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Hematology

Sickle Solubility

Overview of Sickle Solubility Testing

  • Definition: A rapid, qualitative screening test used to detect the presence of Hemoglobin S (HbS) in a blood sample
  • Clinical Significance:
    • Screening for Sickle Cell Disease: Used as a preliminary test to identify individuals who may have sickle cell trait (HbAS) or sickle cell anemia (HbSS)
    • Simple and Inexpensive: Can be performed in resource-limited settings where more sophisticated tests (e.g., hemoglobin electrophoresis, HPLC) are not readily available
  • Limitations:
    • Qualitative Test: Only detects the presence of HbS, does not quantify the amount
    • Cannot Differentiate between HbAS (sickle cell trait) and HbSS (sickle cell anemia)
    • False-Negative Results: Can occur in infants < 6 months old (due to high levels of HbF), in patients with very low hemoglobin levels, or in patients who have received recent blood transfusions
    • False-Positive Results: Can occur in patients with other hemoglobinopathies (e.g., HbC-Harlem) or with hyperglobulinemia
    • Confirmatory Testing Required: Positive results must be confirmed by hemoglobin electrophoresis or HPLC

Principle of the Sickle Solubility Test

  • Solubility of Hemoglobin S:
    • HbS is insoluble in a concentrated phosphate buffer when deoxygenated (oxygen removed)
    • When deoxygenated, HbS molecules polymerize, forming long fibers that cause the red blood cells to sickle
    • The polymerization and sickling process leads to turbidity (cloudiness) of the solution
  • Other Hemoglobins:
    • Normal hemoglobin (HbA) and most other hemoglobin variants remain soluble in the buffer, so the solution remains clear
  • Procedure:
    1. Blood is mixed with a lysing agent to release hemoglobin
    2. A reducing agent is added to deoxygenate the hemoglobin
    3. The hemolysate is added to a concentrated phosphate buffer
    4. The mixture is observed for turbidity
    5. If HbS is present, the solution will become turbid
    6. If HbS is absent, the solution will remain clear

Reagents and Materials

  • Lysing Reagent: Saponin or other reagent to lyse red blood cells and release hemoglobin
  • Reducing Agent: Sodium hydrosulfite (sodium dithionite) to deoxygenate the hemoglobin
  • Phosphate Buffer: Concentrated phosphate buffer (e.g., sodium phosphate) to reduce the solubility of HbS
  • Distilled Water: To prepare reagents and dilute samples
  • Test Tubes or Microcentrifuge Tubes: To mix the reagents and sample
  • Pipettes: To accurately measure reagents and samples
  • Light Source: A light source (e.g., a flashlight or a viewbox) to observe the turbidity of the solution
  • Control Samples: Positive and negative controls with known HbS status

Procedure

  1. Prepare the Reagents: Follow the manufacturer’s instructions to prepare the lysing reagent, reducing agent, and phosphate buffer
  2. Prepare the Sample:
    • Collect blood in an EDTA (purple-top) tube
    • Mix the blood thoroughly
    • Add a specified amount of blood to a test tube or microcentrifuge tube
  3. Lyse the Red Blood Cells:
    • Add the lysing reagent to the blood sample
    • Mix gently and allow the mixture to stand for a few minutes to ensure complete lysis
  4. Deoxygenate the Hemoglobin:
    • Add the reducing agent (sodium hydrosulfite) to the hemolysate
    • Mix gently
  5. Add Phosphate Buffer:
    • Add the concentrated phosphate buffer to the mixture
    • Mix gently
  6. Incubate (Optional):
    • Some protocols recommend incubating the mixture at room temperature for a specific time (e.g., 5-10 minutes)
  7. Observe for Turbidity:
    • Hold the tube in front of a light source and observe the turbidity of the solution
    • Compare the test sample to positive and negative controls
  8. Interpret the Results:
    • Positive Result: The solution is turbid, indicating the presence of HbS
    • Negative Result: The solution remains clear, indicating the absence of HbS

Interpreting Results

  • Positive Result (Turbid Solution): Indicates the presence of HbS
    • Does not differentiate between sickle cell trait (HbAS) and sickle cell anemia (HbSS)
    • Requires confirmation by hemoglobin electrophoresis or HPLC
  • Negative Result (Clear Solution): Indicates the absence of HbS
    • Does not rule out other hemoglobinopathies
    • False-negative results can occur, especially in infants < 6 months old due to high levels of HbF

Factors Affecting Accuracy

  • High Hematocrit:

    • High concentrations of hemoglobin can lead to false-positive results
    • Dilute the sample before performing the test

  • Lipemia:

    • Turbidity from lipemia can interfere with the visual interpretation of the test
    • Centrifuge the sample and use the clear bottom layer for testing

  • High White Blood Cell Count:

    • Can interfere with visual interpretation
    • Lyse the white blood cells before performing the test

  • Recent Transfusion:

    • Can lead to false-negative results if the patient has been recently transfused with blood that does not contain HbS
    • Wait at least 3 months after transfusion before performing the test

  • Hemoglobin Variants Other Than HbS:

    • Some rare hemoglobin variants (e.g., HbC-Harlem) can also cause a positive result
    • Confirm positive results with hemoglobin electrophoresis or HPLC

Quality Control

  • Run Positive and Negative Controls with Each Test Batch:
    • Positive Control: A sample with known HbS
    • Negative Control: A sample with normal hemoglobin (HbA)
  • Use Fresh Reagents:
    • Ensure that the reagents are not expired and have been stored properly
  • Follow the Manufacturer’s Instructions Carefully:
    • Adhere to the recommended sample volumes, reagent concentrations, and incubation times
  • Inspect the Reagents and Equipment:
    • Check for any signs of contamination or deterioration
    • Ensure that the light source is adequate for visualizing the turbidity of the solution

Reporting Results

  • Report the result as “Positive” or “Negative” for HbS
  • Indicate that the test is a screening test and requires confirmation by hemoglobin electrophoresis or HPLC for a definitive diagnosis
  • Include any comments or qualifications, such as “False negative possible in infants < 6 months old” or “Heterozygous result”

Key Terms

  • Sickle Solubility Test: A screening test for sickle cell disease that detects the presence of HbS
  • Hemoglobin S (HbS): The abnormal hemoglobin found in sickle cell disease
  • Deoxygenation: Removal of oxygen
  • Turbidity: Cloudiness or haziness of a solution
  • Sickle Cell Anemia: A severe inherited blood disorder caused by the HbSS genotype
  • Sickle Cell Trait: A heterozygous carrier state for HbS (HbAS)
  • Lysing Agent: A chemical used to rupture red blood cell membranes and release hemoglobin
  • Reducing Agent: A chemical that donates electrons and reduces other substances