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Hematology

Quantitative

Overview of Quantitative Hemoglobin Measurement

  • Definition: Quantitative hemoglobin (HGB) measurement refers to laboratory techniques used to determine the concentration of hemoglobin in a blood sample
  • Clinical Significance:
    • Diagnosis of Anemia: Decreased HGB levels indicate anemia
    • Diagnosis of Polycythemia: Increased HGB levels indicate polycythemia
    • Monitoring Treatment: Used to monitor the response to treatment for anemia or polycythemia
    • Assessing Blood Loss: Helps estimate the severity of blood loss
    • Evaluating Overall Health: Provides information about the oxygen-carrying capacity of the blood
  • Method: Spectrophotometry is the primary method used for quantitative hemoglobin measurement in automated hematology analyzers
  • Reporting Units: Hemoglobin concentration is typically reported in grams per deciliter (g/dL) or grams per liter (g/L)

Principle of Spectrophotometric Hemoglobin Measurement

Spectrophotometry relies on the Beer-Lambert Law, which states that the absorbance of a solution is directly proportional to the concentration of the analyte and the path length of the light beam through the solution:

  • A = εbc
    • A = Absorbance
    • ε = Molar absorptivity (a constant specific to the substance)
    • b = Path length (the distance the light beam travels through the solution)
    • c = Concentration

In the context of hemoglobin measurement:

  • The intensity of the color produced by the converted hemoglobin is directly proportional to the hemoglobin concentration
  • The spectrophotometer measures the absorbance of the solution, and the hemoglobin concentration is calculated based on the Beer-Lambert Law

Detailed Steps of Spectrophotometric Hemoglobin Measurement

  1. Sample Preparation:
    • Whole Blood Collection: Blood is collected in an EDTA (purple-top) tube to prevent clotting
    • Lysis of Red Blood Cells (RBCs):
      • A lysing reagent is added to the whole blood sample to rupture the RBC membranes and release hemoglobin into the solution
      • The lysing reagent also clears the solution, reducing turbidity
      • Different lysing reagents may be used, depending on the specific method used by the analyzer
    • Conversion of Hemoglobin to a Stable Form:
      • The released hemoglobin is converted to a stable, colored compound that can be accurately measured by spectrophotometry
      • The most common method is the cyanmethemoglobin (hemiglobincyanide) method
  2. Cyanmethemoglobin (HiCN) Method:
    • Potassium Ferricyanide:
      • Converts hemoglobin to methemoglobin (Hi), in which the iron is in the ferric (Fe3+) state
    • Potassium Cyanide:
      • Reacts with methemoglobin to form cyanmethemoglobin (HiCN), a stable, colored compound
      • The reaction is as follows: Hb + K3Fe(CN)6 → Hi + KCN → HiCN
    • Absorbance Measurement:
      • The solution is passed through a spectrophotometer, and the absorbance is measured at a specific wavelength (typically 540 nm)
      • A blank (containing the lysing reagent but no blood) is used to zero the spectrophotometer
      • The absorbance is directly proportional to the cyanmethemoglobin concentration, which in turn is directly proportional to the hemoglobin concentration in the original sample
    • Calculation:
      • The hemoglobin concentration is calculated using a calibration curve or a factor derived from the Beer-Lambert Law
  3. Automated Hematology Analyzer Process:
    • Sample Aspiration: The automated analyzer aspirates a small amount of the prepared blood sample
    • Mixing and Incubation: The analyzer mixes the sample with the lysing reagent and allows sufficient time for the reaction to occur
    • Flow Cell: The solution is passed through a flow cell, where the light beam from the spectrophotometer passes through the solution
    • Detection: A photodetector measures the amount of light that passes through the solution
    • Calculation: The analyzer calculates the hemoglobin concentration based on the absorbance and calibration data
    • Reporting: The hemoglobin concentration is displayed and reported on the analyzer’s printout or screen

Interfering Substances and Troubleshooting

Several substances can interfere with spectrophotometric hemoglobin measurement, leading to inaccurate results. It’s crucial to be aware of these interferences and take appropriate corrective actions.

  • Turbidity: Lipemia (excessive lipids in the blood), high white blood cell counts (leukocytosis), or the presence of non-lysed red blood cells can cause turbidity, increasing the absorbance and falsely elevating the hemoglobin result
    • Corrective Actions:
      • Lipemia: Perform a saline replacement procedure or use a lipemia clearing agent
      • High WBC Count: Dilute the sample and multiply the results by the dilution factor
      • Non-Lysed RBCs: Ensure adequate mixing and incubation time with the lysing reagent
  • High White Blood Cell Count (Leukocytosis):
    • Extreme leukocytosis can cause falsely elevated hemoglobin readings.
    • This is due to light scattering caused by the high concentration of cells.
    • Corrective Action:
      • Dilute the sample with an isotonic diluent (e.g., saline) and repeat the measurement.
      • Multiply the result by the dilution factor.
      • A manual hemoglobin method can also be used to confirm the accuracy.
  • Lipemia (High Lipid Levels):
    • Turbidity from high lipid content can falsely elevate the hemoglobin reading.
    • Corrective Actions:
      • Saline Replacement: Replace the lipemic plasma with an equal volume of isotonic saline after centrifugation.
      • Lipemia Clearing Agents: Use commercial reagents to clear the lipemia before analysis.
      • Ultracentrifugation: Remove the lipids by ultracentrifugation.
  • Cold Agglutinins:
    • These antibodies can cause RBCs to clump together, leading to inaccurate cell counts and hemoglobin measurement
      • Corrective Actions:
        • Warm the sample to 37°C to dissociate the agglutinins
        • Repeat the analysis promptly after warming

Quality Control

  • Calibration:

    • Perform regular calibration of the spectrophotometer according to the manufacturer’s instructions
    • Use certified reference materials (calibrators) with known hemoglobin concentrations

  • Control Materials:

    • Run control materials (low, normal, and high levels) at regular intervals (e.g., daily, with each batch of samples)
    • Use controls that are appropriate for the types of samples being analyzed

  • Review Control Results:

    • Review control results and evaluate them using statistical methods (e.g., Levey-Jennings charts, Westgard rules)
    • Take corrective action if control results are outside the acceptable range

Reporting Results

  • Report the hemoglobin concentration in the appropriate units (g/dL or g/L)
  • Include the reference range for the patient’s age and sex
  • Note any abnormal results or flags
  • Document all quality control procedures and corrective actions taken

Key Terms

  • Hemoglobin (Hb): The oxygen-carrying protein in red blood cells
  • Spectrophotometry: A technique for measuring the absorbance or transmittance of light through a solution
  • Beer-Lambert Law: A law that states that the absorbance of a solution is directly proportional to the concentration of the analyte and the path length of the light beam through the solution
  • Lysing Reagent: A chemical used to rupture red blood cell membranes and release hemoglobin
  • Cyanmethemoglobin (HiCN): A stable, colored compound formed by the reaction of hemoglobin with potassium ferricyanide and potassium cyanide
  • Absorbance: A measure of the amount of light absorbed by a solution
  • Path Length: The distance the light beam travels through the solution
  • 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