HAEMOGLOBIN ESTIMATION

 A simple but rather inaccurate method which estimates oxyhaemoglobin and reduced heamoglobi

Place N/10 Hcl up to the mark 20 in the graduated tube. Add 20mm of blood (0.02 ml) with the help of haemoglobinometer pipette. Rinse the pipette 2-3 times mix well and allow to stand for 5 min till the solution becomes dark brownish in color .The solution is diluted drop by drop each time mixing the solution  with a stirring rod until it matches the standard. Read the result from the scale on the graduated tube by observing graduation mark at the lower edge of the meniscus at the top of the liquid column .If the final reading is not made within 10 min after mixing the blood with Hcl N/10  2 % should be deducted from the result obtained. In every anaemic case the quantity of blood has also Hcl  N/10 should be doubled and the result divided by 2. It is best to match in the daylightz

b) CYANMETHAEMOGLOBIN METHOD

                        A reliable method for the estimation of total haemoglobin

i)        Drabkin’s solution

             Sodium bicarbonate               -           1 gm

             Potassium cyanide                 -           0.2 gm

             Potassium kricyanide             -           0.2 gm

             Distilled water (made upto 100 ml)

ii)      Thompson’s inorganic gray solution 

       Chrome alum A.R                  -           16.67 gm

       Copper sulphate A.R             -           33.33 gm

       Cobalt aluminum sulphate     -           39.55 gm

       Potassium dichromate A.R    -           0.12 gm

       Distilled water to 100 ml

Solution 1:

      Dissolve the chemicals in distilled water and make upto 100 ml

Solution 2:

      Dissolve the salts in distilled water to 1000ml .Store separately for 6 weeks. When prepared the solution is slightly pink but gradually turns gray . This solution remains stable at room temperature for many months and can be used as a permanent standard in haemoglobin estimation. When diluted with I part of water to 2 parts of solution it has an optical density of 0.475 equivalent of that of an oxyhaemoglobin solution containing 14.6 gm of haemoglobin per 100ml of blood

      Expel 0.02 ml of blood into 5 ml of Drabkin’s solution and rinse out the pipette several times with the mixture. Stopper the tube with rubber bung and mix by inverting several times. Allow it to stand for 10 min at room temperature. Match against standard with a yellow green filter in colorimeter

       The standard is prepared from blood of unknown haemoglobin content diluted 1:250 with Drabkin’s solution. Standard solution with a haemoglobin content of 5g, 10g, 15g per 100 ml are recommended. Thompson’s inorganic gray solution can be used as a standard if first calculated against standard solution of cyanmethaemoglobin.