ESTIMATION
OF HEMOGLOBIN CONCENTRATION
Hemoglobinometry;
Is the
measurement of the concentration of hemoglobin in the blood
METHODS
of estimation of hemoglobin concentration
Hemoglobin can be estimated by different methods it can be
classified into following categories:
1.
Visual colour
comparisionmethod
2.
Gasometric
method
3.
Spectrophotometric
method
4.
Automated
hemoglobinometry
5.
Non-automated
hemoglobinometry
6.
Other
methods
A. Visual methods,
1.
Sahil’s
method
2.
Dares
method
3.
Hadens
method
4.
Wintrobes
method
5.
Haldanes
method
6.
Tallquists
method
B. Gasometric method
1.
van Slyke method
C. Spectrophotometric method
1.
Oxyhemoglobin
method
2.
Cyanmethemoblobin
method
D.electronic hematology analyszer
Automated hemoglobinometry
E. Non-automated hemoglobinometry
F. Other methods
1.
Alkaline-hematin
method
2.
Specific
gravity method
3.
comparator
method.
VISUAL
METHODS
These methods are more commonly used than
photometric methods. In Sahli’s method, hemoglobin in the blood sample is
converted to acid hematin, which gives brown colour. Since brown is more easily
matched by the human eye than red (the colour of Hb), Sahli’s method for
testing hemoglobin is one of the most acceptable visual methods. However, the
error in visual methods is higher. Therefore, visual methods (especially sahli’s)
are convenient and the cost of estimation is less, they are usually practiced
in hematology laboratories is clinical medicine and for performing practicals
are students in physiology.
INTRODUCTION
Hemoglobin (Hb) is a conjugated protein present
in red blood cells. It carries oxygen from the lungs to the tissues, and carbon
dioxide from the tissues to the lungs. It is made up of heme and globin. The
heme group is an iron complex, containing one iron atom. Iron is essential for
the primary function of the hemoglobin, the transport of oxygen. When reduced
hemoglobin is exposed to oxygen at increased pressure, oxygen is taken up at
the iron atom until each molecule of hemolecule at each iron atom. The Hb
molecule when fully saturated with oxygen, that is, four oxygen molecules
combined with one hemoglobin molecule, is called oxy-hemoglobin. One gram of
hemoglobin carries 1.34 ml of oxygen. Hemoglobin returning with carbon dioxide
from the tissues is called reduced hemoglobin.
TYPE
OF HEMOGLOBIN
Hemoglobins can be broadly divided into normal
and abnormal types.
Normal Hb: Adult Hb, Fetal Hb and Embryonic Hb.
Abnormal Hb: Hb S, Hb C, Hb D, Hb E and Unstable hemoglobins.
NORMAL
HEMOGLOBINS
Adult
hemoglobins
Hemoglobin A (Hb A): About 97 per cent of hemoglobin
of adult red cells is Hb A. It consists of two alpha (a) and two beta (b) chains with the structural formala a2 b2. Hb A is detected in small amounts in the fetus
as early as the eighth week of intrauterine life. During the first few months
of postnatal Life, Hb A almost completely replaces Hb F and the adult pattern
is fully established in six months.
Hemoglobin A2 (Hb A2):
This is the minor hemoglobin in the adult red cells. It has the structural
formal of a2 d2 . Hb A2 is present in very small
amounts at birth and reaches the adult level of 3 per cent during the first
year of life. Its concentration increases in some types of anemia.
Fetal
Hemoglobins
Fetal hemoglobin (Hb F): Hb F is the major
hemoglobin in intrauterine life. It has the structural formala of a2 g2 . Hb F accounts for 70-90 per cent of
hemoglobin at term. It then falls rapidly to 25 per cent in one month, and 5
percent in six months. The adult level of 1 per cent is not reached in some
children until puberty Hb F concentration in adults increases in some types of
anemia, hemoglobinopathies, and some time in leukemia.
