Hemoglobin is the main component of red cell cytoplasm, accounting for about 90 percent of the dry weight of the mature cell. It consists of heme and globin
Understanding the structure of Hemoglobin
Hemoglobin molecule is a tetramer consisting of two pairs of polypeptide similar chains called globin chains. Heme, an iron complex in ferrous form and protoporphyrin, is attached to each of the four chains.The main( 96%) type of adult hemoglobin is called HbA and has 2 alpha globin chains and 2 beta globin chains( α2β2).The gene that codes α globin chain formation is located on chromosome 16 and the gene that codes β globin chain formation is on chromosome 11.In adults, a small amount of HbA2( α2β2) is also present and accounts for less than 3.5%.
Other types of hemoglobins prevail during embryonic and fetal life.These include Gower-1( formerly embryonic), Gower-2 and Hb Portland.An embryonic hemoglobin is replaced by Fetal hemoglobin( α2β2) after the eighth week of development.This remains the predominant hemoglobin until after birth and accounts for 50- 90% of total hemoglobin.After birth, the concentration decreases to less than 2 percent by age 30 weeks.HbA then is the predominant hemoglobin.
|Adult Hb (HbA)||α2β2||Adults|
|Fetal Hb (HbF)||α2γ2||Fetal life|
|Hb Portland||ζ2γ2||Embryonic life|
Functions of Hemoglobin
- Heme can bind oxygen reversibly and carry it to tissues.
- It also facilitates the exchange of carbon dioxide between lungs and tissues. Thus hemoglobin works as the primary medium of oxygen and carbon dioxide exchange.
Measurement or Estimation of Hemoglobin
The following are various methods for estimating hemoglobin in the laboratory.
- Based on color development.
- Sahli’s or acid hematin method
- Cyanmethemoglobin method
- Oxyhemoglobin method
- Alkaline hematin method
2. Based on the measurement of oxygen combining capacity
3. Based on Measurement of iron content
- BASED ON COLOR DEVELOPMENT
The methods commonly used are the Sahli / Hematin acid method and the Cyanmethemoglobin method.
- Sahli’s/acid hematin Method
Principle of Sahli’s acid hematin method
Blood is mixed with N/10 HCl which results in the conversion of Hb to acid hematin brown in color. The solution is diluted until the color matches the brown glass of the comparator box. The Hb concentration is read directly.
Equipment required for Sahli’s acid hematin method
Hemocytometer which consists of
- comparator box which has brown colored glass on either side
- Hb pipette which is marked up to 20 mm 3(0.02ml blood)
- Tube with markings of Hb on one side
- glass rod
Reagents required for Sahli’s acid hematin method
- N/10 HCl
- Distilled water
- Sample (Venous blood collected in EDTA)
Sahli’s acid hematin method procedure
- Start by adding N/10 HCl into the tube upto mark 2g%
- Mix the blood EDTA sample by gentle inversion and fill the pipette with 0.02ml blood. Wipe the external surface of the pipette to remove any excess blood.
- Add the blood into the tube containing HCl. Wash out the contents of the pipette by drawing in and blowing out the acid two to three times. Mix the blood with the acid thoroughly.
- Allow to stand undisturbed for 10min.
- Place the hemoglobinometer tube in the comparator and add distilled water to the solution drop by drop stirring with the glass rod till it’s color matches with that of the comparator glass. While matching the color, the glass rod must be removed from the solution and held vertically in the tube.
- Remove the stirrer and take the reading directly by noting the height of the diluted acid hematin and express in g%. which is the hemoglobin test results
Advantages and Disadvantages of Sahli’s acid hematin method
|Easy to perform||Less accurate|
|Quick||All hemoglobins (oxyhemoglobin, sulfhemoglobin) are not converted to acid hematin and hence the value of Hb obtained is less than the actual value.|
|Inexpensive||The color of acid hematin develops slowly.|
|Can be used as a bedside procedure||Color of acid hematin fades with time and dilution must be done exactly after 10 min when the color development is maximum|
|Does not require technical expertise||Individual variation in matching of color is seen|
|XX||If the matching point is passed, the whole procedure has to be repeated. z Color of glass in the comparator box tends to fade with time|
|XX||Color of glass in the comparator box tends to fade with time|
|XX||Lack of a true standard.|
B. Cyanmethemoglobin method
This is the internationally recommended hemoglobin determination method
Principle of Cyanmethemoglobin method
Blood is diluted in a solution containing cyanide potassium and ferricyanide potassium.The latter converts Hb to methemoglobin converted by potassium cyanide to cyanmethemoglobin( HiCN).The solution absorbance is measured in a spectrophotometer at 540 nm wavelength or in a colorimeter using a yellow green filter.
Equipments and reagents required for Cyanmethemoglobin method
- Hb pipette
- Spectrophotometer or hemoglobin colorimetric
- Sample used is Venous blood collected in EDTA
- Drabkin’s solution of pH7.0-7.4 which contains the following
|Potassium cyanide||50 mg|
|Potassium ferricyanide||200 mg|
|Nonionic detergent||1 m|
|Potassium dihydrogen phosphate||140 mg|
|Distilled water||1 L|
Some important notice to take note about Drabkin’s solution
- The solution should be pale yellow in color.
