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Hematology

Differentials

Overview of Differentials and Morphology Evaluation

  • Definition: The process of identifying, classifying, and quantifying cells in a blood or body fluid sample, as well as evaluating their morphological characteristics (size, shape, color, nuclear and cytoplasmic features)
  • Purpose:
    • Diagnose and monitor a wide range of hematologic and non-hematologic disorders
    • Assess the relative proportions of different cell types
    • Identify abnormal cells (e.g., blasts, atypical lymphocytes)
    • Detect morphological abnormalities in cells
    • Evaluate the response to therapy
  • Methods:
    • Manual Differential: Microscopic examination of a stained peripheral blood smear or body fluid cytospin preparation
    • Automated Differential: Automated hematology analyzers use various technologies to identify and classify cells

Peripheral Blood Smear Preparation and Staining

  • Blood Collection:
    • Collect blood in EDTA (purple-top) tubes for CBC and peripheral blood smear preparation
    • Ensure proper collection technique and avoid clots
  • Smear Preparation:
    • Wedge Smear Technique: The most common method for preparing blood smears
      1. Place a small drop of blood near one end of a clean glass slide
      2. Hold a second slide (spreader slide) at a 30-45 degree angle to the first slide
      3. Bring the spreader slide back to contact the blood drop, allowing the blood to spread along the edge of the spreader slide
      4. Push the spreader slide forward in a smooth, continuous motion, creating a thin film of blood
      5. Allow the smear to air dry completely
    • Factors Affecting Smear Quality:
      • Angle of the spreader slide
      • Speed of spreading
      • Size of the blood drop
      • Blood viscosity
  • Staining:
    • Wright Stain or Wright-Giemsa Stain: The most commonly used stains for blood smears
    • Staining Procedure:
      1. Flood the air-dried smear with Wright or Wright-Giemsa stain for 3-5 minutes
      2. Add an equal volume of buffer (distilled water or a phosphate buffer) to the slide and mix gently for 5-10 minutes
      3. Wash the slide gently with buffer
      4. Allow the slide to air dry completely
    • Proper Staining Technique:
      • Use fresh stain and buffer
      • Use appropriate staining and washing times
      • Avoid overstaining or understaining
      • Check buffer pH

Manual Differential Count

  • Principle:
    • A trained medical laboratory scientist examines the stained peripheral blood smear under a microscope and identifies, classifies, and counts 100 or more leukocytes
    • The results are expressed as percentages of each cell type and can be converted to absolute counts
  • Procedure:
    1. Preparation is Key
      • Use a Well-Prepared and Stained Peripheral Blood Smear: As mentioned earlier, the wedge smear technique is common. You want a thin area of the blood, well stained, with minimal artifacts. This is important as poor technique can lead to poor test results
      • Good Lighting and Microscope Settings: A bright and properly aligned microscope is essential. Make sure you have a good oil immersion lens (100x objective) and that your microscope is routinely serviced
    2. Finding the “Counting Area”
      • Scan at Low Power (10x Objective): Before you get started, you’ll want to get a big picture view. With the 10x, use the coarse and fine focus adjustments until you find the “counting area.” This is the region of the smear:
        • RBCs are evenly distributed
        • RBCs are mostly monolayered (not piled on top of each other)
        • Cells are not distorted
        • It’s usually found between the thickest part (near where you applied the blood) and the thinnest part (the feathered edge)
    3. Systematic Counting Technique
      • Use a Consistent Pattern: The goal is to count every cell in a way that avoids bias or double-counting. Here are a few common approaches:
        • Meandering (Serpentine) Pattern: Start at one edge of the counting area and move across the smear in a zigzag or winding pattern. When you reach the edge, shift down slightly and continue counting in the opposite direction
        • Battlement (Greek Key) Pattern: Count cells along the edges of a defined area (like a square or rectangle) and then move inward, creating a “battlement” effect.
      • Count at 40x or 50x Objective: Once you’ve found a good spot to count, switch to a higher power (100x)
    4. Cell Identification
      • Rely on Cellular Morphology at 100x: Accurately identifying cells is what it is all about!
      • Count at least 100 WBCs: This is the number needed to create a good count in the smear.
    5. Repeat as Needed
      • If the Peripheral Blood Smear shows few WBC, you can count fewer locations. If it is very very little, you can try to make a better slide or ask for another blood sample.
  • Sources of Error:
    • Uneven Cell Distribution: Scan the smear to ensure even cell distribution
    • Cell Clumping or Overlap: Choose an area with well-separated cells
    • Staining Artifacts: Use proper staining techniques and fresh reagents
    • Inaccurate Cell Identification: Proper training and experience are essential
    • High nRBC Count: When nucleated red blood cells (nRBCs) are present, a corrected WBC count must be calculated: Corrected WBC = (Uncorrected WBC x 100) / (100 + Number of nRBCs per 100 WBCs)

Body Fluid Cytospin Preparation and Staining

  • Cytospin Preparation:

