprocal of the dilution (i.e., 10/9 or 1.1), WBC and platelet counts are multiplied by 1.1 to obtain accurate counts
• Another source of blood for films is from finger and heel punctures, but some platelet clumping must be expected if films are made directly from a drop of finger-stick or heel-stick blood
• Wedge film technique


  Probably the easiest to master, the most convenient and most commonly used method for making peripheral blood films
• Wedge film preparation


  Place a drop of blood on one slide, use another slide as a spreader to create a wedge-shaped film
• Features of a well-made wedge peripheral blood film

  • Two-thirds to three-fourths the length of the slide, finger shaped, smooth without irregularities, whole drop of blood is picked up and spread
• Automated slide making and staining


  The Sysmex SP-10 is an automated slide-making and staining system
• Regardless of film preparation method, before staining, all blood films and bone marrow smears should be dried as quickly as possible to avoid drying artifact
• Staining of peripheral blood films

  Pure Wright stain or a Wright-Giemsa stain (Romanowsky stain) is used, these are polychrome stains because they contain both eosin and methylene blue
• Methanol in the stain fixes the cells to the slide
• Drying of Films


  1. Dry blood films and bone marrow smears as quickly as possible to avoid drying artifact

  2. Blowing breath on a slide causes RBCs to become echinocytic or develop water artifact
• Wright stain


  Pure Wright stain or a Wright-Giemsa stain (Romanowsky stain) used for staining peripheral blood films and bone marrow smears
• Purpose of staining blood films

  To make the cells more visible and allow their morphology to be evaluated
• Staining of cells or cellular components

  1. Methanol in the stain fixes the cells to the slide

  2. Oxidized methylene blue and eosin form a thiazine-eosinate complex, which stains neutral components

  3. Free methylene blue stains acidic (and basophilic) cellular components

  4. Free eosin stains basic (and eosinophilic) components
• Slides must be completely dry before staining or the thick part of the blood film may come off the slide in the staining process
• Water or drying artifact

  Can give a moth-eaten look to the RBCs, appear as a heavily demarcated central pallor, or appear as refractive (shiny) blotches on the RBCs
• Factors contributing to water or drying artifact

  • Humidity in the air as the slide dries
  • Very high ratio of plasma to RBCs in extremely anemic patients
  • Water absorbed from the air into the alcohol-based stain
• Manual Wright staining technique

  1. Slides placed on a staining rack with film side facing upward

  2. Wright stain filtered or poured directly from the bottle through a filter onto the slide

  3. Stain left on the slide for 1-3 minutes to fix the cells

  4. Approximately equal amount of buffer added to the slide

  5. Mixture allowed to remain on the slide for 3 minutes or more

  6. Slide rinsed with a steady but gentle stream of neutral-pH water, back of slide wiped, and slide air-dried in a vertical position
• Manual Wright staining technique

  • Desirable for staining peripheral blood films containing very high WBC counts
  • Time can be easily lengthened to enhance the staining required by the increased numbers of cells