- When measured in a spectrometer against water blank at a wavelength of 540 nm, the absorbance must be zero.
- The solution is unstable when exposed to light and can be stored in brown borosilicate bottles at room temperature for several months.
- If room temperature is higher than 30 ° C, the solution should be stored in a refrigerator, but must be brought to room temperature before being used
- Also The solution should never be frozen.
- The solution’s pH must be checked each month.
- Discard the solution if it is found to be turbid / if pH is out of range / it is not zero absorbance at 540 nm.
- Do not mouth pipette Drabkin mouth solution.
Cyanmethemoglobin method procedure
- Take 5ml of Drabkin’s solution in a test tube.
- Mix the blood sample by gentle inversion and draw 0.02ml of blood into the Hb pipette. Wipe the outer surface of the pipette to remove excess blood.
- Place the pipette into the tube containing Drabkin’s solution and slowly expel the blood into the solution. Mix well and let it stand undisturbed for 5min.
- Measure the absorbance of this solution at 540 nm in a spectrophotometer after adjusting the OD at 0 by using Drabkin’s solution as blank.
- Calculate the hemoglobin concentration using a standard curve.
Advantages and Disadvantages of Cyanmethemoglobin method
|All forms of Hb except sulfhemoglobin are converted to HiCN.||Diluted blood has to stand for a period of time to ensure complete conversion of Hb.Abnormal plasma proteins cause turbidity when blood is diluted with Drabkin’s solution.|
|Visual error is not there as no color matching is required.||Potassium cyanide is a poisonous substance and that is why Drabkin’s solution must never be pipetted by mouth.|
|Cyanmethemoglobin solution is stable and it’s color does not fade with time so readings may not be taken immediately.||The rate of conversion of blood containing carboxyhemoglobin is slowed considerably. Prolonging the reaction time to 30min can overcome this problem.|
|Absorbance may be measured soon after dilution.||A high leukocyte count also causes turbidity on dilution of blood. Centrifuging the diluted blood can help overcome the turbidity.|
|A reliable and stable reference standard is available from World Health Organisation for direct comparison.||XXX|
Preparing a Standard curve for Hemoglobin estimation by Cyanmethemoglobin method
In a laboratory that tests several samples a day, it is more convenient to prepare a standard Hb curve.
An international HiCN standard of the WHO is available as sealed 10 ml ampoules on the market. This solution is stable for years. The exact concentration of Hb in the solution is indicated on the label.
How to prepare an HICN standard curve
- Make serial dilutions of the reference solution with Drabkin’s solution eg 1 in 2, 1 in 4, 1 in 8 and so on.The Hb concentration of each dilution is also known as the Hb present in the solution.
- Take the OD of each colorimeter dilution against a blank of Drabkin’s solution.
- Plot the OD on a linear graph paper against the Hb concentration.The absorbance is concentrated on the vertical axis and the hemoglobin concentration on the horizontal axis. The points should lie in a straight line passing through the origin.
- A reading table and the corresponding Hb value can be prepared from this graph. This is more convenient than the graph when a large number of readings are required. The corresponding Hb value can be read directly from the table after the sample OD has been taken.
C. OXYHEMOGLOBIN METHOD
In this method,Hb is converted with ammonia to oxyhemoglobin and the color of the solution is measured in a photocolorimeter.
The benefits of this method are that it is simple, fast and reliability is not affected by increased bilirubin levels.
However, oxyhemoglobin solution fades very quickly. The method does not give satisfactory results in carboxyhemoglobin, methemoglobin, and sulfhemoglobin
D. ALKALINE HEMATIN METHOD
In this method,Blood is converted to alkaline hematin by adding alkali such as sodium hydroxide and the color measured in a 540 nm colorimeter.This method gives a real Hb estimate and is not affected by plasma proteins and lipids.However,It is not routinely used because it is less accurate than the cyanmethemoglobin method and certain forms of Hb such as HbF are resistant to alkali denaturation.
2. MEASUREMENT OF HEMOGLOBIN OXYGEN CARRYING CAPACITY
The oxygen combining blood capacity is 1,34 ml oxygen per gm Hb. This is a measure of the Hb function. It is not routinely used because it is not practical and results are 2 percent lower than other methods.
3. MEASUREMENT OF IRON CONTENT OF HEMOGLOBIN
The iron content can be measured and converted to Hb using the 0.347 mg iron 100 g Hb formula. However, it is not practical for routine use.
5. USING HEMOGLOBINOMETER
Hemoglobin Meters are available based on the cyanmethemoglobin / oxyhemoglobin method.It is a hemoglobin lab test instrument and hemoglobin test meter home use. The instrument has a filter and a scale built and provides direct reading of Hb in g / dl. They are used for field surveys. It is essential that the instrument is often calibrated.
Normal values of Hb (normal haemoglobin level in male and female hb range)
|Males||15 ± 2 g/dl|
|Females||13.5 ± 1.5 g/dl|
WHO Anemia HB Values (hemoglobin anemia range)
Clinical significance of Hb measurement
- Hemoglobin estimate is used as an anemia screening test.This is a frequent abnormality in our population.Anemia is not a diagnosis by itself, and when detected, the underlying cause must be detected.Therefore, accurate Hb estimates (normal haemoglobin level in blood) are essential for further testing to determine the cause and the patient being treated accordingly.