Determination of Hemoglobin

Mr. Topesh Patle

AIM: Determination of Hemoglobin

Principle of Sahli’s method

  • When blood is added to 0.1 N hydrochloric acid, hemoglobin is converted to brown colored acidhematin.
  • The resulting colour after dilution is compared with standard brown glass reference blocks of a Sahli hemoglobinometer.

 

Materials Required

 

  1. Blood Sample: EDTA or Fresh venous blood.
  2. Reagents:
    • Hydrochloric Acid (HCl): Concentration of about 0.1 N.
    • Distilled Water: For dilution and washing.
  3. Glassware:
    • Test tubes 
    • Pipettes
    • A graduated cylinder
  4. Hemoglobinometer
  5. Colour Comparison Chart: A device or printed chart to compare the colour of the hemolysate against known standards.

 

Procedure

Step 1: Preparation of Blood Sample

  1. Collect the Blood:
    • Collect venous blood into a clean test tube or container using an aseptic technique.
    • If using an anticoagulant (like EDTA), ensure the blood remains fluid for accurate measurement.

Step 2: Hemolysis of Red Blood Cells

  1. Add Hydrochloric Acid:
    • Measure about 2 mL of blood and transfer it to a test tube.
    • Carefully add 2-3 drops of 0.1 N hydrochloric acid to the blood sample. This acid will lyse the red blood cells, releasing haemoglobin into the solution.
  2. Mix the Solution:
    • Gently invert the test tube or mix the blood and acid solution with a pipette.
    • Allow the mixture to stand at room temperature for 5-10 minutes. The solution will turn red as the haemoglobin is released and dissolved.

Step 3: Preparation of the Standard

  1. Prepare a Standard Solution:
    • If a standard solution is unavailable, prepare a standard haemoglobin solution with a known concentration (e.g., 15 g/dL).
    • This can be done by dissolving a specific amount of purified haemoglobin in distilled water to create a colour standard for comparison.

Step 4: Color Comparison

  1. Dilution (if necessary):
    • If the hemolysate is too concentrated, dilute it with distilled water to achieve a suitable concentration for comparison. The typical dilution ratio is about 1:10, but this can be adjusted based on the depth of colour.
  2. Use a Second Test Tube:
    • Transfer an equal volume (2 mL) of the standard haemoglobin solution into a separate test tube for comparison.
  3. Comparison of Colors:
    • Place both test tubes (the hemolysate and the standard solution) against a white background or light source to enhance visibility.
    • Compare the colour of the hemolysate with the standard solution or colour comparison chart. Look for the closest match in colour intensity.

Step 5: Interpretation of Results

  1. Record the Hemoglobin Level:
    • Based on the comparison, estimate the haemoglobin concentration in the blood sample. The concentration is typically expressed in grams per deciliter (g/dL).
    • Use the colour intensity of the hemolysate to determine the haemoglobin level by referencing the standard.

 

Advantages

  • Simple and Cost-Effective: Requires minimal equipment and can be performed in basic laboratory settings.
  • Quick Procedure: Results can be obtained relatively quickly.

 

Disadvantages

  • Subjective Measurement: Color comparison can be affected by lighting conditions and personal interpretation, leading to potential inaccuracies.
  • Limited Precision: Less accurate than advanced methods, especially in hemoglobinopathies or abnormal haemoglobin forms.
  • Interferences: Other blood components can influence the colour, impacting the accuracy of the results.

 

Principle of the Cyanmethemoglobin Method

  • The Cyanmethemoglobin method is based on converting haemoglobin (Hb) in the blood to a stable, coloured complex known as cyanmethemoglobin.
  • This conversion occurs through the action of specific reagents, primarily potassium ferricyanide and potassium cyanide.

Materials Required

  1. Reagents:
    • Drabkin’s Solution: A mixture containing:
      • Potassium ferricyanide (K₃[Fe(CN)₆]): Converts haemoglobin to cyanmethemoglobin.
      • Potassium cyanide (KCN): Stabilizes the cyanmethemoglobin complex.
    • Distilled Water
  2. Glassware:
    • Test tubes.
    • Pipettes (for accurate measurement of liquids).
    • Cuvettes (for spectrophotometric measurement).
  3. Spectrophotometer: For measuring the absorbance at a specific wavelength (540 nm).
  4. Standard Haemoglobin Solution: For calibration and comparison.

 

Procedure

Step 1: Preparation of Blood Sample

  1. Collect the Blood:
    • Use an aseptic technique to collect venous blood into a clean test tube containing an anticoagulant (like EDTA) to prevent clotting.

Step 2: Preparation of Hemolysate

  1. Add Drabkin’s Solution:
    • Measure 5 mL of Drabkin’s solution and add it to a test tube.
    • Add 20 µL (0.02 mL) of the blood sample to the Drabkin solution test tube.
  2. Mix the Solution:
    • Mix the solution by inverting the test tube or using a vortex mixer to ensure thorough mixing. This process lyses the red blood cells and converts haemoglobin to cyanmethemoglobin.
  3. Allow Reaction to Occur:
    • Let the mixture stand for 10-15 minutes at room temperature. This allows for the complete conversion of haemoglobin to cyanmethemoglobin.

Step 3: Measurement

  1. Set Up the Spectrophotometer:
    • Turn on the spectrophotometer and allow it to warm up if necessary.
    • Calibrate the spectrophotometer using distilled water as a blank. Set the wavelength to 540 nm.
  2. Measure the Absorbance:
    • Transfer the prepared solution to a cuvette.
    • Place the cuvette in the spectrophotometer and record the absorbance at 540 nm.

