Automated Blood Cell Analysis

AUTOMATED BLOOD CELL ANALYSIS

Principles of Hema-Analizers

  • Electrical Impedance and Optical Light Scatter

    • Two most commonly used principles in hema-analyzers:

    • Electrical Impedance

    • Optical Light Scatter

Basic Components of Hema-Analyzers

  • Hydraulics

    • Components include:

    • Aspirating unit

    • Dispensers

    • Diluters

    • Mixing chambers

    • Aperture baths or flow cells

    • Hemoglobinometer

  • Pneumatics

    • Generates the vacuums and pressures required to operate valves and move samples through the hydraulics system.

  • Electrical System

    • Controls operational sequences of the total system, includes electronic analyzers and computing circuitry for processing data generated.

MUST KNOW IN ELECTRICAL IMPEDANCE

  1. Known as Low Voltage Direct Current (DC) resistance or “Coulter Principle.”

  2. Radio Frequency (RF) or Alternating Current (AC) resistance is sometimes modified to be used with DC electronic impedance.

  3. Common machines using this principle include SYSMEX and Coulter.

  4. In Coulter, results are generated in triplicate.

  5. The number of electrical pulses is directly proportional to the number of cells counted.

  6. The height or amplitude of electrical pulses correlates with the size or volume of the cells.

  7. Data obtained from points 5 and 6 are plotted on a histogram (a graphical representation).

  8. Cell Size/Volume is plotted on the X-AXIS of the histogram.

  9. Cell Number is plotted on the Y-AXIS of the histogram.

HISTOGRAMS

  • Histograms represent cell frequencies versus sizes.

  • In a homogeneous cell population, the curve typically takes the shape of a symmetrical bell or Gaussian distribution.

  • 1:50,000 - RBC dilution factor in automated hema-analyzer.

  • 1:500 - WBC dilution factor in automated hema-analyzer.

Principle of Electrical Impedance

  • Electrical impedance involves detecting and measuring changes in electrical resistance produced by cells as they pass through a small aperture.

  • Cells are suspended in an electrically conductive diluent like saline and are pulled through an aperture in a glass tube.

  • Blood cells are non-conductive but suspended in a conductive diluent.

  • The relationship of electrical pulses and cell counts is directly proportional:

    • Directly proportional to the number of cells counted.

    • The height of electrical pulses is also directly proportional to the cell size or volume.

Oscilloscope Screens

  • Used to display pulses generated during electrical impedance measurement.

Coincident

  • Passage of multiple cells simultaneously through the orifice leads to artificially large pulses, resulting in falsely increased cell volumes and decreased counts.

Hydrodynamic Focusing

  • Technique used to avoid issues associated with rigid apertures in electrical impedance measurements.

Back Wash or Sweep Flow Mechanism

  • This mechanism prevents the recirculation of cells back into the sensing zone, thereby editing out anomalously shaped pulses electronically.

MUST KNOW IN RADIOFREQUENCY

  1. Used in conjunction with DC electronic impedance.

  2. Measures cell interior density or complexity with a high voltage AC resistance.

  3. An increased pulse height of RF is directly proportional to the cell interior density.

Interior Density of a Cell

  • Determined by:

    1. N:C ratio

    2. Nuclear density

    3. Cytoplasmic granulation

MUST KNOW IN OPTICAL SCATTER

  1. Optical scatter systems (flow cytometers) use a focused sample stream directed through a quartz flow cell past a light source (laser or non-laser).

  2. Example non-laser light sources:

    • Tungsten halogen lamp.

  3. Detection of scatter relates to light rays converted into electrical signals by photodetectors at specific angles.

  4. Scatter properties at different angles can be plotted to create two-dimensional cytograms or scatterplots.

Three Independent Processes in Optical Light Scatter

  1. Diffraction - Bending of light around corners at small angles.

  2. Refraction - Bending of light due to speed changes at intermediate angles.

Blocker Bars

  • Prevent non-scattered light from entering the detector and are used to collect scattered light.

Different Angles of Light Scatter for Cellular Analysis

  1. Forward Light Scatter (0°):

    • Diffraction relates to the volume of the cell.

  2. Forward Low-Angle Light Scatter (2-3°):

    • Indicates size or volume.

  3. Forward High Angle (5°-15°):

    • Measures the refractive index of cellular components.

  4. Orthogonal or Side Angle Light Scatter (90°):

    • Produces data based on reflection/refraction of internal components, correlating with internal complexity.

Differential Scatter

  • Combination of forward low-angle and forward high-angle light scatter.

Reticulocyte Count

  • The last manual cell-counting procedure to be automated and a major focus of hematology analyzer advancements.

  • Auramine O: A supravital fluorescent stain used in Sysmex R-3000/3500 reticulocyte analyzer.

Commercial Calibrators

  • Whole-blood calibration method has almost completely been replaced by using assayed reference methods.

High MPV Values

  • Associated with higher-risk cardiovascular disease and assessing patient thrombosis risk.

Key Parameters from Histograms

  • Directly Obtained:

    • WBC count, RBC count, Hemoglobin.

