Haemoglobin by Cyanomethemoglobin Method
Summary
TLDRThe 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
🧬 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.
🧪 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.
Mindmap
Keywords
💡Hemoglobin estimation
💡Sine Meth method
💡Spectrophotometer
💡Absorbance
💡Potassium cyanide
💡pH
💡EDTA
💡Standard curve
💡Dilution factor
💡Turbidity
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
you
[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
you
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