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SOP

Sickling Test (Sodium Metabisulfite Method)

SOP for screening for the presence of Hemoglobin S (HbS) by inducing deoxygenation and observing red blood cell sickling under microscopy.

Last verified 1 month ago 2 min read Haematology Manual Haematology Microscopy Hemoglobinopathy #anemia #hemoglobinopathy #Sickling Test #HbS #Sickle Cell #sodium metabisulfite
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Document Control
Version
1.0
Effective
Aug 28, 2026
Reviewed
Aug 29, 2026
Department
Hematology
Principle

Sodium metabisulfite is a reducing agent that removes oxygen from the blood sample. Deoxygenated Hemoglobin S (HbS) is poorly soluble and polymerizes into long, rigid rods. This polymerization distorts the red blood cell membrane, causing the cell to assume a characteristic sickle or crescent shape, which is visible under a microscope.

Standard Operating Procedure: Sickling Test

1. Purpose and Scope

To screen for the presence of Hemoglobin S (HbS) by chemically inducing deoxygenation, which causes HbS-containing red blood cells to assume a sickle shape.

2. Specimen Requirements

  • Type: Whole blood.
  • Anticoagulant: EDTA or capillary blood.
  • Stability: Test within 6 hours of collection.
  • Rejection: Clotted samples.

3. Safety and Precautions

  • Standard PPE. Sodium metabisulfite is an irritant; handle with care.

4. Equipment and Reagents

  • 2% Sodium metabisulfite solution (freshly prepared or commercially stabilized).
  • Clean glass slides, coverslips, vaseline or petroleum jelly.
  • Light microscope with 40x and 100x objectives.

5. Step-by-Step Procedure

  1. Place 1 drop of 2% sodium metabisulfite solution on a clean glass slide.
  2. Add 1 drop of well-mixed patient blood and mix gently with an applicator stick.
  3. Place a coverslip over the mixture and seal the edges with vaseline to create an airtight environment (preventing re-oxygenation).
  4. Allow the slide to stand at room temperature for 15-30 minutes.
  5. Examine under the microscope using the 40x objective, then confirm with 100x oil immersion.
  6. Look for characteristic elongated, crescent, or holly-leaf shaped red blood cells (sickled cells).

6. Quality Control

  • Run a known HbS positive and HbA (normal) negative control with each new batch of reagent.
  • Ensure the sodium metabisulfite solution is fresh (oxidized solution will yield false negatives).

7. Reporting Results

  • Positive: Sickled cells observed. (Note: Does not distinguish between Sickle Cell Trait (HbAS) and Sickle Cell Disease (HbSS)).
  • Negative: No sickled cells observed after 30 minutes.
  • Recommend Hemoglobin Electrophoresis for definitive diagnosis.

Frequently Asked Questions

No. It only indicates the presence of HbS. Hemoglobin electrophoresis or HPLC is required to differentiate between HbAS (trait) and HbSS (disease).
Sealing prevents atmospheric oxygen from re-entering the sample. Re-oxygenation can cause sickled cells to revert to their normal shape, leading to a false-negative result.