  • Derived from RBC Histogram:

    • MCV and RDW (Mean Corpuscular Volume and Red Cell Distribution Width).

  • Derived from Platelet Histogram:

    • MPV and PDW (Mean Platelet Volume and Platelet Distribution Width.

  • Computed Parameters:

    • Hematocrit, MCH (Mean Corpuscular Hemoglobin), MCHC (Mean Corpuscular Hemoglobin Concentration).

Differential Counts

  • Three-part: Lymphocytes, Monocytes, Granulocytes.

  • Five-part: Neutrophils, Lymphocytes, Monocytes, Eosinophils, Basophils.

  • Six-part: Includes immature granulocytes (e.g., RODAKS).

Current and Resistance

  • Described by Ohm’s Law.

MUST KNOW IN BLOOD CELL HISTOGRAM

  1. Particles with cell size of 36 to 360 fl are counted as RBC.

  2. Particles with cell size of 2 to 20 fl are counted as platelets.

  3. WBC HISTOGRAM:

    • Increased RBC size shifts the curve right.

    • Decreased RBC size shifts the curve left.

    • Bimodal histogram distributions can occur in conditions like cold agglutinin disease.

Positive Instrumental Errors

  • Include bubbles, extraneous electrical pulses, and aperture plugs (BEA).

Negative Instrumental Errors

  • Caused by excessive lysing of RBCs.

RDW (Red Cell Distribution Width)

  1. Normal RDW indicates homogeneous erythrocytes and minimal anisocytosis.

  2. Increased RDW indicates heterogeneous erythrocytes and significant anisocytosis.

MUST KNOW IN MPV (MEAN PLATELET VOLUME)

  1. MPV measures average platelet volume.

  2. In EDTA-anti-coagulated blood, platelets swell, increasing MPV by 20% within the first hour.

  3. MPV stabilizes for at least 12 hours but should be based on specimens 1 to 4 hours old.

  4. There is an inverse relationship between MPV and platelet count.

Disorders Associated with Increased MPV

  • Idiopathic thrombocytopenic purpura, post-splenectomy, sickle cell anemia, disseminated intravascular coagulation (DIC), myeloproliferative disorders, heterozygous thalassemia.

Disorders Associated with Decreased MPV

  • Aplastic anemia, megaloblastic anemia, Wiskott-Aldrich syndrome, post-chemotherapy.

PLATELET DISTRIBUTION WIDTH (PDW)

  • PDW measures the uniformity of platelet size.

  • Normal PDW is less than 20%; increased in aplastic and megaloblastic anemias and chronic myelogenous leukemia.

FLOW CYTOMETRY

Overview

  • Designed to measure physical properties of cells based on their capability to deflect light.

  • Provides rapid, simultaneous analysis of multiple parameters across many cells.

Specimen Types

  • Utilized mostly for bone marrow, peripheral blood, and lymphoid tissue for diagnosing hematological malignancies.

  • Peripheral blood and bone marrow specimens should be processed within 1 to 2 days of collection.

  • Heparin is the preferred anticoagulant in BM and peripheral specimens.

Specimen Transportation

  • Must be transported at room temperature.

Stains and Dyes in Flow Cytometry

  • Propidium Iodide or 7-Aminoactinomycin A are fluorescent dyes used to test specimen viability.

Multicolor Flow Cytometry

  • Enables the simultaneous analysis of multiple markers for diagnosis and monitoring of hematologic disorders.

Principle

  • Particles are stained and suspended in fluid, passing one-by-one in front of a light source (e.g., laser).

  • As illuminated, they emit fluorescent signals registered by detectors, yielding digital outputs analyzed by flow cytometry software.

  • Forward scatter (FS or FSC) measures particle size, side scatter (SS or SSC) reflects surface complexity and internal structures.

Parts of Flow Cytometer

  • Components include:

    • Fluidics

    • Light source (typically laser)

    • Detection system

    • Computer to control data collection, analysis, and sorting.

Common Light Sources and Dyes

  • Laser: Examples include Argon laser.

  • Fluorescent Dyes: Acridine orange, thioflavin T, pyronin Y, phycoerythrin (PE), fluorescein isothiocyanate (FITC); often used for dual color analysis.

Fluorochrome Mechanism

  • Absorbs light at one wavelength; emits at a longer wavelength.

  • Linked to monoclonal antibodies to determine binding to cell surface.

Clusters and Gates

  • Cell populations share similar physical properties form clusters on data displays produced by flow cytometers.

  • Gates define electronic boundaries to delineate cell clusters.

Applications of Flow Cytometry

  • Used for diagnosing and monitoring immunodeficiencies, diagnosing PNH, monitoring sepsis, stem cell enumeration and transplantation, tissue typing, and classifying hematologic neoplasia via immunophenotyping.

Flow Cytometry in Detecting Malarial Parasites

  • Erythrocytes stained with acridine orange allow for distinguishing between infected and uninfected cells.

  • Uninfected mature erythrocytes do not fluoresce, while infected ones do due to DNA presence.

Quantitative and Qualitative Flow Cytometers

  • Flow cytometers may range between quantitative clinical versions and qualitative systems derived from research applications.