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Laboratory Operations

Hematology Instrumentation

Let’s talk about the absolute workhorse of the hematology lab: the automated hematology analyzer. I want you to think of this incredible machine not just as a piece of equipment, but as a high-speed cellular census bureau. Its job is to take a tiny sample of a patient’s blood and, in less than a minute, conduct a detailed survey of millions of cells, giving us a vital snapshot of the patient’s health. It is a symphony of physics, electronics, and fluid dynamics, but the principles behind its magic are elegant and understandable

The analyzer’s primary mission is to perform the Complete Blood Count (CBC) with a White Blood Cell Differential. This report is the bedrock of hematology, and it’s built upon three core technological pillars that work in perfect harmony

Core Principle 1: Electrical Impedance (The Coulter Principle)

This is the classic, foundational method for counting cells. Imagine a single-lane tollbooth on a highway. The only way to count the cars is to make them go through one at a time. The Coulter Principle does exactly this with cells

  • The Setup: The instrument creates a very precise dilution of the blood sample. This diluted sample is drawn through a tiny hole, or aperture, that connects two chambers. A constant, low-voltage electrical current is passed through this aperture
  • The Action: Cells are very poor conductors of electricity compared to the saline-based diluent they are suspended in. As each individual cell is pulled through the aperture, it momentarily interrupts, or impedes, the flow of the electrical current
  • The Result: The instrument’s electronic detectors register each interruption as a single pulse
    • The number of pulses generated is directly proportional to the cell count. This is how we get our Red Blood Cell (RBC) count and Platelet (PLT) count
    • The size (amplitude) of the pulse is directly proportional to the volume of the cell. A big cell creates a big pulse; a small cell creates a small pulse. This is how the instrument calculates the Mean Corpuscular Volume (MCV) for red cells and the Mean Platelet Volume (MPV)

Core Principle 2: Optical Scatter & Fluorescence (The Flow Cytometry Method)

While impedance is great for counting cells of different sizes, it can’t tell the difference between two cells that are the same size but have different internal structures, like a large lymphocyte and a small neutrophil. To perform the WBC differential, modern analyzers use technologies borrowed directly from flow cytometry

  • The Setup: Using hydrodynamic focusing, the instrument forces the cells to line up in a perfect single-file line as they are injected into a stream of sheath fluid. This string of cells is then sent through the path of a highly focused laser beam
  • The Action: As each cell zips through the laser, it scatters the light in predictable ways. Specialized detectors are positioned to measure this scatter
    • Forward Scatter (FSC): Light scattered at a low angle, almost in a straight line with the laser, is proportional to the cell’s size
    • Side Scatter (SSC): Light bounced off at a 90-degree angle is proportional to the cell’s internal complexity (e.g., the presence of granules, the shape of the nucleus)
  • The Result: By measuring the FSC and SSC for every single cell, the instrument can plot them on a graph. Different cell types will naturally fall into different clusters based on their unique size and complexity signatures. Lymphocytes are small and simple. Monocytes are large and slightly complex. Granulocytes (neutrophils, eosinophils, basophils) are large and highly complex. Some analyzers use additional measurements like fluorescence (by adding special dyes that bind to RNA/DNA) or conductivity to further separate the granulocyte populations, yielding a highly accurate 5-part or 6-part differential

Core Principle 3: Spectrophotometry for Hemoglobin

The analyzer needs a way to measure hemoglobin that is independent of the RBC count. For this, it reverts to the classic principle of spectrophotometry

  • The Action: A separate aliquot of the blood sample is mixed with a lysing reagent. This chemical blows up all the red blood cells, releasing their hemoglobin into the solution. The reagent then converts the hemoglobin into a stable, colored compound (like cyanmethemoglobin in the reference method, though most modern analyzers use a cyanide-free version)
  • The Measurement: The instrument shines a light of a specific wavelength through this colored solution and measures its absorbance. According to Beer’s Law, the amount of light absorbed is directly proportional to the concentration of the hemoglobin. This gives us our final, highly accurate hemoglobin (Hgb) value

Visualizing the Data: Histograms and Scatterplots

A huge part of the analyzer’s job is to present this data visually. Two key graphics are generated with every run:

  • Histograms: These are size distribution graphs for RBCs and platelets. The x-axis represents the cell volume (in femtoliters), and the y-axis represents the number of cells counted at that size. A normal RBC histogram is a symmetrical, bell-shaped curve. A wide, short curve would indicate a high RDW (anisocytosis)
  • Scatterplots (Scattergrams): This is the visual representation of the WBC differential, plotting size vs. complexity. Each dot on the plot represents a single cell, and the distinct clusters allow us, the laboratorians, to visually QC the differential and spot abnormalities immediately

When Automation Needs Human Help: The Manual Differential

No machine is perfect. The analyzer is programmed with sophisticated algorithms to recognize when it sees something it can’t classify or that doesn’t fit a normal pattern. When this happens, it generates a “flag.” A flag is the instrument’s way of raising its hand and saying, “I need a human expert to look at this.” Common flags include “Immature Granulocytes,” “Blasts?,” “Variant Lymphocytes,” or “Platelet Clumps.”

When we see a flag, we revert to the gold standard: we make a peripheral blood smear, stain it, and use our eyes and a microscope to perform a manual differential. The microscope is the ultimate arbiter, and our morphology skills are the final, essential component of the hematology workflow

Key Terms

  • Electrical Impedance (Coulter Principle): A method for counting and sizing cells based on the detection of changes in electrical resistance as cells pass, one by one, through a small aperture
  • Hydrodynamic Focusing: A fluid dynamics technique that uses a sheath fluid to align cells into a single-file stream, essential for accurate analysis in flow-based systems
  • Optical Scatter: The technology used for the WBC differential, where a cell’s size (Forward Scatter) and internal complexity (Side Scatter) are determined by how it scatters light from a laser beam
  • Histogram: A graphical representation of the size distribution of a cell population (e.g., RBCs or platelets), plotting cell volume against the relative number of cells
  • Scatterplot (Scattergram): A two-dimensional dot plot generated by the analyzer that displays the WBC populations based on two or more measured parameters, typically size and complexity
  • Flag: An alert message from a hematology analyzer indicating that a result is outside of set limits or that an abnormality may be present, often requiring a manual peripheral smear review
  • Mean Corpuscular Volume (MCV): The average volume of a red blood cell, measured in femtoliters (fL). It is derived from the amplitude of the pulses generated during impedance counting