Complete steps for Haemoglobin estimation using Drabkin's Method

What is Haemoglobin estimation?

Haemoglobin estimation is a laboratory procedure that involves measuring the amount of hemoglobin in blood. Haemoglobin estimation is intended for:

Screening for anemia
Confirming or monitoring anemia or polycythemia

Structure of Haemoglobin and its function

Haemoglobin is an iron containing protein molecule found in red blood cells which transports oxygen from the lungs to the body’s tissues. The haemoglobin molecule is roughly spherical and is made up of two pairs of dissimilar subunits. Four polypeptide chains (globins), each wrapped in a specific way around its own heme group, make up the haemoglobin molecule. A heme group consists of an iron atom in the ferrous state (Fe 2+) and a porphyrin ring.

The oxygen-binding site of haemoglobin is the heme pocket available in each of the four polypeptide chains; a single atom of oxygen forms a reversible bond with the ferrous iron at each of these sites, therefore a molecule of haemoglobin binds four oxygen molecules; the end result is oxyhemoglobin (O2Hb).

The oxygen delivery function of Hb, that is its ability to “pick up” oxygen at the lungs and “release” it to tissue cells is made possible by minute conformational changes in quaternary structure that occur in the hemoglobin molecule and which alter the affinity of the heme pocket for oxygen. haemoglobin has two quaternary structural states: the deoxy state (low oxygen affinity) and the oxy state (high oxygen affinity).

A range of environmental factors determine the quaternary state of haemoglobin and so its relative oxygen affinity. The microenvironment in the lungs favors the oxy-quaternary state, and thus haemoglobin has high affinity for oxygen here.

A small amount (20 %) of CO2 is transported from the tissues to the lungs loosely bound to the N-terminal amino acid of the four globin polypeptide units of hemoglobin; the end product of this combination is carbaminohemoglobin. Though, most CO2 is transported as bicarbonate in blood plasma.

The erythrocyte conversion of CO2 to bicarbonate, necessary for this mode of CO2 transport, results in the production of hydrogen ions (H+). These hydrogen ions are buffered by deoxygenated hemoglobin.

By difference, the microenvironment of the tissues induces the conformational change in Hb structure that reduces its affinity for oxygen, thus allowing oxygen to be released to tissue cells.

In capillary blood flowing through the tissues oxygen is unleashed from hemoglobin and goes into tissue cells. Carbon dioxide diffuses out of tissue cells into red blood cells, where the red-cell enzyme carbonic anhydrase enables its reaction with water to form carbonic acid.

The carbonic acid breaks down to bicarbonate (which goes into the blood plasma) and hydrogen ions, which complexes with the now deoxygenated hemoglobin. The blood flows to the lungs, and in the capillaries of the lung alveoli the above pathways are reversed. Bicarbonate enters red blood cells and here combine with hydrogen ions, released from hemoglobin, to form carbonic acid.

This breaks down to carbon dioxide and water. The carbon dioxide diffuses from the blood into the alveoli of the lungs and is eliminated in expired air. As oxygen diffuses from the alveoli to capillary blood and complexes with hemoglobin.

Types of Haemoglobin

Three kinds of normal haemoglobin molecules exist:

Haemoglobin A (α2 β2),
Haemoglobin A2 (α2 δ2
Hemoglobin F (α2 γ2).

The haemoglobins present in healthy adults are:

Hb A (96-98 %),
Hb A2 (1.5- 3.2%) and
Hb F (0.5 -0.8%).

In fetuses, the major Hb is F. It is possible to use manual, semi-automated, or automated techniques to determine haemoglobin concentration and other blood components. Manual techniques are generally low cost with regard to equipment and reagents but are labor intensive.

Although usually available in only trace amounts, there are three species of hemoglobin which cannot bind oxygen These include:

Methemoglobin (MetHb or Hi),
Sulfhemoglobin (SHb)
Carboxyhemoglobin (COHb)

They are thus functionally deficient, and increased amounts of any of these hemoglobin species, usually the result of exposure to specific drugs or environmental toxins, can seriously compromise oxygen delivery.

Automated techniques entail high capital costs but permit rapid performance of a large number of tests by a smaller number of laboratory workers. Automated techniques are more precise, but their accuracy depends on correct calibration and the use of reagents that are usually specific for the particular analyzer. Many laboratories now use automated techniques, certain manual techniques are necessary as reference for standardization of the methods.

Measurement of Haemoglobin (Hb) concentration or Hemoglobin estimation in a whole blood sample is a basic screening test for anemia and polycythemia. The haemoglobin concentration of the solution may be estimated by measurement of it color, by its power of combining with oxygen or carbon monoxide, or by its iron content.

The methods mostly used are color or light-intensity matching techniques. Ideally, for assessing clinical anemia, a functional estimation of haemoglobin should be carried out by measurement of oxygen capacity, but this is hardly practical in the routine haematology laboratory.

It gives results that are at least 2% lower than those given by the other methods. The iron content of haemoglobin can be estimated accurately, but again the method is impractical for routine use. Estimations based on iron content are generally taken as authentic, iron bound to inactive pigment is included.

Haemoglobin estimation using Drabkin’s Method

The haemiglobincyanide (Cyanmethaemoglobin) method is the internationally recommended method for determining the haemoglobin concentration of blood.

Principle estimation using Drabkin’s Method

Blood is diluted in Drabkin’s solution, a solution containing potassium cyanide and potassium ferricyanide. Potassium ferricyanide oxidizes hemoglobin to methaemoglobin. Methaemoglobin then combines with potassium cyanide to form Cyanmethaemoglobin (HiCN). The absorbance of the solution is measured in a spectrophotometer at a wave length of 540 nm against Drabkin’s solution as a blank. The result is calculated using the formula provided below and it is expressed in gm/dl.

Sample required for Haemoglobin estimation

Venous or capillary blood collected into an EDTA tube. Alternatively, free flowing capillary blood may be added directly to the diluting fluid and measured

Procedure for Haemoglobin estimation using Drabkin’s Method

Pipette 4ml of Drabkin’s solution into a test tube.
Pipette 20 μl (0.02 ml) of properly mixed blood.
Clean outside of pipette and wash out the blood in the tube containing the diluent.
Mix and leave for 5-10 minutes for the reaction to complete.
Using drabkin’s solution as a blank, read the absorbance in the spectrophotometer at wavelength 540 nm.

Calculation:

Use the following formula:

𝐴×64500×𝐷𝐼𝐿
Hb in g/dl = ————————- = 𝐴 × 29.3
44×𝐷×1000×10

Where:

A = Reading of absorbance of Hb solution
64500 = Molecular wt. of Hb
44 = Millimolar extinction coefficient
D = Thickness of cuvette
1000 = Conversion factor of mg to gm
DIL = Dilution Factor = 200