Hemoglobin
Bart’s (Hb Barts): This is
the minor hemoglobin present in fetal life. It consists of four gamma (g) chains g4 . Hb Bart’s concentration increases in fetal
life in thalassemia.
Embryonic
hemoglobins
These hemoglobins are confined to the very
early stage (the embryonic stage) of development. There are three embryonic
hemoglobins: 1)Hb Gower 1 (consisting of two zeta and two epsilon chains: V2 e2 ), 2) Hb Gower 2 (consisting of two alpha and
two epsilon chains: a2 e2 ) and 3) Hb Portland (consisting of two zeta
and two gamma chains:V2 g2).
ABNORMAL
HEMOGLOBINS
There are four clinically important abnormal
hemoglobins: Hb S, Hb C, Hb D, and Hb E. These are present in different
hereditary hemoglobinopathies. The most commonly encountered hemoglobin is Hb S
which consists of a2 b2 but in the beta chain valine is substituted
for glutamic acid at the sixth position. Hb S is present in sickle cell anemia.
Unstable
hemoglobins are
hemoglobin variants that undergo denaturation and precipitate in the red cells
at Heinz bodies. Unstable hemoglobins are present in a type of congenital
nonspherocytic hemolytic anemia.
HEMOGLOBIN
COMPLEXES
Hb can combine with other substances besides
oxygen, some normally and some abnormally. Some of these commonly encountered
complexes are carbaminohemoglobin, carboxyhemoglobin, methemoglobin,
sulfhemoglobin, and cyanmethemoglobin.
Carboxyhemoglobin
When hemoglobins combine with carbon monoxide
(CO), carboxyhemoglobin is formed. Hemoglobin has a much greater affinity for
CO than for oxygen. Therefore, it readily combines with CO even when CO is
present in low concentrations. Fortunately the formation of carboxyhemoglobin
is reversible, so, once CO is removed from the blood, the hemoglobin combines
with oxygen. Carboxyhemoglobin is found in very low concentrations in normal
persons, but in smokers its concentration ranges from 1-10g/dl, which impairs
oxygen transport from lungs to tissues.
Methemoglobin
Methemoglobin is an abnormal Hb in which iron
is oxidized from its ferrous to ferric state. Therefore, it is incapable of
carrying oxygen. Normally it is present in low concentrations, but its
formation increases in the presence of certain chemicals or drugs. The
formation of methemoglobin is also reversible.
Sulfhemoglobin
This is an abnormal Hb complex formed by the
action of some drugs and chemicals such as sulfonamides. Once it is formed, it
is irreversible and remains in the carrier RBC. It is incapable of transporting
oxygen.
Cyanmethemoglobin
(hemoglobin-cyanide)
This is formed by the action of a chemical
called cyanide (for example. Potassium cyanide, KCN). The combination is
reversible. Hemiglobincyanide is the methemoglobin bonded to cyanide ions. Note: To measure accurately the total
Hb in the blood, it is essential to prepare a stable derivative that will
contain all the a Hb forms (complexes) that are present in the blood. All forms
of circulating hemoglobin are readily converted to hemoglobin-cyanide
(cyanmethemoglobin), except for sulfhemoglobin which is normally not present in
the blood. Therefore, the cyanmethemoglobin
method is the most accurate method for the determination of hemoglobin.
HEMOGLOBIN
DERIVATIES
When red blood cells are destroyed in the
tissue macrophage system, hemoglobin is degraded into heme and globin. Globin
returns to the body’s metabolic pool where its amino acids are subsequently
reutilised. The porphyrin ring of heme is cleaved by the microsomal enzyme,
heme oxidase, yielding biliverdin. by
biliverdin reductase.
NORMAL
VALUES
Adult males: 14-18(16± 2) g / dl of blood
Adult females: 12-16 (14 ± 2) g /dl of blood
In newborns, hemoglobin concentration is
normally 16-22 g/dl. It decreases to 9-14 g/dl by about two months of age. By
ten years of age, the normal hemoglobin concentration will be 12-14 g/dl. There
may be a slight decrease in hemoglobin level after 50 years of age.
