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Hematology

Automated

Overview of Automated Cell Counts

  • Definition: Automated cell counts are performed using sophisticated instruments called hematology analyzers, which rapidly and accurately count and characterize blood cells in whole blood and body fluids
  • Advantages over Manual Methods:
    • Increased Speed and Efficiency: Automated analyzers can process a large number of samples quickly and efficiently
    • Improved Accuracy and Precision: Automated methods generally provide more accurate and precise results than manual methods
    • Reduced Labor Costs: Automated analyzers reduce the need for manual cell counts, freeing up laboratory personnel for other tasks
    • Standardization: Automated methods provide more standardized and reproducible results
    • Ability to Measure Additional Parameters: Automated analyzers can measure a wide range of parameters beyond just cell counts (e.g., cell size, hemoglobin concentration, cell differentials)
  • Limitations:
    • Cost: Automated analyzers are expensive to purchase and maintain
    • Complexity: Requires trained personnel to operate and maintain the instruments
    • Interference: Certain substances or conditions can interfere with automated cell counts, leading to inaccurate results
    • Need for Validation: Automated methods for body fluid cell counts must be validated before clinical use

Principles of Automated Cell Counting Technologies

  • Electrical Impedance (Coulter Principle):
    • Principle: Cells are suspended in an electrolyte solution and pass through a small aperture. As each cell passes through the aperture, it displaces a volume of electrolyte, causing a change in electrical impedance
    • Cell Counting: The number of impedance changes is proportional to the number of cells
    • Cell Sizing: The magnitude of the impedance change is proportional to the cell volume
    • Advantages: Simple and reliable method for cell counting and sizing
    • Limitations: Cannot differentiate between different cell types based on size alone; susceptible to interference from non-cellular particles
  • Optical Scatter:
    • Principle: Cells pass through a focused light beam (laser or tungsten-halogen lamp), and the amount and angle of light scattered by each cell are measured
    • Forward Scatter (FS): Measures the amount of light scattered in the forward direction; related to cell size
    • Side Scatter (SS): Measures the amount of light scattered at a 90-degree angle; related to cell granularity and internal complexity
    • Cell Differentiation: Different cell types scatter light in different ways, allowing the analyzer to differentiate between RBCs, WBCs, and platelets
    • Advantages: Can differentiate between different cell types and provide information about cell structure
    • Limitations: Can be affected by interfering substances (e.g., lipemia, protein)
  • Flow Cytometry:
    • Principle: Cells are stained with fluorescent dyes that bind to specific cell surface markers or intracellular components and are then passed through a laser beam. The fluorescence intensity and scatter patterns are measured
    • Cell Identification: Cells are identified and quantified based on their fluorescence and scatter properties
    • Immunophenotyping: Can identify specific cell populations based on their expression of cell surface markers (e.g., CD4+ T cells, CD8+ T cells, B cells)
    • Advantages: High sensitivity and specificity for cell identification; can measure multiple parameters simultaneously
    • Limitations: More complex and expensive than other methods
  • Cytochemical Staining:
    • Principle: The analyzer uses cytochemical stains to differentiate cell types based on their enzymatic activity or other chemical properties
    • Example:
      • Myeloperoxidase (MPO) stain: Used to differentiate myeloblasts (positive for MPO) from lymphoblasts (negative for MPO) in acute leukemia
    • Advantages: Can provide additional information for cell classification
    • Limitations: Requires specific reagents and controls

