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Questions and Answers
What is the most critical time frame for measuring the absorbance of a test sample after its initial dilution?
What is the most critical time frame for measuring the absorbance of a test sample after its initial dilution?
Which condition can cause turbidity, leading to a falsely high result in the absorbance measurement?
Which condition can cause turbidity, leading to a falsely high result in the absorbance measurement?
What should be done to correct the effects of lipaemia causing turbidity?
What should be done to correct the effects of lipaemia causing turbidity?
How should cyanmethhemoglobin reagent be stored to prevent degradation from light exposure?
How should cyanmethhemoglobin reagent be stored to prevent degradation from light exposure?
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Which of the following statements is true regarding the handling of the haemoglobin reagent?
Which of the following statements is true regarding the handling of the haemoglobin reagent?
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What effect does carboxyhaemoglobin have on absorbance measurement in heavy smokers?
What effect does carboxyhaemoglobin have on absorbance measurement in heavy smokers?
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To correct for cells containing Hb S and Hb C that may resist haemolysis, what dilution method should be used?
To correct for cells containing Hb S and Hb C that may resist haemolysis, what dilution method should be used?
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What should be done with any unused portion of a cyanmethhemoglobin standard after its ampoule is opened?
What should be done with any unused portion of a cyanmethhemoglobin standard after its ampoule is opened?
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What is the role of potassium ferricyanide in the cyanmethemoglobin method?
What is the role of potassium ferricyanide in the cyanmethemoglobin method?
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What is the consequence of allowing the cyanmethemoglobin reagent to freeze?
What is the consequence of allowing the cyanmethemoglobin reagent to freeze?
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What is the importance of measuring absorbance at 540 nm during hemoglobin determination?
What is the importance of measuring absorbance at 540 nm during hemoglobin determination?
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What is the proper dilution ratio for the blood sample in the hemoglobin determination method?
What is the proper dilution ratio for the blood sample in the hemoglobin determination method?
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What condition must be ensured for the diluent when preparing the hemoglobin sample?
What condition must be ensured for the diluent when preparing the hemoglobin sample?
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What happens if the pH of the reagent is outside the range of 7.0–7.4?
What happens if the pH of the reagent is outside the range of 7.0–7.4?
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What should be done if the absorption measurement at 540 nm against water as a blank is other than zero?
What should be done if the absorption measurement at 540 nm against water as a blank is other than zero?
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Which of the following factors is NOT necessary for the stable pigment cyanmethemoglobin to form?
Which of the following factors is NOT necessary for the stable pigment cyanmethemoglobin to form?
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Study Notes
Hemoglobin Determination Method
- Cyanmethaemoglobin (haemoglobincyanide) method is the reference method for determining hemoglobin concentration.
- This method involves converting hemoglobin to cyanmethemoglobin using Drabkin's solution, a reagent containing potassium cyanide and potassium ferricyanide.
- The absorbance of cyanmethemoglobin is measured at 540 nm using a spectrophotometer.
Drabkin's Solution
- Drabkin's solution is a reagent used to convert hemoglobin to cyanmethemoglobin for measurement.
- It should be clear and pale yellow in color, with zero absorbance at 540 nm when measured against water.
- Store Drabkin's solution in a brown borosilicate glass bottle at room temperature for several months.
- If the ambient temperature is above 30°C, store the solution in the refrigerator but bring it to room temperature before use.
- Do not freeze Drabkin's solution.
- Discard the solution if it becomes turbid, the pH is outside the 7.0–7.4 range, or if it has an absorbance other than zero at 540 nm against a water blank.
Method
- To determine hemoglobin concentration, perform a 1 in 201 dilution of blood by adding 20 µl of blood to 4 ml of Drabkin's solution.
- Allow the solution to stand at room temperature for at least 5 minutes to ensure complete conversion of hemoglobin to cyanmethemoglobin.
- Measure the absorbance of the sample in a spectrophotometer at 540 nm against a reagent blank.
- Measure the absorbance of the test sample within 6 hours of its initial dilution.
- Compare the absorbance of a HiCN standard to a reagent blank using the same spectrophotometer used for the patient sample.
- Store the HiCN standard in the dark and discard any unused solution at the end of the day.
Calculation of Hemoglobin Concentration
- The hemoglobin concentration is calculated based on the absorbance of the sample and the standard curve.
- The standard curve is constructed by plotting the absorbance of known hemoglobin concentrations against their corresponding values.
- The absorbance of the sample is used to determine the corresponding hemoglobin concentration from the standard curve.
Sources of Error
- Drabkin's solution is sensitive to light and should be stored in a brown bottle or in a dark place.
- High WBC count (greater than 20 X 109/L) or high platelet count (greater than 700 X 109/L) can cause turbidity and falsely high results.
- Lipaemia can also cause turbidity and falsely high results.
- Cells containing Hb S and Hb C may be resistant to haemolysis, causing turbidity.
- Carboxyhaemoglobin takes 1 hour to convert to cyanmethaemoglobin, potentially causing erroneous results in samples from heavy smokers.
- Cyanide in the reagent is highly toxic and must be used cautiously.
- Acidification of cyanide in the reagent releases highly toxic hydrogen cyanide gas.
- Do not discard reagent-sample solutions into sinks, use a licensed waste disposal service.
Safety Precautions
- Drabkin's solution contains cyanide and is highly toxic.
- Handle the reagent with caution and avoid contact with skin and eyes.
- Consult the safety data sheet provided by the manufacturer.
- Do not discard reagent-sample solutions into sinks.
- Use a licensed waste disposal service for disposal.
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Description
Test your knowledge on the cyanmethaemoglobin method for hemoglobin determination. This quiz covers the principles, usage, and handling of Drabkin's solution in the measurement process. Enhance your understanding of this important laboratory technique and its applications.
