Estimation of Haemoglobin, Sahli's method, Cyanmethaemoglobin Method, Calculations, Normal Values

 

Estimation of Haemoglobin: -

The purpose of haemoglobin is to determine the Oxygen carrying capacity of blood. The results of haemoglobin help in detecting disease which cause deficiency in or excess of haemoglobin. Haemoglobin estimation helps to detect the changes in the haemoglobin concentration before and after operation and blood transfusion.

Several Methods are used to determine the haemoglobin in the forms of Sahli’s method, Cyanmethaemoglobin method, Carboxyhaemoglobin method based on their Oxygen carrying capacity of blood.

Most common methods are used in laboratories Sahli's method and Cyanmethaemoglobin method. But the problem in accurate measurement of Hb content  is the non availability of suitable standard. Like as Sahli’s  method use artificial standard.

In 1966  the International Committee for Standardization in Haematology (ICSH) recommended Cyanmethaemoglobin, this prepared according to specification and as a suitable standard. Cyanmethaemoglobin is very stable and standard is available also. Some visual method  such as Sahli’s  method use artificial standard.

click here to watch:-  What is Haemoglobin (Hb) 

click here to watch :-    Forms of haemoglobin

click here to watch :-  Synthesis of Haemoglobin

click here to watch  :- Degradation and complexes of Haemoglobin

 1.    Acid Haematin Method (Sahli’s):-

This is the visual method of Haemoglobin estimation.  It is not accurate method.  In this method use artificial standard that is not very accurate. These methods are rare used now  a days.  These methods are now used only in the areas of the world, where electricity is not available.

Principle:-

·        Haemoglobin is converted in to acid haematin by the action of hydrochloric acid.

·        The brown colour of the compound is matched visually against a Brown glass standard in a comparator.

Specimen:-

Capillary blood, Venous blood collected in EDTA or Oxalate anticoagulant.

 

Haemoglobin Apparatus:-

·        Sahli’s graduate  tube and haemoglobinometer consisting of the comparator with the glass standards.

·        One side of the tube gives the reading in the grans of/100ml of blood.

·        The other side of the tube gives the reading as percentage.

 

Reagent:-

1.        0.1 N Hydrochloric acid.

2.        Haemoglobin pipette marked at 20cu.mm (microlitre) or 0.02ml.

 

Technique:- 

·        Fill the graduated tube mark up to 20 with 0.1n Hydrochloric acid.

·        Fill Hb pipette up to the 20 cu.mm , hold the pipette horizontal when sucking blood to fill the pipette.

·        Wipe off the blood on the outside of pipette with a piece of gauze.

·        Empty the pipette in to the acid present in the tube, avoid bubbles.

·        Rinse the pipette 2-3 times by drawing in and discharging the fluid.

·        Mix the acid haematin solution in the tube.

·        Stand it for 5-10 minutes in sahli comparator.

·        After 10 minutes start adding 0.1N HCL drop by drop.

·        Mix well until the colour matches that of the standard.

·        Read the volume of the solution in the graduated tube, and express the reading as gm/dl or as percentage.

 

2.       Cyanmethaemoglobin Method:-

This is the colorimetric method. This method is more accurate than Sahli's method.

 

Principle:-

The haemoglobin is treated with the reagent containing  Potassium ferricyanide, potassium cyanide and potassium dihydrogen phosphate.

The ferricyanide forms methaemoglobin which is converted to Cyanmethaemoglobin by the cyanide.

 

Equipments:-

·        Test tube

·        Drabkin’s Solution

·        Pipette (0-20 microlitre)

·        Gauze piece

·        Colorimeter.

 

Reagents:-

1.        Drabkin’s solution:-

                                      Potassium cyanide  -             500mg

                                      Potassium ferricyanide         200mg

                                       Potassium  dihydrogen         140mg

                                                              Phosphate

                                      Distilled water                          1 litre

                                       pH                                              7.0-7.4

 

This solution store in dark bottle. Keeps for several months.

 

2.          Cyanmethaemoglobin standard:-

The concentration is indicated on the label and is usually about 60mg/100ml.

 

Technique:-

 

·        Take 5ml Drabkin’s solution in a glass tube.

·        Add 0.02ml (20microlitre) of blood in the solution.

·        Mix well and allow it to stand for 10 minutes.

·        Red the absorbance in the colorimeter at 540 nm. (nanometers) on green filter with the drabkin’s solution as blank.

·        Read the absorbance of the standard in same way.

 

Calculations:-

Haemoglobin in gm/dl  = 

              Reading of test / Reading of standard  x dilution factor x                      

                                                                 concentration of Standard/1000

 

Normal Values of haemoglobin:-

 

Male      -      13.0 – 18.0 gm/dl

Female   -     12.0 – 16.0 gm/dl

Children -    At Birth      –    13.0 – 24.0  gm/dl

                     <1 month    –   10.0 - 20.0   gm/dl

                      1-2 months  -   10.0 - 18.0   gm/dl

                     2-5 months   –   9.0 - 14.0    gm/dl

                     5-12 months –   10.5- 13.5   gm/dl

                     1 - 5 years     -   11.0 - 14.0  gm/dl

                      5-12 years   -    11.5 – 14.5 gm/dl

                     12- 18 years   -  14.0 - 15.0 gm/dl

Click here to watch :- What is haematology

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