Haemoglobin by Cyanomethemoglobin Method

Summary

The video script explains the cyanmethemoglobin method for hemoglobin estimation, an internationally recognized technique. It involves diluting blood in a reagent with potassium cyanide and potassium ferrocyanide, which converts hemoglobin to cyanmethemoglobin. The absorbance is measured at 540 nm using a spectrophotometer or a calorimeter. The script details the equipment, reagents, and procedure, including preparing a standard curve for accurate hemoglobin concentration calculation. It highlights the method's advantages, such as eliminating visual error and providing a stable reference standard, and cautions against the use of poisonous potassium cyanide.

Takeaways

• 🧪 Hemoglobin estimation is performed using the internationally recommended cyanmethemoglobin method.
• 💉 The method involves diluting blood in a reagent containing potassium cyanide and potassium ferrocyanide, converting hemoglobin to cyanmethemoglobin.
• 📏 Absorbance is measured at a wavelength of 540 nm using a spectrophotometer or a colorimeter with a yellow-green filter.
• ⚗️ The reagent solution must be stored in brown borosilicate bottles, kept at room temperature, and shielded from light for stability.
• 📊 Hemoglobin concentration is calculated by comparing the absorbance of the sample to a known standard using a specific formula or a standard curve.
• 🔬 A standard curve is created by measuring the absorbance of various dilutions of a standard solution, which is used to determine hemoglobin concentration.
• 📉 The hemoglobin values are plotted on a graph, with concentration on the horizontal axis and absorbance on the vertical axis, forming a straight line.
• 📅 A new calibration curve must be prepared whenever the test method, equipment, or reagent lot changes.
• 💡 This method eliminates visual errors as no color matching is required, making it more accurate.
• ⚠️ Potassium cyanide is poisonous, and special care must be taken to avoid exposure or ingestion during the testing process.

Q & A

What is the principle behind hemoglobin estimation by the sine meth hemoglobin method?

The principle involves diluting blood in a reagent solution containing potassium cyanide and potassium ferrocyanide. Hemoglobin is converted to meth hemoglobin, which is further converted to sine meth hemoglobin by potassium cyanide. The absorbance is then measured using a spectrophotometer at 540 nm or a calorimeter with a yellow-green filter.

What equipment is required for the hemoglobin estimation process?

The equipment required includes a pipette, a spectrophotometer or a calorimeter, and a cuvette. These are used to measure the absorbance of the sine meth hemoglobin solution.

What are the key reagents used in the sine meth hemoglobin method?

The key reagents include potassium cyanide and potassium ferrocyanide, which are part of the reagent solution, and hemoglobin standard solution, which is used for calibration and comparison.

How should the reagent solution be stored, and what are the conditions for maintaining its stability?

The reagent solution must be stored in brown borosilicate bottles to protect it from light, which can cause instability. It can be stored at room temperature for several months. However, it should be kept in the refrigerator if the room temperature exceeds 30°C and must be brought to room temperature before use.

What is the importance of checking the pH of the reagent solution, and what is the ideal pH range?

The pH of the reagent solution must be checked monthly to ensure accuracy, as an incorrect pH can affect the hemoglobin estimation. The ideal pH range is between 7.0 and 7.4.

How is the sample prepared for hemoglobin estimation?

A 0.02 mL sample of EDTA-treated whole blood or capillary blood is drawn into a pipette, wiped clean, and expelled into the reagent solution. The mixture is allowed to stand undisturbed for 15 minutes before measuring its absorbance.

What is the formula used to calculate the hemoglobin concentration in a sample?

The concentration of hemoglobin in the sample is calculated using the formula: Hemoglobin concentration = (Absorbance of sample / Absorbance of standard) × Concentration of standard.

How is a standard curve for hemoglobin estimation prepared?

A standard curve is prepared by creating several dilutions of the hemoglobin standard solution and measuring the absorbance of each dilution against a blank solution. The hemoglobin concentration in each dilution is plotted against the absorbance to form the calibration curve.

What are the advantages of the sine meth hemoglobin method?

Advantages include the conversion of all hemoglobin types (except sulfhemoglobin) to sine meth hemoglobin, elimination of visual error since no color matching is required, and availability of a reliable and stable WHO reference standard for comparison.

What are some disadvantages of the sine meth hemoglobin method?

Disadvantages include the use of potassium cyanide, a toxic substance, and the prolonged reaction time for samples containing carboxyhemoglobin. Additionally, abnormal plasma proteins and high leukocyte counts can cause turbidity, which may affect accuracy.