FUNCTION
Hemoglobin serves two important functions.
1. It transports oxygen from the lungs to the
tissues by forming oxyhemoglobin, and carbon dioxide from the tissues to the
lungs by forming carbaminohemoglobin. When fully saturated 1 g of hemoglobin carries 1.34 ml of oxygen.
2.
Hemoglobin
acts as a buffer in maintaining blood pH.
SAHLI’S
ACID HEMATIN METHOD
Principle
Hemoglobin is converted to acid hematin by the action of HCL. The acid hematin solution is further diluted until its colour matches exactly with that of the permanent standard of the comparator block. The hemoglobin concentration is read directly from the calibration tube.
Hemoglobin is converted to acid hematin by the action of HCL. The acid hematin solution is further diluted until its colour matches exactly with that of the permanent standard of the comparator block. The hemoglobin concentration is read directly from the calibration tube.
Requirements
1. Sahli’s hemoglobinometer
It contains a comparator, hemoglobin tube,
hemoglobin pipette, and stirrer.
Comparator
At the middle there is a slot
which accommodates the hemoglobin tube. Non-fading standard brown tinted glass
pieces are provided on either side of the slot for colour matching. An opaque
white glass is fitted at the back to provide uniform illumination.
Hemoglobin
tube It is graduated on one side in gram per
cent (g%), from 2-24, and on the other side as percentage (%), from 20-140.
This tube is called as Sahli-Adams tube.
Hemoglobin
Pipette The
pipette bears only one mark indicating 20 cu mm (0.2ml). There is no bulb in
this pipette.
Stirrer It is thin glass rod used for stirring the
solution.
2. N / 10 HCl
3. Distilled water
4. Dropper
5. Materials for a sterile
finger prick
Procedure
- Clean the
hemoglobinometer tube and pipette and ensure that they are dry.
- Fill the
hemoglobinometer tube with N / 10 HCl up to its lowest mark (10 per cent
or 2 g%) with the help of a dropper.
- Prick the finger with
all aseptic precautions, and discard the first drop of blood Note: The prick should be deep
enough to give spontaneous flow of blood. Do not squeeze the finger to
make the drop of blood.
- Allow a large drop of
blood to form on the fingertip, and then dip and tip of the
hemoglobinometer pipette into the blood-drop and such blood up to 20 cu mm
mark of the pipette, Note:
While sucking blood into the pipette care should be taken to prevent entry
of air bubbles. This is done by not lifting the tip of pipetting. If an
air bubble enters, remove and discard the blood and make another drop of
blood to repipette. If blood is sucked about the 20 cu mm mark of the
pipette, bring down the blood column to the mark by tapping the pipette
against the finger, but not by using any absorbent material like cotton
wool.
- Wipe the tip of the
pipette. Immediately transfer the 0.02ml of blood from the pipette into
the hemoglobinometer tube containing N / 10 HCl by immersing tip of the
pipette in the acid solution and blowing out blood from the pipette. Rinse
the pipette two to three times by drawing up and blowing out the acid
solution. Withdraw the pipette from the tube. Note: Make sure that no solution remains in the pipette.
- Leave the solution in
the tube in the hemoglobinometer, for about ten minutes (for maximum
conversion of hemoglobin to acid hematin, which occurs in the first ten
minutes).
- After ten minutes,
dilute the acid hematin by adding distilled water drop by drop. Mix it
with the stirrer. Match the colour of the solution in the tube with the
standards of the comparator. Note:
After addition of every drop of distilled water, the solution should be
mixed and the colour of the solution should be compared with the standard.
While matching, take care to hold the stirrer above the level of the
solution. But, remember that at no stage should the stirrer by taken out
of the tube.
- If the colour of the
test solution is darker, then continue dilution till it matches with that
of the standard.
- Note. The reading when
the colour of the solution exactly matches with the standard and express
the hemoglobin content as g% Note:
The reading of the lower meniscus of the solution should be noted as the
result. One more drop of distilled water should be added and the colour
should be observed to check the result. The colour will be lighter than
the standard if the previous reading was accurate.
