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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 & 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