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Hematology

Hemoglobin

Overview of Hemoglobin (Hb) Analysis

  • Definition: Hemoglobin analysis involves a range of laboratory tests used to measure and characterize hemoglobin, the oxygen-carrying protein found in red blood cells (RBCs)
  • Clinical Significance: Used to:
    • Diagnose anemias
    • Detect hemoglobinopathies (inherited disorders of hemoglobin structure or synthesis)
    • Monitor treatment of hemoglobin disorders
  • Types of Hemoglobin Analysis:
    • Quantitative: Measures the total amount of hemoglobin in the blood
    • Qualitative: Identifies and characterizes different types of hemoglobin
  • Methods:
    • Quantitative: Automated hematology analyzers
    • Qualitative: Hemoglobin electrophoresis, high-performance liquid chromatography (HPLC), and specialized tests (e.g., sickle solubility test)

Quantitative Hemoglobin Measurement

  • Principle:
    • Automated hematology analyzers measure hemoglobin concentration using spectrophotometry
    • RBCs are lysed (broken open) to release hemoglobin
    • Hemoglobin is converted to a stable colored compound (e.g., cyanmethemoglobin)
    • The analyzer measures the absorbance (optical density) of the solution at a specific wavelength
    • The absorbance is directly proportional to the hemoglobin concentration (Beer-Lambert Law)

Qualitative Hemoglobin Analysis

Qualitative methods are used to identify and characterize different types of hemoglobin

  • Hemoglobin Electrophoresis:
    • Principle: Separates hemoglobin variants based on their electrical charge and migration patterns in an electric field
  • High-Performance Liquid Chromatography (HPLC):
    • Principle: Separates hemoglobin variants based on their interaction with a stationary phase in a column, followed by detection using spectrophotometry
  • Sickle Solubility Test:
    • Principle: Hemoglobin S (HbS) is insoluble in a concentrated phosphate buffer when deoxygenated, causing the solution to become turbid.

Interpreting Results and Correlating with Clinical Findings

  • Consider the Patient’s Age, Ethnicity, and Clinical History: These factors can provide valuable clues for interpreting hemoglobin analysis results
  • Correlate with RBC Indices and Peripheral Blood Smear Findings: RBC indices (MCV, MCH, MCHC) and peripheral blood smear morphology can help narrow down the differential diagnosis
  • Consider all Test Results: Look at all test results together to determine the potential cause of the disease

Key Terms

  • Hemoglobin (Hb): The oxygen-carrying protein in red blood cells
  • Hemoglobinopathy: A genetic disorder affecting the structure or synthesis of hemoglobin
  • Hemoglobin Variant: An abnormal hemoglobin molecule
  • Hemoglobin Electrophoresis: A technique to separate and identify hemoglobin types
  • High Performance Liquid Chromatography (HPLC): A chromatographic method for hemoglobin separation
  • Sickle Solubility Test: A screening test for sickle cell disease
  • Hemolysate: Lysed red blood cells used for hemoglobin analysis
  • Densitometry: A technique for quantifying the amount of each hemoglobin type on an electrophoretic gel
  • Point Mutation: Change that occurs during replication where a single base is either inserted, deleted, or replaced
  • Turbidity: Cloudiness or haziness of a solution