OTHER
METHODS
Gasometric
method
Gasometric method of estimation of hemoglobin
by using van Slyke apparatus is the most accurate method. But it is not used
routinely in clinical laboratories because it is time-consuming and the process
of estimation is complex. It is used as a reference method to obtain the
hemoglobin concentration in blood samples used for standardization of
hemoglobin estimation procedures. This is the preferred method for research.
Spectrophotometric
method
These methods are rapid and give accurate
results.
- Oxyhemoglobin method
Ammonium hydroxide (0.04ml /
dl) is used to hemolyse the red cells and convert the hemoglobin to
oxyhemoglobin for measurement in the spectrophotometer. This conversion is
complete and immediate and the resulting colour is stable.
- Cyanmethemoglobin method
Modified Drabkin’s reagent is
used in this method. Drabkin’s reagent contains sodium bicarbonate, potassium
ferricyanide, and potassium cyanide. This reagent takes at least ten minutes
for complete conversion of hemoglobin to cyanmethemoglobin. It also produces
turbid solutions caused by protein precipitation or incomplete hemolysis. In
modified Drabkin’s reagent, potassium phosphate is used for sodium bicarbonate,
which shortens the conversion time to three minutes, and minimizes turbidity
and enhances red cell lysis.
Automated
hemoglobinometry
Various automated techniques have been employed
to measure hemoglobin. Automatic pipettors and dilutors are used for pipetting
and diluting blood in many procedures. Hemoglobin estimation done by an
automated instrument applies the same principle as that described for the
manual methods.
Nonautomated
hemoglobinometry
Disposable, self-filling, self-measuring
diluting micropipettes are commercially available for the determination of
hemoglobin. One such system is the Unopette. These systems are easy to use and
are available with a series of different diluting fluids for different
purposes.
The Unopette
system for hemoglobin determination consists of a self-filling,
self-measuring pipette attached to a plastic holder. The pipette is filled with
the blood automatically by capillary action. A plastic container called a
reservoir is filled with modified Drabkin’s regent. The pipette containing
blood is inserted into the regent reservoir, emptied and rinsed according to
the manufacturer’s instruction. The blood is mixed well with the reagent and is
then ready to be read in the spectrophotometer.
Alkaline
hematin method
The alkaline hematin method is a useful
ancillary method under special circumstances as it gives a true estimate of
total hemoglobin including methemoglobin and sulfhemoglobin. A true solution is
obtained, and plasma proteins and lipids have little effect on the colour. The
principle is to convert hemoglobin into alkaline hematin, which is in the true
solution. There are two methods; the standard method, and the acid alkaline
method.
Specific
gravity method
This method uses the principle that when a drop
of whole blood is dropped into a solution of copper sulfate, which has a given
specific gravity, the drop will maintain its own density for approximately 15
seconds. The density of the drop is directly proportional to the amount of
hemoglobin in that drop. If that drop is denser than the specific gravity of
the solution, the drop will sink to the bottom; if not, it will float on the
surface. It is not a quantitative test. However, it is a quick, easy, and a
reasonably accurate technique to screen blood donors for possible anemia. It is
also used to detect hematocrit.
Comparator
method
This is a visual method similar to that of the
acid hematin method, except that the diluent used is an alkali solution
(ammonia solution 0.04 percent). After mixing with dilute ammonia solution, the
intensity of the colour of the hemolyzed solution of red blood cells is
compared against a standard colour disc in the comparator. This method has all
the disadvantage of Sahli’s acid hematin method.
Tallquist
method
This method involves direct disual matching of
the red colour of a drop of whole fresh blood on a filter paper with colour
standards on a paper. This technique is totally unsatisfactory with a high
degree of error, through it is one of the quickest methods.
Haldane
method
In this method, hemolysis of red cells is
produced by mixing blood with a hypotonic solution like distilled water. Carbon
monoxide is added to the mixture. The colour of the solution is compared with
the standard one.
