Sunday, March 3, 2013

ESTIMATION OF HEMOGLOBIN PERCENTAGE


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.

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
  1. Clean the hemoglobinometer tube and pipette and ensure that they are dry.
  2. 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.
  3. 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.
  4. 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.
  5. 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.
  6. 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).
  7. 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.
  8. If the colour of the test solution is darker, then continue dilution till it matches with that of the standard.
  9. 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.

    1. 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.

    1. 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.



































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.

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
  1. Clean the hemoglobinometer tube and pipette and ensure that they are dry.
  2. 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.
  3. 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.
  4. 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.
  5. 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.
  6. 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).
  7. 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.
  8. If the colour of the test solution is darker, then continue dilution till it matches with that of the standard.
  9. 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.

    1. 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.

    1. 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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