    • Used to concentrate cells from a fluid sample onto a slide for microscopic examination
    • Procedure:
      1. Mix the Fluid Sample: Gently mix the body fluid sample to ensure even cell distribution
      2. Prepare the Cytospin:
        • Load a known volume of the fluid sample into a cytospin funnel
        • Attach the funnel to a microscope slide
        • Place the assembly into a cytocentrifuge
      3. Centrifugation:
        • Centrifuge the sample at a specific speed and time to concentrate the cells onto the slide
      4. Air Dry:
        • Carefully remove the slide from the cytocentrifuge and allow it to air dry completely

  • Staining:

    • Wright or Wright-Giemsa Stain: The most commonly used stains
    • Follow the same staining procedure as for peripheral blood smears
    • Ensure that the staining is appropriate for the type of cells being examined

Body Fluid Cell Differential

  • Cerebrospinal Fluid (CSF):

    • Cell Types:
      • Lymphocytes (predominant cell type in normal CSF)
      • Monocytes
      • Neutrophils (rare in normal CSF)
      • Ependymal Cells (cells lining the ventricles of the brain)
    • Abnormal Cells:
      • Blast cells (suggestive of leukemia or lymphoma)
      • Malignant cells (metastatic cancer)
    • Interpreting the Cell Differential:
      • Increased neutrophils: Suggests bacterial meningitis
      • Increased lymphocytes: Suggests viral meningitis or other inflammatory conditions
      • Presence of blasts or malignant cells: Suggests leukemia, lymphoma, or metastatic cancer

  • Serous Fluids (Pleural, Peritoneal, Pericardial):

    • Cell Types:
      • Mesothelial cells (cells lining the serous membranes)
      • Lymphocytes
      • Macrophages
      • Neutrophils (may be present in inflammation or infection)
    • Abnormal Cells:
      • Malignant cells (metastatic cancer)
      • Blast cells (rare)
    • Interpreting the Cell Differential:
      • Increased neutrophils: Suggests bacterial infection or inflammation
      • Increased lymphocytes: Suggests viral infection, tuberculosis, or autoimmune disease
      • Presence of malignant cells: Suggests metastatic cancer

  • Synovial Fluid:

    • Cell Types:
      • Synovial lining cells (cells lining the joint space)
      • Monocytes/macrophages
      • Lymphocytes (few)
      • Neutrophils (should be minimal in a normal sample)
    • Abnormal Cells:
      • Crystals (urate crystals in gout, calcium pyrophosphate dihydrate (CPPD) crystals in pseudogout)
      • Cartilage debris
      • Bacteria
    • Interpreting the Cell Differential:
      • Increased neutrophils: Suggests bacterial arthritis
      • Presence of crystals: Suggests crystal-induced arthritis (e.g., gout, pseudogout)

Cell Identification

  • Develop expertise in identifying different cell types based on their:
    • Size
    • Nuclear Morphology (shape, chromatin pattern, nucleoli)
    • Cytoplasmic Characteristics (color, granules, vacuoles)
    • Use a reliable cell atlas or reference guide
  • Accurate cell identification is essential for performing accurate differential counts

Automated Cell Differentials

  • Automated hematology analyzers can perform cell differentials using various technologies:
    • Electrical Impedance: Cells are classified based on their size
    • Optical Scatter: Cells are classified based on their light scattering properties
    • Flow Cytometry: Cells are stained with fluorescent dyes and classified based on their fluorescence and scatter properties
  • Advantages of Automated Differentials:
    • Increased Speed and Efficiency
    • Improved Precision
    • Reduced Labor Costs
  • Limitations of Automated Differentials:
    • Inability to Identify all Cell Types: Some abnormal or immature cells may not be accurately identified by the analyzer
    • Interference: Certain substances can interfere with the automated cell counts and differentials
  • Manual Review Criteria: Peripheral blood smears should be reviewed manually when the automated differential flags abnormal cells or if the results do not correlate with the patient’s clinical condition

Reporting Results

  • Report the cell counts and percentages of each cell type
  • Include a description of any morphological abnormalities observed
  • Note any flags or comments generated by the instrument
  • For body fluid cell differentials, include a description of the fluid and the source of the fluid
  • Correlate the laboratory findings with the patient’s clinical information

Key Terms

  • Peripheral Blood Smear: A blood sample spread thinly on a slide for microscopic examination
  • Differential Count: Enumeration and classification of different types of leukocytes
  • Morphology: The study of cell structure and form
  • Anisocytosis: Variation in red blood cell size
  • Poikilocytosis: Variation in red blood cell shape
  • Hypochromia: Decreased hemoglobin content in red blood cells (pale color)
  • Polychromasia: Increased blueness of red blood cells due to residual RNA
  • Spherocytes: Spherical-shaped red blood cells
  • Schistocytes: Fragmented red blood cells
  • Target Cells: Red blood cells with a bullseye appearance
  • Auer Rods: Rod-shaped inclusions in myeloblasts
  • Blasts: Immature blood cells seen in acute leukemia
  • Atypical Lymphocytes: Activated lymphocytes with abnormal morphology
  • Megaloblasts: Abnormal, large erythroid precursors
  • Hypersegmented Neutrophils: Neutrophils with 5 or more lobes
  • Cytospin Preparation: A method for concentrating cells from a fluid sample onto a slide
  • Supernatant: The fluid component of a sample after centrifugation
  • Quality Control: Procedures used to monitor the accuracy and precision of laboratory testing