Hemoglobinometry;
Is the
measurement of the concentration of hemoglobin in the blood
METHODS
of estimation of hemoglobin concentration
Hemoglobin can be estimated by different methods it can be
classified into following categories:
1.
Visual colour
comparisionmethod
2.
Gasometric
method
3.
Spectrophotometric
method
4.
Automated
hemoglobinometry
5.
Non-automated
hemoglobinometry
6.
Other
methods
A. Visual methods,
1.
Sahil’s
method
2.
Dares
method
3.
Hadens
method
4.
Wintrobes
method
5.
Haldanes
method
6.
Tallquists
method
B. Gasometric method
1.
van Slyke method
C. Spectrophotometric method
1.
Oxyhemoglobin
method
2.
Cyanmethemoblobin
method
D.electronic hematology analyszer
Automated hemoglobinometry
E. Non-automated hemoglobinometry
F. Other methods
1.
Alkaline-hematin
method
2.
Specific
gravity method
3.
comparator
method.
VISUAL
METHODS
These methods are more commonly used than
photometric methods. In Sahli’s method, hemoglobin in the blood sample is
converted to acid hematin, which gives brown colour. Since brown is more easily
matched by the human eye than red (the colour of Hb), Sahli’s method for
testing hemoglobin is one of the most acceptable visual methods. However, the
error in visual methods is higher. Therefore, visual methods (especially sahli’s)
are convenient and the cost of estimation is less, they are usually practiced
in hematology laboratories is clinical medicine and for performing practicals
are students in physiology.
INTRODUCTION
Hemoglobin (Hb) is a conjugated protein present
in red blood cells. It carries oxygen from the lungs to the tissues, and carbon
dioxide from the tissues to the lungs. It is made up of heme and globin. The
heme group is an iron complex, containing one iron atom. Iron is essential for
the primary function of the hemoglobin, the transport of oxygen. When reduced
hemoglobin is exposed to oxygen at increased pressure, oxygen is taken up at
the iron atom until each molecule of hemolecule at each iron atom. The Hb
molecule when fully saturated with oxygen, that is, four oxygen molecules
combined with one hemoglobin molecule, is called oxy-hemoglobin. One gram of
hemoglobin carries 1.34 ml of oxygen. Hemoglobin returning with carbon dioxide
from the tissues is called reduced hemoglobin.
TYPE
OF HEMOGLOBIN
Hemoglobins can be broadly divided into normal
and abnormal types.
Normal Hb: Adult Hb, Fetal Hb and Embryonic Hb.
Abnormal Hb: Hb S, Hb C, Hb D, Hb E and Unstable hemoglobins.
NORMAL
HEMOGLOBINS
Adult
hemoglobins
Hemoglobin A (Hb A): About 97 per cent of hemoglobin
of adult red cells is Hb A. It consists of two alpha (a) and two beta (b) chains with the structural formala a2 b2. Hb A is detected in small amounts in the fetus
as early as the eighth week of intrauterine life. During the first few months
of postnatal Life, Hb A almost completely replaces Hb F and the adult pattern
is fully established in six months.
Hemoglobin A2 (Hb A2):
This is the minor hemoglobin in the adult red cells. It has the structural
formal of a2 d2 . Hb A2 is present in very small
amounts at birth and reaches the adult level of 3 per cent during the first
year of life. Its concentration increases in some types of anemia.
Fetal
Hemoglobins
Fetal hemoglobin (Hb F): Hb F is the major
hemoglobin in intrauterine life. It has the structural formala of a2 g2 . Hb F accounts for 70-90 per cent of
hemoglobin at term. It then falls rapidly to 25 per cent in one month, and 5
percent in six months. The adult level of 1 per cent is not reached in some
children until puberty Hb F concentration in adults increases in some types of
anemia, hemoglobinopathies, and some time in leukemia.
Hemoglobin
Bart’s (Hb Barts): This is
the minor hemoglobin present in fetal life. It consists of four gamma (g) chains g4 . Hb Bart’s concentration increases in fetal
life in thalassemia.
