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Cyanmethemoglobin Method Hemoglobin Det'n

Cyanmethemoglobin Method Hemoglobin Determination

Short Summary:

The video explains the cyanmethemoglobin method, a standard hematological test for measuring hemoglobin levels in blood. It involves converting hemoglobin into a stable form called cyanmethemoglobin, which is then measured using a spectrophotometer. The method is widely recommended by international organizations and is used in routine hematology labs. The video details the procedure, necessary reagents, potential sources of error, and how to address them.

Detailed Summary:

Section 1: Introduction

• The video introduces the cyanmethemoglobin method as a standard test for hemoglobin determination.
• It highlights the method's recommendation by the International Committee for Standardization in Hematology and the National Committee for Clinical Laboratory Standards.
• The method is also known as the hemiglobin cyanide method.

Section 2: Materials, Reagents, and Equipment

• The video lists the necessary materials, reagents, and equipment:
  • Cyanmethemoglobin reagent (Drabkin's reagent)
  • Serological or micropipettes
  • Test tubes
  • Spectrophotometer
• It mentions that the cyanmethemoglobin reagent is commercially available and contains potassium ferrocyanide, potassium cyanide, sodium bicarbonate, and a surfactant.

Section 3: Specimen and Principle

• The video explains that the test can be performed on EDTA anticoagulated blood or capillary blood.
• It describes the principle of the method:
  • Drabkin's reagent oxidizes hemoglobin to methemoglobin.
  • Potassium cyanide converts methemoglobin to cyanmethemoglobin, a stable compound.
  • The absorbance of cyanmethemoglobin at 540 nanometers is directly proportional to the hemoglobin concentration.

Section 4: Procedure

• The video provides a step-by-step guide for the procedure:
  • Pipette 5 ml of Drabkin's reagent into two test tubes (blank and sample).
  • Add 20 microliters of blood to the sample tube.
  • Rinse the pipette with Drabkin's reagent to ensure all blood is transferred.
  • Mix well and allow the solution to stand for at least 10 minutes.
  • Read the absorbance of the blank tube at 540 nanometers and adjust to 0.
  • Read the absorbance of the sample tube at 540 nanometers.
  • Calculate the hemoglobin concentration using a formula that involves the absorbance of the sample and a standard.

Section 5: Sources of Error

• The video discusses potential sources of error:
  • Turbidity in the Drabkin's reagent can lead to falsely high results.
  • Light exposure can degrade the reagent.
  • The reagent is highly toxic due to the presence of cyanide and must be handled with caution.
  • High white blood cell or platelet counts can cause turbidity.
  • Lipidemia can also cause turbidity.
  • Hemoglobin S and C can resist hemolysis and cause turbidity.
  • Abnormal globulins can precipitate in the reagent.
  • Carboxyhemoglobin takes time to convert to cyanmethemoglobin, potentially leading to erroneous results in heavy smokers.

Section 6: Addressing Errors

• The video provides solutions for addressing the mentioned errors:
  • Discard turbid or darkened Drabkin's reagent.
  • Store the reagent in an amber bottle or dark place.
  • Handle the reagent with caution and dispose of it properly.
  • Centrifuge the sample and measure the supernatant if turbidity is present.
  • Correct for lipidemia by adding patient plasma to the reagent.
  • Dilute the sample with distilled water if hemoglobin S or C is present.
  • Add potassium carbonate to the reagent if abnormal globulins precipitate.
  • Allow sufficient time for carboxyhemoglobin conversion in heavy smokers.

Conclusion:

The video concludes by summarizing the cyanmethemoglobin method for hemoglobin determination, emphasizing its importance in routine hematology and highlighting the potential sources of error and their solutions.