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Hematology

Scatter Plots

While histograms provide a one-dimensional view of cell size, the Scatter Plot (also known as a Scattergram, Cytogram, or Dot Plot) provides a two-dimensional or three-dimensional analysis of cell populations. By plotting two different physical or chemical properties against each other on X and Y axes, automated analyzers can separate complex cell populations that overlap in size, specifically enabling the 5-Part Differential (Neutrophils, Lymphocytes, Monocytes, Eosinophils, and Basophils)

Principles of Generation

Scatter plots are generated using Flow Cytometry principles combined with optical light scatter, fluorescence, or conductivity. As cells pass single-file through a flow cell, they are interrogated by a laser beam. The light that bounces off the cell is measured by photodetectors at specific angles

  • Forward Scatter (FSC): Light that continues in the forward direction (0° to 10°). The amount of shadow cast by the cell is directly proportional to the Cell Size. Large cells (Monocytes) generate high forward scatter; small cells (Lymphocytes) generate low forward scatter
  • Side Scatter (SSC): Light that bounces off the cell at a 90° angle. This light is reflected and refracted by internal structures. It measures Internal Complexity (Granularity and Nuclear Lobulation)
    • Neutrophils/Eosinophils: Have granules and multi-lobed nuclei, resulting in High Side Scatter
    • Lymphocytes: Have no granules and a round nucleus, resulting in Low Side Scatter
  • Fluorescence (SFL): Some analyzers (e.g., Sysmex XN series) use a polymethine dye that penetrates the cell membrane and stains nucleic acids (RNA/DNA). The amount of fluorescence emitted correlates to the cell’s Metabolic Activity or maturity. Immature cells (Reticulocytes, Blasts) have high RNA content and fluoresce brightly

Anatomy of the Scatter Plot

Although specific axes differ by manufacturer (e.g., Beckman Coulter plots Volume vs. Scatter; Sysmex plots Fluorescence vs. Side Scatter), the topographical arrangement of normal cells generally follows a predictable logic based on cell morphology

The Lymphocyte Cluster

  • Location: typically found in the Bottom Left quadrant
  • Characteristics: These are small cells (Low Forward Scatter) with round, simple nuclei and no granules (Low Side Scatter). They form a tight, dense cluster. If the cluster is “smeared” upward into the Monocyte region, it may indicate Variant Lymphocytes

The Monocyte Cluster

  • Location: Found in the Middle/Upper Left or distinct “bridge” area
  • Characteristics: These are the largest normal cells (High Forward Scatter) but have moderate complexity due to their vacuoles and folded nuclei (Medium Side Scatter). They often sit “above” the lymphocytes

The Neutrophil Cluster

  • Location: Found in the Middle/Bottom Right quadrant
  • Characteristics: These cells have moderate size but high granularity. Their high side scatter pushes them to the right side of the graph. This is usually the largest, most dense cloud in a normal patient

The Eosinophil Cluster

  • Location: Found in the Far Right quadrant
  • Characteristics: Eosinophils contain dense, refractive crystalline granules. They scatter light more intensely than any other cell type. They usually appear as a smaller, distinct cluster to the right of the neutrophils

The “Ghost” Region

  • Location: The extreme Bottom Left (below Lymphocytes)
  • Characteristics: This area represents “noise.” It contains particles that are not WBCs, such as Lyse-Resistant RBCs, Giant Platelets, or Nucleated RBCs (nRBCs). Modern analyzers use “gating” to exclude this area from the total WBC count, but a heavy population here will trigger an interference flag

Clinical Interpretation of Abnormal Patterns

The true power of the scatter plot lies in its ability to visualize abnormal populations that do not fit into the standard normal clusters. The analyzer uses “Floating Discriminators” (Dynamic Gating) to draw boxes around the clusters. When cells fall outside these boxes, specific flags are generated

The Left Shift (Immature Granulocytes)

  • The “IG” Region: Immature granulocytes (Metamyelocytes, Myelocytes, Promyelocytes) are larger than mature neutrophils and have higher RNA content. On fluorescence-based plots, they appear Above the Neutrophil cluster
  • Interpretation: A widening of the neutrophil cluster extending upward suggests a “Left Shift” due to infection or inflammation

The Blast Region (Stem Cell Flag)

  • Location: Blasts are distinct because they are large (High Forward Scatter) and have high RNA activity (High Fluorescence), but they have very little cytoplasm and no granules (Low Side Scatter)
  • Pattern: They typically appear in the Upper Left quadrant, often bridging the gap between Lymphocytes and Monocytes. This generates a “Blasts?” or “Abnormal Lymph/Blast” flag. Because they lack granules, they do not appear on the right side of the graph

Atypical (Variant) Lymphocytes

  • Pattern: Reactive lymphocytes are larger than normal lymphs and have more cytoplasm (higher RNA). They appear as a “comet tail” or smear extending Upward and Rightward from the normal Lymphocyte cluster. They often merge into the Monocyte area, causing the analyzer to flag “Variant Lymphs?” or potentially miscount them as Monocytes

Manufacturer-Specific Technologies

It is important to understand the nuance of the specific instrument in your laboratory, as the methodology dictates the scatter plot appearance

  • Sysmex (Fluorescence Flow Cytometry)
    • Axes: Y = Side Fluorescence (RNA/DNA), X = Side Scatter (Complexity)
    • Advantage: Excellent separation of nucleated red blood cells (nRBCs) and distinction between reactive lymphs and blasts due to RNA staining
  • Beckman Coulter (VCS Technology)
    • Axes: Y = Volume (Impedance), X = Light Scatter (Granularity) (with Z-axis = Conductivity)
    • Advantage: Uses a specialized reagent to shrink cells; the scatter plot (DF1) shows Volume vs. Scatter. Eosinophils are separated by their high scatter; Lymphs and Monos are separated by volume
  • Abbott (MAPSS)
    • Axes: Utilizes multiple angles of scatter including 90-degree depolarized scatter
    • Advantage: Depolarized side scatter is specific for Eosinophil granules, allowing for a very clean separation of Eosinophils from Neutrophils even without fluorescence

Troubleshooting with Scatter Plots

The laboratory scientist often consults the scatter plot to validate data before releasing it

  • Non-Lysis: If the “Ghost” region in the bottom left is extremely dense and extends into the Lymphocyte box, the sample likely contains Lyse-Resistant RBCs (common in liver disease or hemoglobinopathies). The WBC count will be falsely elevated. Corrective action involves manual dilution or extended lysis time
  • Old Blood: As blood ages (24+ hours), Neutrophils undergo apoptosis and lose their granules (degranulation). On the scatter plot, the Neutrophil cluster shifts to the Left (lower side scatter) and may merge with Monocytes. This leads to a falsely low Neutrophil count and falsely high Monocyte count (Pseudomonocytosis)