Automated Hematology Analyzer Parameters

  • Red Blood Cell (RBC) Parameters:
    • RBC Count: Number of red blood cells per unit volume of blood (e.g., x 10^12/L)
    • Hemoglobin (HGB): Concentration of hemoglobin in whole blood (g/dL)
    • Hematocrit (HCT): Percentage of blood volume occupied by red blood cells (%)
    • Mean Corpuscular Volume (MCV): Average volume of individual red blood cells (fL)
      • MCV = (HCT / RBC) x 10
    • Mean Corpuscular Hemoglobin (MCH): Average amount of hemoglobin per red blood cell (pg)
      • MCH = HGB / RBC x 10
    • Mean Corpuscular Hemoglobin Concentration (MCHC): Average concentration of hemoglobin in a given volume of red blood cells (g/dL)
      • MCHC = HGB / HCT x 100
    • Red Cell Distribution Width (RDW): A measure of the variation in red blood cell size (anisocytosis) (%)
  • White Blood Cell (WBC) Parameters:
    • WBC Count: Total number of white blood cells per unit volume of blood (e.g., x 10^9/L)
    • Differential Count: Percentages and absolute numbers of each type of leukocyte:
      • Neutrophils
      • Lymphocytes
      • Monocytes
      • Eosinophils
      • Basophils
    • Immature Granulocytes (IG): Percentage and absolute number of immature granulocytes (e.g., metamyelocytes, myelocytes, promyelocytes) - used to assess for “left shift”
  • Platelet Parameters:
    • Platelet Count: Number of platelets per unit volume of blood (e.g., x 10^9/L)
    • Mean Platelet Volume (MPV): Average volume of individual platelets (fL)
    • Platelet Distribution Width (PDW): Measure of the variation in platelet size
  • Reticulocyte Parameters:
    • Reticulocyte Percentage (%): Percentage of RBCs that are reticulocytes
    • Absolute Reticulocyte Count: Number of reticulocytes per unit volume of blood (e.g., x 10^9/L)
    • Immature Reticulocyte Fraction (IRF): Percentage of reticulocytes that are the least mature
    • Reticulocyte Hemoglobin Content (CHr or Ret-He): Hemoglobin content of reticulocytes

Body Fluid Cell Counts

  • Fluids Analyzed:
    • Cerebrospinal fluid (CSF)
    • Synovial fluid
    • Serous fluids (pleural, peritoneal, pericardial)
    • Other body fluids (e.g., ascites, bronchoalveolar lavage)
  • Parameters Measured:
    • Red Blood Cell (RBC) Count
    • Total Nucleated Cell (TNC) Count: Includes all nucleated cells (e.g., WBCs, malignant cells)
    • Cell Differential: May be performed manually or by automated methods to identify the types of nucleated cells present
  • Automated Methods for Body Fluid Cell Counts:
    • Some hematology analyzers are validated for use with certain body fluids
    • These analyzers typically use:
      • Electrical impedance
      • Optical scatter
      • Flow cytometry
    • Cell Differentials: Some analyzers can perform automated cell differentials on body fluids using flow cytometry or other techniques
  • Manual Methods:
    • Manual Cell Counts: May be necessary if the automated method is not validated for a particular fluid or if the automated results are questionable
    • Cytospin Preparation: Cells are concentrated onto a slide using a cytocentrifuge, allowing for better visualization and differentiation

Quality Control (QC) and Troubleshooting

  • Instrument Calibration:
    • Perform regular calibration according to the manufacturer’s instructions
  • Control Materials:
    • Run control materials at regular intervals (e.g., daily, with each batch of samples)
    • Use controls that are appropriate for the types of samples being analyzed (e.g., whole blood controls, body fluid controls)
    • Record control results and evaluate them using statistical methods (e.g., Levey-Jennings charts, Westgard rules)
  • Maintenance:
    • Perform routine maintenance on the analyzer according to the manufacturer’s recommendations
    • This includes cleaning, replacing reagents, and performing preventative maintenance procedures
  • Troubleshooting:
    • Investigate flags and alarms generated by the instrument
    • Check the sample for clots, hemolysis, or lipemia
    • Repeat the test on a fresh sample
    • Perform manual cell counts to confirm automated results
    • Consult with a supervisor or pathologist if the cause of the discrepancy cannot be determined

Reporting Results

  • Report the cell counts in the appropriate units (e.g., cells/μL, x 10^9/L)
  • Include the reference range for each parameter
  • Note any abnormal results or flags
  • Document all quality control procedures and corrective actions taken
  • For body fluid cell counts, include a description of the fluid (e.g., CSF, synovial fluid) and the source of the fluid

Key Terms

  • Automated Cell Count: Enumeration of cells using automated hematology analyzers
  • Electrical Impedance (Coulter Principle): A method for counting and sizing cells
  • Optical Scatter: A method for characterizing cells based on light scattering properties
  • Flow Cytometry: A technique for identifying and quantifying cells based on their surface markers and other characteristics
  • Calibration: The process of adjusting an instrument to ensure accurate and reliable results
  • Control Materials: Samples with known values that are used to assess the accuracy and precision of an instrument
  • Quality Control: Procedures used to monitor the accuracy and reliability of laboratory testing
  • Body Fluid: A fluid other than blood (e.g., cerebrospinal fluid, synovial fluid, serous fluid)
  • Reticulocyte: Immature red blood cell
  • Flags and Alarms: Indications that instrument is reporting abnormal results