Step 4: Calculation

  1. Standard Curve:
    • Prepare a standard curve using known concentrations of haemoglobin. Measure their absorbance at 540 nm and plot the results to create a standard curve.
  2. Calculate Haemoglobin Concentration:
    • Use the absorbance value from the test sample and compare it to the standard curve to determine the haemoglobin concentration in grams per deciliter (g/dL).

Advantages

  • High Accuracy: The method provides precise and reliable measurements of haemoglobin levels.
  • Wide Applicability: Can differentiate between various haemoglobin types with additional testing.
  • Standardization: Well-established method with standardized protocols.

Disadvantages

  • Handling of Cyanide: Potassium cyanide is toxic, requiring careful handling and disposal.
  • Time-Consuming: Takes longer than some point-of-care tests, requiring 15-20 minutes to complete.
  • Interferences: Certain substances (e.g., elevated bilirubin or lipemia) can affect absorbance measurements.

Errors Involved in Haemoglobin Estimation

1. Pre-Analytical Errors

  • Sample Collection:
    • Aseptic Technique: Not using an aseptic technique can lead to contamination. Blood samples should be collected using sterile equipment and proper skin antisepsis.
    • Vascular Complications: Incorrect venipuncture can result in hemolysis. Use a gentle approach to avoid damaging red blood cells.
  • Storage Conditions:
    • Time Delay: If blood samples are not processed promptly, haemoglobin can degrade, leading to inaccurate results. It is recommended to analyze samples within 1-2 hours of collection.
    • Temperature: If processing is delayed, samples should be kept at 2-8°C. Prolonged exposure to room temperature can cause hemolysis.
  • Anticoagulant Choice:
    • EDTA vs. Citrate: An inappropriate anticoagulant can alter the sample’s haemoglobin behaviour. EDTA is commonly used for haematological tests.

 

2. Analytical Errors

  • Reagent Preparation:
    • Concentration Issues: Incorrect preparation of Drabkin’s solution can lead to inaccurate results. Regular checks on the reagent concentration are vital.
    • Expiration: Using expired reagents can result in ineffective hemolysis or the formation of the cyanmethemoglobin complex.
  • Inadequate Mixing:
    • Incomplete Hemolysis: If the blood sample is not thoroughly mixed with the reagent, the hemolysis may be incomplete, affecting the accuracy of the measurement.
  • Spectrophotometer Calibration:
    • Wavelength Accuracy: Ensure that the spectrophotometer is calibrated to the correct wavelength (540 nm) for optimal measurement of cyanmethemoglobin.
    • Absorbance Range: Regularly check the instrument’s absorbance range to ensure it functions within the specified limits.
  • Interference from Other Substances:
    • Bilirubin and Lipemia: High levels of bilirubin or lipids can interfere with the colourimetric measurement, leading to falsely elevated or decreased haemoglobin readings. Pre-treatment of samples may be necessary in such cases.

 

3. Post-Analytical Errors

  • Data Interpretation:
    • Reference Ranges: Results can be misinterpreted if the laboratory does not use appropriate reference ranges based on the tested population.
    • Variability: Variability in results may arise from differences in methodology or equipment between laboratories.
  • Human Error:
    • Pipetting Errors: Inaccurate pipetting can lead to errors in sample and reagent volumes. Ensure proper techniques and use calibrated pipettes.
    • Calculation Mistakes: Errors in calculating haemoglobin concentration from absorbance values can skew results.

Standardization of Instruments for Hemoglobin Estimation

1. Calibration of Spectrophotometer

  • Blank Calibration:
    • Use distilled water or a blank of the reagent solution (Drabkin’s solution) to set the baseline absorbance to zero. This step ensures that only the absorbance from the haemoglobin is measured.
  • Standard Curve Preparation:
    • Create a standard curve using several known haemoglobin concentrations (e.g., 0, 5, 10, 15 g/dL). Measure the absorbance of these standards to establish a relationship between absorbance and concentration.
    • Plot the absorbance against haemoglobin concentration to derive the equation for future calculations.

 

2. Quality Control

  • Control Samples:
    • Use quality control samples with known haemoglobin concentrations alongside patient samples. Regular testing of controls helps monitor the accuracy and precision of the method.
    • Analyze control samples daily to ensure the system is functioning correctly.
  • Documentation:
    • Maintain a log of all control measurements, calibration data, and any adjustments made to the equipment or procedures. This documentation is crucial for tracking performance over time.

 

3. Routine Maintenance

  • Cleaning:
    • Regularly clean cuvettes and the spectrophotometer to prevent contamination and ensure consistent results. Follow the manufacturer’s recommendations for cleaning procedures.
  • Professional Servicing:
    • Schedule periodic servicing and calibrating of the spectrophotometer by qualified technicians to maintain accuracy and reliability.

 

4. Training of Personnel

  • Standard Operating Procedures (SOPs):
    • Develop and implement clear SOPs for all aspects of haemoglobin testing, including specimen collection, reagent preparation, measurement, and interpretation.
    • Ensure that all laboratory personnel know and adhere to these SOPs.
  • Training Programs:
    • Provide regular training for staff on the latest techniques, equipment use, and error-reduction strategies. Continuous education helps reduce human error and improve overall laboratory performance.

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