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.

Outlines

00:00

🧬 Hemoglobin Estimation by Sine Meth Hemoglobin Method

This paragraph introduces the Sine Meth Hemoglobin method, an internationally recognized technique for determining hemoglobin levels in blood. The process involves diluting blood in a reagent solution containing potassium cyanide and potassium ferrocyanide, which converts hemoglobin to methemoglobin and then to Sine Meth Hemoglobin. The absorbance of the solution is measured using a spectrophotometer at 540 nanometers or a calorimeter with a yellow-green filter. The equipment used includes pipettes, calorimeters, or spectrophotometers. The paragraph also discusses the importance of maintaining the pH of the solution, its light sensitivity, and storage conditions. It advises against freezing the solution and emphasizes safety precautions, such as not pipetting by mouth due to the presence of the toxic substance potassium cyanide. The paragraph concludes with instructions on using a hemoglobin standard solution for calibration, which is commercially available and should be stored in brown bottles to protect it from light.

05:01

🧪 Preparation of a Standard Curve for Hemoglobin Estimation

The second paragraph delves into the preparation of a standard curve for hemoglobin estimation using the Sine Meth Hemoglobin method. It outlines the process of creating dilutions of a hemoglobin standard solution with drop cancellation reagent in test tubes, each with varying dilution factors. The paragraph explains how to calculate the hemoglobin concentration in each dilution by multiplying the dilution factor with the known strength of the standard. The procedure for measuring the absorbance of each dilution using a spectrophotometer and plotting the results on a graph is described. The graph, with hemoglobin concentration on the x-axis and absorbance on the y-axis, should be linear and pass through the origin. The paragraph also highlights the importance of recalibrating the curve when there are changes in the test method, calorimeter, or reagent lot. It concludes with the advantages of the method, such as the conversion of all forms of hemoglobin except sulfhemoglobin and the elimination of visual error, as well as the disadvantages, including the use of the poisonous substance potassium cyanide and issues with turbidity caused by abnormal plasma proteins or high leukocyte counts.

Keywords

💡Hemoglobin estimation

Hemoglobin estimation refers to the process of determining the concentration of hemoglobin in blood. In the video, this is done using the Sine Meth method, which is internationally recommended. The method involves diluting blood in a reagent solution and measuring the absorbance of the resulting solution to calculate hemoglobin levels. This is crucial for diagnosing conditions like anemia.

💡Sine Meth method

The Sine Meth method is a specific technique for hemoglobin estimation. It involves converting hemoglobin to methemoglobin and then to Sine Methemoglobin using potassium cyanide and potassium ferrocyanide. The video script describes this method as the internationally recommended approach, highlighting its importance in medical diagnostics.

💡Spectrophotometer

A spectrophotometer is an instrument used to measure the absorbance of light by a solution. In the context of the video, it is used to measure the absorbance of the solution at a specific wavelength (540 nanometers) to determine the concentration of hemoglobin. This device is essential for the accuracy of the Sine Meth method.

💡Absorbance

Absorbance is a measure of how much light a solution absorbs. In the video, the absorbance of the solution is measured after the blood has been treated with reagents. The absorbance value is then used to calculate the hemoglobin concentration, making it a key parameter in the Sine Meth method.

💡Potassium cyanide

Potassium cyanide is a chemical compound used in the Sine Meth method to convert methemoglobin to Sine Methemoglobin. The video script cautions that it is a poisonous substance, emphasizing the need for careful handling and safety precautions in the laboratory.

💡pH

pH is a measure of the acidity or alkalinity of a solution. The video mentions that the pH of the reagent solution must be checked and maintained between 7 to 7.4 for accurate hemoglobin estimation. This is important because the chemical reactions involved in the Sine Meth method are sensitive to pH changes.

💡EDTA

EDTA (Ethylenediaminetetraacetic acid) is a chelating agent used in the video to collect blood samples. It prevents coagulation of blood, allowing for accurate hemoglobin estimation. The script specifies using EDTA whole blood for the procedure, highlighting its role in sample preparation.

