Cyanmethemoglobin Method for Estimation of Hemoglobin
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Cell Biology

Cyanmethemoglobin Method for Estimation of Hemoglobin

Learn the cyanmethemoglobin method for accurate hemoglobin estimation with a step-by-step procedure, essential equipment, and reagents. Improve lab results with this reliable technique.

By Dayyal Dg.
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Blood Hemoglobin Measurement by Cyanmethemoglobin Method
Cyanmethemoglobin method for blood hemoglobin measurement.

Hemoglobin (Hb) is a protein in red blood cells (RBCs) responsible for carrying oxygen and carbon dioxide between the lungs and tissues. It also helps maintain blood pH. Hemoglobin is made up of four heme molecules, each attached to a globin chain. In adults, three types of hemoglobin are present:

  • HbA (96-98%): Composed of two alpha (α) and two beta (β) globin chains.
  • HbA2 (1.5-3.5%): Composed of two alpha (α) and two delta (δ) globin chains.
  • HbF (<1%): Composed of two alpha (α) and two gamma (γ) globin chains.

Hemoglobin can bind with other substances, both normally and abnormally, forming different compounds:

  • Oxyhemoglobin: Hemoglobin bound to oxygen.
  • Carboxyhemoglobin: Hemoglobin bound to carbon monoxide (CO).
  • Carbaminohemoglobin: Hemoglobin bound to carbon dioxide (CO2).
  • Methemoglobin: Hemoglobin with oxidized iron (from ferrous to ferric state).
  • Sulfhemoglobin: Hemoglobin bound to sulfur.
  • Cyanmethemoglobin: Hemoglobin bound to cyanide ions.

What is cyanmethemoglobin method?

The cyanmethemoglobin method is a widely used laboratory technique for measuring hemoglobin concentration. In this method, hemoglobin is converted into cyanmethemoglobin by reacting with potassium cyanide and potassium ferricyanide in Drabkin’s solution, which lyses red blood cells. The absorbance of the resulting cyanmethemoglobin solution is then measured at 540 nm using a spectrophotometer. This method is known for its precision, stability, and compatibility with most hemoglobin variants, making it a reliable choice for accurate hemoglobin quantification. It is recommended by the International Committee for Standardization in Hematology for its standardized results and high reproducibility.

History of Cyanmethemoglobin Method for Hemoglobin Estimation

The cyanmethemoglobin method for hemoglobin estimation was developed by Dr. Leonard Drabkin in the mid-20th century. Drabkin, an American hematologist and biochemist, introduced this method as a more accurate and reliable alternative to existing hemoglobin measurement techniques. His work with potassium cyanide and potassium ferricyanide solutions, which lysed red blood cells to create a stable cyanmethemoglobin complex, became foundational in hematology.

This method was later adopted by the International Committee for Standardization in Hematology (ICSH) as the preferred standard due to its precision and effectiveness in transforming all major forms of hemoglobin (except sulfhemoglobin) into a single, measurable compound. Drabkin’s contribution revolutionized clinical diagnostics by providing a dependable technique for hemoglobin estimation used worldwide today.

Advantages of Cyanmethemoglobin Method for Hemoglobin Estimation

The cyanmethemoglobin method is the preferred technique for hemoglobin estimation due to its significant advantages, including accuracy, standardization, and cost-effectiveness:

  1. Accuracy and Precision: Converts most hemoglobin types into a single stable compound for consistent results.
  2. Broad Detection: Measures all hemoglobin forms except sulfhemoglobin.
  3. Standardization: Globally recognized as the reference method by ICSH.
  4. Ease of Use: Compatible with automated spectrophotometric analyzers.
  5. Stability: Cyanmethemoglobin remains stable for hours, reducing processing errors.
  6. Cost-Effectiveness: Uses affordable reagents like Drabkin’s solution, ideal for routine testing

Principle of Cyanmethemoglobin Method

The principle of the cyanmethemoglobin method involves converting all hemoglobin types in a blood sample into cyanmethemoglobin, a stable compound, using potassium cyanide and potassium ferricyanide in Drabkin’s solution. The absorbance of cyanmethemoglobin is measured at 540 nm using a spectrophotometer, and the hemoglobin concentration is determined by comparing this absorbance to a standardized solution. This method ensures precise, consistent, and reliable hemoglobin estimation.

Step 1: Conversion of Hemoglobin to Methemoglobin

Equation: Hb + K₃Fe(CN)₆ → MetHb + K₄Fe(CN)₆

In this reaction, potassium ferricyanide (K₃Fe(CN)₆) oxidizes hemoglobin (Hb) to methemoglobin (MetHb).

Step 2: Formation of Cyanmethemoglobin (Hemiglobincyanide)

Equation: MetHb + KCN → Cyanmethemoglobin (Hemiglobincyanide)

Here, methemoglobin (MetHb) reacts with potassium cyanide (KCN) to form cyanmethemoglobin, the stable colored compound measured in the assay.

Required Equipment

  1. Spectrophotometer or photoelectric colorimeter.
  2. Pipette 5 ml.
  3. Sahli’s pipette.

Reagents

  1. Drabkin’s Solution (pH 7.0-7.4):
    • Potassium ferricyanide 200 mg
    • Potassium cyanide 50 mg
    • Potassium dihydrogen phosphate140 mg
    • Non-ionic detergent 1 ml
    • Distilled water 1000 ml
  2. Cyanmethemoglobin standard solution with a verified hemoglobin value.