Embryonic
hemoglobins
These hemoglobins are confined to the very
early stage (the embryonic stage) of development. There are three embryonic
hemoglobins: 1)Hb Gower 1 (consisting of two zeta and two epsilon chains: V2 e2 ), 2) Hb Gower 2 (consisting of two alpha and
two epsilon chains: a2 e2 ) and 3) Hb Portland (consisting of two zeta
and two gamma chains:V2 g2).
ABNORMAL
HEMOGLOBINS
There are four clinically important abnormal
hemoglobins: Hb S, Hb C, Hb D, and Hb E. These are present in different
hereditary hemoglobinopathies. The most commonly encountered hemoglobin is Hb S
which consists of a2 b2 but in the beta chain valine is substituted
for glutamic acid at the sixth position. Hb S is present in sickle cell anemia.
Unstable
hemoglobins are
hemoglobin variants that undergo denaturation and precipitate in the red cells
at Heinz bodies. Unstable hemoglobins are present in a type of congenital
nonspherocytic hemolytic anemia.
HEMOGLOBIN
COMPLEXES
Hb can combine with other substances besides
oxygen, some normally and some abnormally. Some of these commonly encountered
complexes are carbaminohemoglobin, carboxyhemoglobin, methemoglobin,
sulfhemoglobin, and cyanmethemoglobin.
Carboxyhemoglobin
When hemoglobins combine with carbon monoxide
(CO), carboxyhemoglobin is formed. Hemoglobin has a much greater affinity for
CO than for oxygen. Therefore, it readily combines with CO even when CO is
present in low concentrations. Fortunately the formation of carboxyhemoglobin
is reversible, so, once CO is removed from the blood, the hemoglobin combines
with oxygen. Carboxyhemoglobin is found in very low concentrations in normal
persons, but in smokers its concentration ranges from 1-10g/dl, which impairs
oxygen transport from lungs to tissues.
Methemoglobin
Methemoglobin is an abnormal Hb in which iron
is oxidized from its ferrous to ferric state. Therefore, it is incapable of
carrying oxygen. Normally it is present in low concentrations, but its
formation increases in the presence of certain chemicals or drugs. The
formation of methemoglobin is also reversible.
Sulfhemoglobin
This is an abnormal Hb complex formed by the
action of some drugs and chemicals such as sulfonamides. Once it is formed, it
is irreversible and remains in the carrier RBC. It is incapable of transporting
oxygen.
Cyanmethemoglobin
(hemoglobin-cyanide)
This is formed by the action of a chemical
called cyanide (for example. Potassium cyanide, KCN). The combination is
reversible. Hemiglobincyanide is the methemoglobin bonded to cyanide ions. Note: To measure accurately the total
Hb in the blood, it is essential to prepare a stable derivative that will
contain all the a Hb forms (complexes) that are present in the blood. All forms
of circulating hemoglobin are readily converted to hemoglobin-cyanide
(cyanmethemoglobin), except for sulfhemoglobin which is normally not present in
the blood. Therefore, the cyanmethemoglobin
method is the most accurate method for the determination of hemoglobin.
HEMOGLOBIN
DERIVATIES
When red blood cells are destroyed in the
tissue macrophage system, hemoglobin is degraded into heme and globin. Globin
returns to the body’s metabolic pool where its amino acids are subsequently
reutilised. The porphyrin ring of heme is cleaved by the microsomal enzyme,
heme oxidase, yielding biliverdin. by
biliverdin reductase.
NORMAL
VALUES
Adult males: 14-18(16± 2) g / dl of blood
Adult females: 12-16 (14 ± 2) g /dl of blood
In newborns, hemoglobin concentration is
normally 16-22 g/dl. It decreases to 9-14 g/dl by about two months of age. By
ten years of age, the normal hemoglobin concentration will be 12-14 g/dl. There
may be a slight decrease in hemoglobin level after 50 years of age.