💡Standard curve

A standard curve is a graphical representation used to determine the concentration of an unknown sample by comparing its absorbance to that of known standards. In the video, a standard curve is prepared using a hemoglobin standard solution to facilitate the calculation of hemoglobin concentration in blood samples.

💡Dilution factor

The dilution factor is a ratio used to calculate the concentration of hemoglobin in diluted blood samples. The video script describes how different dilutions of the standard solution are made to create a standard curve. The dilution factor is calculated by dividing the volume of the standard solution by the total volume of the diluted solution.

💡Turbidity

Turbidity refers to the cloudiness or haziness of a solution, which can interfere with the accurate measurement of absorbance. The video mentions that abnormal plasma proteins and high leukocyte counts can cause turbidity. To overcome this, the script suggests centrifuging the diluted blood, which helps in clarifying the solution before measurement.

Highlights

Hemoglobin estimation by the cyanmethemoglobin method is internationally recommended.

Blood is diluted in a reagent solution containing potassium cyanide and potassium ferrocyanide.

Methemoglobin is converted to cyanmethemoglobin by potassium cyanide.

Absorbance is measured at 540 nanometers using a spectrophotometer or a calorimeter with a yellow-green filter.

The pH of the solution must be maintained between 7 to 7.4 and checked monthly.

The reagent solution is unstable if exposed to light and should be stored in brown bottles.

The solution should be clear and pale yellow when measured against water as a blank.

If room temperature exceeds 30°C, the solution should be refrigerated and brought to room temperature before use.

The solution must never be frozen and should be discarded if found to be turbid.

Hemoglobin standard solution is commercially available and must be stored in a brown bottle due to photosensitivity.

Procedure involves taking 5 mL of Drabkin's solution in a test tube and mixing with blood sample.

The absorbance of the blood-reagent mixture is measured at 540 nanometers after a 15-minute静置.

Hemoglobin concentration is calculated using a formula or standard curve.

A standard curve is prepared by diluting the standard solution with Drabkin's solution in a series of tubes.

The concentration of hemoglobin in each dilution is calculated based on the dilution factor and strength of the standard.

A calibration curve is plotted with hemoglobin concentration on the x-axis and absorbance on the y-axis.

A new calibration curve must be prepared when there are changes in the test method, calorimeter, or reagent lot.

The cyanmethemoglobin method has the advantage of converting all forms of hemoglobin except sulfhemoglobin.

The method eliminates visual error as no color matching is required.

Potassium cyanide is a poisonous substance, so Drabkin's solution must never be ingested.

Abnormal plasma proteins and high leukocyte counts can cause turbidity in the diluted blood.

Transcripts

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[Music]