Specimen

Blood samples may be obtained via skin puncture or from EDTA-anticoagulated venous blood.

Cyanmethemoglobin Method Procedure for Hemoglobin Estimation

Step-by-Step Procedure:

  1. Prepare the Blood Sample: Mix 5 ml of Drabkin’s solution with 20 μl of blood to create a 1:25 dilution. Stir the mixture and let it sit for at least 5 minutes to fully convert hemoglobin to cyanmethemoglobin.
  2. Measure Absorbance: Transfer the mixture to a cuvette and measure absorbance at 540 nm using a spectrophotometer. Alternatively, use a photoelectric colorimeter with a yellow-green filter. Ensure the measurement is taken against Drabkin’s solution as a reference.
  3. Calculate Hemoglobin Concentration: Use the following formula to calculate hemoglobin concentration:
Hemoglobin in gm/dl = (Absorbance of test sample ÷ Absorbance of standard) × concentration of standard × (Dilution factor ÷ 100)
Pipetting Protocol for the Cyanmethemoglobin Method: Volumes of Drabkin’s Reagent, Hemoglobin Standard, and Sample for Blank, Standard, and Test Preparations.
ComponentBlankStandardTest
Drabkin’s Reagent 5 ml 5 ml 5 ml
Hemoglobin Standard 20 µl
Sample 20 µl

Calibration Table and Graph Preparation

For efficient hemoglobin quantification, a calibration graph is essential, especially in laboratories processing high sample volumes. Here’s how to prepare the graph:

  1. Prepare Standard Solutions: Use commercially available adulterated cyanmethemoglobin standards or create a serial dilution of standard cyanmethemoglobin in Drabkin’s solution.
  2. Plot the Graph: On linear graph paper, plot the hemoglobin concentration (horizontal axis) against the corresponding absorbance (vertical axis). The points should form a straight line passing through the origin.
  3. Generate the Calibration Table: This graph can be used to create a calibration table, which correlates absorbance values with hemoglobin concentrations, aiding in rapid and accurate data interpretation.

Factors Affecting Cyanmethemoglobin Method Accuracy

  1. The cyanmethemoglobin solution remains stable, allowing flexible timing for absorbance readings without affecting accuracy.
  2. High white blood cell counts (TLC > 25,000/μl), abnormal plasma proteins (e.g., in Waldenström’s macroglobulinemia or multiple myeloma), and lipemic blood (hypertriglyceridemia) can cause errors in hemoglobin measurements.

FAQs

  1. What is the cyanmethemoglobin method?

    The cyanmethemoglobin method is a precise laboratory technique for measuring hemoglobin levels in blood. It works by converting all forms of hemoglobin into a stable compound called cyanmethemoglobin, providing accurate results. This method is globally endorsed by the International Committee for Standardization in Hematology.

  2. How does the cyanmethemoglobin method work?

    The cyanmethemoglobin method works by mixing blood with Drabkin’s solution, potassium cyanide, and potassium ferricyanide. Potassium ferricyanide converts hemoglobin to methemoglobin, which then reacts with potassium cyanide to form cyanmethemoglobin. The absorbance of the resulting solution is measured at 540 nm using a spectrophotometer to determine hemoglobin concentration.

  3. Who developed the cyanmethemoglobin method?

    The cyanmethemoglobin method was developed by Dr. H.A. Drabkin in the early 20th century. He also formulated Drabkin’s solution, which is now a standard reagent for hemoglobin estimation.

  4. What precautions should be taken when using the cyanmethemoglobin method?

    When using the cyanmethemoglobin method, handle potassium cyanide with care due to its toxicity. Follow laboratory safety protocols, including wearing personal protective equipment (PPE) and working in a well-ventilated area.

  5. What is hemiglobincyanide?

    Hemiglobincyanide, or cyanmethemoglobin, is a stable compound formed when hemoglobin reacts with potassium ferricyanide and potassium cyanide in Drabkin’s solution. It enables precise spectrophotometric measurement of hemoglobin levels and is widely used in hematology due to its stability and compatibility with various hemoglobin types.

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Reference(s)

  1. Cheesbrough, Monica. “District Laboratory Practice in Tropical Countries.” 2nd ed., Cambridge University Press, 2006, isbn: 9780511543470.
  2. SM, Lewis., et al. “Dacie and Lewis’s Practical Haematology.” 9th ed., Churchill Livingstone, 25 Oct. 2001, isbn: 9780443063770.

Cite this page:

Dayyal Dg.. “Cyanmethemoglobin Method for Estimation of Hemoglobin.” BioScience. BioScience ISSN 2521-5760, 25 August 2016. <https://www.bioscience.com.pk/en/topics/cell-biology/cyanmethemoglobin-hemoglobin-cyanide-method-for-estimation-of-hemoglobin>. Dayyal Dg.. (2016, August 25). “Cyanmethemoglobin Method for Estimation of Hemoglobin.” BioScience. ISSN 2521-5760. Retrieved February 02, 2025 from https://www.bioscience.com.pk/en/topics/cell-biology/cyanmethemoglobin-hemoglobin-cyanide-method-for-estimation-of-hemoglobin Dayyal Dg.. “Cyanmethemoglobin Method for Estimation of Hemoglobin.” BioScience. ISSN 2521-5760. https://www.bioscience.com.pk/en/topics/cell-biology/cyanmethemoglobin-hemoglobin-cyanide-method-for-estimation-of-hemoglobin (accessed February 02, 2025).
  • Posted by Dayyal Dg.

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