FUNCTION
Hemoglobin serves two important functions.
1. It transports oxygen from the lungs to the
tissues by forming oxyhemoglobin, and carbon dioxide from the tissues to the
lungs by forming carbaminohemoglobin. When fully saturated 1 g of hemoglobin carries 1.34 ml of oxygen.
2.
Hemoglobin
acts as a buffer in maintaining blood pH.
SAHLI’S
ACID HEMATIN METHOD
Principle
Hemoglobin is converted to acid hematin by the action of HCL. The acid hematin solution is further diluted until its colour matches exactly with that of the permanent standard of the comparator block. The hemoglobin concentration is read directly from the calibration tube.
Hemoglobin is converted to acid hematin by the action of HCL. The acid hematin solution is further diluted until its colour matches exactly with that of the permanent standard of the comparator block. The hemoglobin concentration is read directly from the calibration tube.
Requirements
1. Sahli’s hemoglobinometer
It contains a comparator, hemoglobin tube,
hemoglobin pipette, and stirrer.
Comparator
At the middle there is a slot
which accommodates the hemoglobin tube. Non-fading standard brown tinted glass
pieces are provided on either side of the slot for colour matching. An opaque
white glass is fitted at the back to provide uniform illumination.
Hemoglobin
tube It is graduated on one side in gram per
cent (g%), from 2-24, and on the other side as percentage (%), from 20-140.
This tube is called as Sahli-Adams tube.
Hemoglobin
Pipette The
pipette bears only one mark indicating 20 cu mm (0.2ml). There is no bulb in
this pipette.
Stirrer It is thin glass rod used for stirring the
solution.
2. N / 10 HCl
3. Distilled water
4. Dropper
5. Materials for a sterile
finger prick
Procedure
- Clean the
hemoglobinometer tube and pipette and ensure that they are dry.
- Fill the
hemoglobinometer tube with N / 10 HCl up to its lowest mark (10 per cent
or 2 g%) with the help of a dropper.
- Prick the finger with
all aseptic precautions, and discard the first drop of blood Note: The prick should be deep
enough to give spontaneous flow of blood. Do not squeeze the finger to
make the drop of blood.
- Allow a large drop of
blood to form on the fingertip, and then dip and tip of the
hemoglobinometer pipette into the blood-drop and such blood up to 20 cu mm
mark of the pipette, Note:
While sucking blood into the pipette care should be taken to prevent entry
of air bubbles. This is done by not lifting the tip of pipetting. If an
air bubble enters, remove and discard the blood and make another drop of
blood to repipette. If blood is sucked about the 20 cu mm mark of the
pipette, bring down the blood column to the mark by tapping the pipette
against the finger, but not by using any absorbent material like cotton
wool.
- Wipe the tip of the
pipette. Immediately transfer the 0.02ml of blood from the pipette into
the hemoglobinometer tube containing N / 10 HCl by immersing tip of the
pipette in the acid solution and blowing out blood from the pipette. Rinse
the pipette two to three times by drawing up and blowing out the acid
solution. Withdraw the pipette from the tube. Note: Make sure that no solution remains in the pipette.
- Leave the solution in
the tube in the hemoglobinometer, for about ten minutes (for maximum
conversion of hemoglobin to acid hematin, which occurs in the first ten
minutes).
- After ten minutes,
dilute the acid hematin by adding distilled water drop by drop. Mix it
with the stirrer. Match the colour of the solution in the tube with the
standards of the comparator. Note:
After addition of every drop of distilled water, the solution should be
mixed and the colour of the solution should be compared with the standard.
While matching, take care to hold the stirrer above the level of the
solution. But, remember that at no stage should the stirrer by taken out
of the tube.
- If the colour of the
test solution is darker, then continue dilution till it matches with that
of the standard.
- Note. The reading when
the colour of the solution exactly matches with the standard and express
the hemoglobin content as g% Note:
The reading of the lower meniscus of the solution should be noted as the
result. One more drop of distilled water should be added and the colour
should be observed to check the result. The colour will be lighter than
the standard if the previous reading was accurate.