hemoglobin estimation by sine meth

hemoglobin method this is the

internationally recommended method for

determining hemoglobin principle blood

is diluted in the reagent solution

containing potassium cyanide and

potassium ferrocyanide the latter

converts hemoglobin to meth hemoglobin

which is converted to sine meth

hemoglobin by potassium cyanide the

absorbance of the solution is then

measured in a spectrophotometer at a

wavelength of 540 nanometers or in a

calorimeter using a yellow-green filter

equipment pipette calorimeter or

spectrophotometer the principle applied

for the estimation of hemoglobin is the

same in both the wavelengths available

for the estimation differ reagents

required drop cancellation the pH of the

solution must be checked every month and

should be maintained between 7 to 7.4

the solution is unstable if exposed to

light and can be stored at room

temperature in Brown borosilicate

bottles for several months the solution

should be clear and pale yellow in color

when measured against water as a blank

in a spectrometer at a wavelength of 540

Nm the absorbance must be adjusted to 0

if the room temperature is higher than

30 degree centigrade the solution should

be stored in a refrigerator but brought

to room temperature before use the

solution must never be frozen discard

the solution I found to be turbot if pH

is outside range

do not pip it drop cancel ushion by

mouth

hemoglobin standard solution this is

available commercially at a specific

concentration or strength it is stored

in a brown bottle as it is

photosensitive exposure to light causes

deterioration in the strength of the

standard the concentration of the

standard used in this demonstration is

14 point 8 gram percent sample EDTA

whole blood venous sample or capillary

sample procedure take 5 milliliters of

trap cancellation in a test tube mix

blood sample by gentle inversion and

draw point 0 2 milliliters of blood into

the hipot wipe the outer surface of the

pipette with the tissue paper to remove

excess blood place the pipit into the

tube containing drop cancellation and

slowly expel the blood into the solution

mix well

and let it stand undisturbed for 15

minutes measure the absorbance of this

solution at 540 nanometers in a

spectrophotometer after adjusting the

optical density at zero by using Ratkin

solution as blank calculate the

hemoglobin concentration in the sample

by using this formula or a standard

curve concentration of hemoglobin in

sample is equal to absorbance of sample

divided by absorbance of standard

multiplied by concentration of standard

preparation of a standard curve will be

discussed shortly concentration of

standard will be available on the kit

insert or the vial of a standard

solution measured its absorbance or OD

against a blank of drop concession

preparation of standard curve for

hemoglobin estimation by sine myth

hemoglobin method let's learn to prepare

a calibration curve for hemoglobin

estimation by sine myth hemoglobin

method in a laboratory where several

samples are tested in a day this is an

important exercise to standardize the

test method for this you will require

drop concession and a hemoglobin

standard the concentration of the

standard used in this demonstration is

14 point 8 gram percent a w-h-o

international reference sign hemoglobin

standard is also available commercially

as 10 milliliters sealed ampoules and a

stable for years make several dilutions

of the standard solution with drop

concession in the first test tube take 5

milliliters of drop concession in the

second test tube makes one milliliter of

standard with 4 milliliters of drop

concession the dilution on the standard

is thus 1 in 5 or 0.2 let's call this

the dilution factor in the third test

tube makes 2 milliliters of standard

three milliliters of draftkings solution

the dilution factor here will be 0.4 in

the fourth test tube

mix three milliliters of standard with

two milliliters of drop concession the

dilution factor is 0.6 in the fifth test

tube mix four milliliters of standard

with one milliliter of drop concession

here the dilution factor is 0.8 in the

sixth test you take five milliliters of

standard solution only as this is pure

standard the illusion factor can be

taken as one as a strength of the

standard is known that is fourteen point

eight gram per deciliter the value of

hemoglobin in each tradition can be

calculated by multiplying the dilution

factor with the strength of the standard

thus these values will be zero two point

nine five point nine eight point nine

eleven point eight fourteen point eight

round off to the nearest decimal point

take the test tube with neat wrapkin

solution and transfer the solution to

the cubit place the cubit on the

spectrophotometer and set the OD to zero

at a wavelength of 540 nanometers now

measure the OD of each dilution in the

spectrophotometer against the blank of

drop cancel ushion taking a clean cubit

for each dilution record Audrey values

in a table as shown this table shows

volume of standard in east elution

volume of drop cancel ushion dilution

factor OD values and value of hemoglobin

in each dilution of standard this

information can be plotted on a graph

with the concentration of hemoglobin in

grams per deciliter in standard plotted

on the horizontal axis and corresponding

absorbance values plotted against the

vertical axis this graph can be plotted

on an excel sheet or manually on a graph

paper

the

wines should be in a straight line that

passed through the origin

after audio of the sample is taken the

corresponding hemoglobin value can be

directly read by plotting on the graph

for example if the OD of a test sample

is 0.32 after plotting it on the graph

the corresponding hemoglobin

concentration is 14 point 8 grams per

deciliter a new calibration curve must

be prepared

whenever the test method calorimeter or

cubed type reagent lot is changed

advantages of sine meat hemoglobin

method all forms of hemoglobin except

salfi ma globin are converted to sign

meat hemoglobin visual error is

eliminated as no color matching is

required a reliable and stable reference

standard is available from w-h-o for

direct comparison disadvantages

potassium cyanide is a poisonous

substance and that is the reason why

drop concision must never be repeated by

mouth the rate of conversion of blood

containing carboxyhemoglobin is slowed

considerably prolonging the reaction

time to 30 minutes can overcome this

problem abnormal plasma proteins cause

turbidity when blood is diluted with

wrapkin solution a high leukocyte count

also causes turbidity on dilution of

blood centrifuging the diluted blood can

help overcome the turbidity

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