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Hematology

Histograms

The histogram is the graphical fingerprint of the cellular population. While the numerical values (RBC count, MCV, PLT count) provide quantitative data, the histogram provides qualitative data regarding the uniformity (anisocytosis) and sub-populations of the cells. Competency in interpreting these graphs allows the laboratory scientist to detect interferences, identify dual populations (e.g., transfusions), and validate abnormal flags before a manual smear review is performed

Principles of Histogram Construction

Histograms are frequency distribution curves created primarily by Impedance technology

  • X-Axis: Represents the Size of the cell (Volume in femtoliters, fL)
  • Y-Axis: Represents the Relative Number (Frequency) of cells at that specific size
  • Thresholds (Discriminators): The analyzer uses electronic or digital “gates” to determine where one cell population ends and another begins. These gates are flexible (floating) in modern analyzers to account for pathological changes

The Red Blood Cell (RBC) Histogram

The RBC histogram describes the size distribution of the erythrocytes

  • Normal Range: The curve should be a symmetrical “Gaussian” (Bell) curve peaking between 80 and 100 fL (the normal MCV)
  • Discriminators
    • Lower Discriminator (LD): Set around 25–36 fL. Separates RBCs from Platelets (which are smaller)
    • Upper Discriminator (UD): Set around 200–250 fL. Defines the upper limit of counting
  • The RDW (Red Cell Distribution Width): This parameter is mathematically derived from the width of the histogram at the 20% height level. A wide curve indicates high anisocytosis (high RDW)

Abnormal RBC Histogram Patterns

  1. Shift to the Left (Microcytosis)
    • The entire curve shifts toward the Y-axis (lower fL)
    • Causes: Iron Deficiency Anemia, Thalassemia
  2. Shift to the Right (Macrocytosis)
    • The entire curve shifts away from the Y-axis (higher fL)
    • Causes: Megaloblastic Anemia (B12/Folate deficiency), Liver Disease, Reticulocytosis
  3. Bimodal Distribution (Two Peaks)
    • The curve looks like a camel’s back (two humps). This indicates Dimorphic: RBC population
    • Causes: Recent blood transfusion (donor cells + patient cells), treated Iron Deficiency (new healthy cells + old microcytic cells), or Sideroblastic Anemia
  4. Tail on the Right
    • The curve does not return to the baseline at the Upper Discriminator
    • Causes: Cold Agglutinins (clumps of RBCs read as large single cells), Rouleaux
  5. Elevation at the Left (Lower Discriminator)
    • The curve starts high on the Y-axis instead of starting at zero
    • Causes: Giant Platelets: (large platelets overlapping into RBC zone), Microcytes, or RBC Fragments (Schistocytes)

The Platelet (PLT) Histogram

The platelet histogram is generated from the same aperture bath as RBCs but focuses on the smaller size range

  • Normal Range: A right-skewed curve peaking between 2 and 20 fL
  • Fitted Curve: Because large platelets often overlap with small RBCs or debris, the analyzer uses a “log-normal” mathematical fit to smooth the curve and extrapolate the count. It does not simply count pulses; it models the population

Abnormal Platelet Histogram Patterns

  1. Interference at the Lower End (< 2 fL)
    • High “take-off” at the far left
    • Causes: Electronic noise, dust, or cell debris (e.g., from lysed RBCs or cytoplasmic fragments)
  2. Interference at the Upper End (> 20 fL)
    • The curve does not return to baseline before the RBC region begins
    • Causes: Giant Platelets: (Macrothrombocytes), Microcytic RBCs (severe iron deficiency), or RBC Fragmentation (Schistocytes). This overlap creates a “Platelet Clumps” or “RBC Fragments” flag

The WBC Histogram (3-Part Differential)

In analyzers using impedance for WBC differentials (after lysis), the cells are sorted by size. The lytic agent strips the cytoplasm, causing membrane shrinkage around the nucleus and granules

  • X-Axis: Size (fL)
  • Valleys (Troughs): The analyzer expects valleys to separate the populations. If the line does not drop to the baseline between peaks, it triggers an interference flag

The Three Zones

  1. Lymphocyte Peak (35 – 90 fL): The first peak on the left. Represents small, mature lymphocytes
  2. Mononuclear Region (90 – 160 fL): The “valley” or middle plateau. Represents Monocytes, Eosinophils, and Basophils. (Sometimes called the “Mondo” or “Mid” region)
  3. Granulocyte Peak (160 – 450 fL): The large peak on the right. Represents Neutrophils

Abnormal WBC Histogram Patterns

  1. R1 Flag (Far Left of Lymphocyte Peak)
    • An elevation before the lymphocyte peak (at the < 35 fL mark)
    • Meaning: The analyzer is detecting particles that are smaller than WBCs but larger than background noise
    • Causes: Nucleated RBCs (nRBCs): (most common), Giant Platelets, Clumped Platelets, or Lyse-Resistant RBCs (e.g., in Sickle Cell or HbC disease)
  2. R2 Flag (Between Lymphs and Monos)
    • Bridge/Elevation between the first and second populations
    • Causes: Variant (Atypical) Lymphocytes, Plasma Cells, or Blasts (which are often larger than normal lymphs)
  3. R3 Flag (Between Monos and Granulocytes)
    • Bridge/Elevation between the middle and right populations
    • Causes: Eosinophilia, Basophilia, or Immature Granulocytes (Metamyelocytes/Myelocytes)
  4. R4 Flag (Far Right of Granulocyte Peak)
    • Extension of the curve past the upper limit
    • Causes: High Absolute Granulocyte count (Neutrophilia) or interference

Scatterplots (Scattergrams)

In 5-part differential analyzers (Flow Cytometry), histograms are replaced or supplemented by Scatterplots

  • X-Axis: Usually Side Scatter (Complexity/Granularity)
  • Y-Axis: Usually Forward Scatter (Size) or Fluorescence (RNA/DNA)
  • Interpretation
    • Neutrophils: High Scatter (Granular) + Medium Size
    • Eosinophils: Very High Scatter (Dense granules) + Medium Size
    • Lymphocytes: Low Scatter (Agranular) + Small Size
    • Monocytes: Medium Scatter (Vacuoles) + Large Size
    • “Ghost” Region: An area of very low scatter/size usually represents unlysed RBCs or nRBCs (interference)