OTHER
METHODS
Gasometric
method
Gasometric method of estimation of hemoglobin
by using van Slyke apparatus is the most accurate method. But it is not used
routinely in clinical laboratories because it is time-consuming and the process
of estimation is complex. It is used as a reference method to obtain the
hemoglobin concentration in blood samples used for standardization of
hemoglobin estimation procedures. This is the preferred method for research.
Spectrophotometric
method
These methods are rapid and give accurate
results.
- Oxyhemoglobin method
Ammonium hydroxide (0.04ml /
dl) is used to hemolyse the red cells and convert the hemoglobin to
oxyhemoglobin for measurement in the spectrophotometer. This conversion is
complete and immediate and the resulting colour is stable.
- Cyanmethemoglobin method
Modified Drabkin’s reagent is
used in this method. Drabkin’s reagent contains sodium bicarbonate, potassium
ferricyanide, and potassium cyanide. This reagent takes at least ten minutes
for complete conversion of hemoglobin to cyanmethemoglobin. It also produces
turbid solutions caused by protein precipitation or incomplete hemolysis. In
modified Drabkin’s reagent, potassium phosphate is used for sodium bicarbonate,
which shortens the conversion time to three minutes, and minimizes turbidity
and enhances red cell lysis.
Automated
hemoglobinometry
Various automated techniques have been employed
to measure hemoglobin. Automatic pipettors and dilutors are used for pipetting
and diluting blood in many procedures. Hemoglobin estimation done by an
automated instrument applies the same principle as that described for the
manual methods.
Nonautomated
hemoglobinometry
Disposable, self-filling, self-measuring
diluting micropipettes are commercially available for the determination of
hemoglobin. One such system is the Unopette. These systems are easy to use and
are available with a series of different diluting fluids for different
purposes.
The Unopette
system for hemoglobin determination consists of a self-filling,
self-measuring pipette attached to a plastic holder. The pipette is filled with
the blood automatically by capillary action. A plastic container called a
reservoir is filled with modified Drabkin’s regent. The pipette containing
blood is inserted into the regent reservoir, emptied and rinsed according to
the manufacturer’s instruction. The blood is mixed well with the reagent and is
then ready to be read in the spectrophotometer.
Alkaline
hematin method
The alkaline hematin method is a useful
ancillary method under special circumstances as it gives a true estimate of
total hemoglobin including methemoglobin and sulfhemoglobin. A true solution is
obtained, and plasma proteins and lipids have little effect on the colour. The
principle is to convert hemoglobin into alkaline hematin, which is in the true
solution. There are two methods; the standard method, and the acid alkaline
method.
Specific
gravity method
This method uses the principle that when a drop
of whole blood is dropped into a solution of copper sulfate, which has a given
specific gravity, the drop will maintain its own density for approximately 15
seconds. The density of the drop is directly proportional to the amount of
hemoglobin in that drop. If that drop is denser than the specific gravity of
the solution, the drop will sink to the bottom; if not, it will float on the
surface. It is not a quantitative test. However, it is a quick, easy, and a
reasonably accurate technique to screen blood donors for possible anemia. It is
also used to detect hematocrit.
Comparator
method
This is a visual method similar to that of the
acid hematin method, except that the diluent used is an alkali solution
(ammonia solution 0.04 percent). After mixing with dilute ammonia solution, the
intensity of the colour of the hemolyzed solution of red blood cells is
compared against a standard colour disc in the comparator. This method has all
the disadvantage of Sahli’s acid hematin method.
Tallquist
method
This method involves direct disual matching of
the red colour of a drop of whole fresh blood on a filter paper with colour
standards on a paper. This technique is totally unsatisfactory with a high
degree of error, through it is one of the quickest methods.
Haldane
method
In this method, hemolysis of red cells is
produced by mixing blood with a hypotonic solution like distilled water. Carbon
monoxide is added to the mixture. The colour of the solution is compared with
the standard one.
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