Estimation of haemoglobin by sahli’s acid haematin method

 

Introduction:

 

Hemoglobin estimation also known as Hg. Hemoglobin is a protein used by red blood cells to distribute oxygen to other tissues and cells in the body. It is made up of heme, which contains iron atoms plus the red pigment, porphyrin, (responsible for giving the blood its red color) and globin a chain of amino acids.

Normal range Varies with altitude.

·       Male     -              8.1 to 11.2 m mole/L     (13 to 18 gm./ dl.)

·       Female -            7.4 to 9.9 mmol/L        (12 to 16 gm./dL)

·       Child     -              7.1 to 8.4 mmol/L         (11.5 to 13.5 gm./dL)

·       Newborns-       10.5 to 13.7 mmol/L     (17 to 22 gm./dl)

Functions of hemoglobin:

 

1. Transport of oxygen 

2. Transport of CO₂  

3. It acts as a buffer  

4. Bilirubin formation

Decreased hemoglobin may indicate:

a) Anemia of various types.

b) Blood loss - e.g. from traumatic injury, surgery or bleeding colon cancer.

c) Gastrointestinal tract diseases resulting in blood loss e.g. parasitic infections, colitis,  haemorrhoids, ulcers.

d) Erythropoietin deficiency e.g. from kidney disease.

e) Red blood cell destruction associated with transfusion reaction

f) Lead poisoning

g) Malnutrition and the associated nutritional deficiencies of iron, folic acid, vitamin B-12 & vitamin B-6

h) Abnormal hemoglobin e.g. sickle cell anemia & thalassemia.

Increased hemoglobin may indicate:

a) Dehydration - This can cause a falsely high hemoglobin reading which disappears when proper fluid balance is restored, e.g., as in severe diarrhea. 

b) Polycythemia Vera - a condition in which there is a long-term increase in red blood cells and other types of cells.

c)  Congenital heart disease

d)  Malabsorption problem and 

e)  Lung Diseases eg:- Emphysema , Severe asthma, pulmonary fibrosis - scarring of the lung. on the other hand, pulmonale - failure of the right side of the heart caused by prolonged high blood pressure in the pulmonary artery and right ventricle of the heart.

f)  Smokers may have elevated hemoglobin levels.

g)  Testosterone supplementation

 

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Principle:

Hemoglobin present in a sample of blood is converted into acid hematic by addition of 0.1 N HCl to the blood and its hemoglobin content is determined by matching the solution against non-fading glass having a standard color.

Requirements:

Sahli’s haemoglobinometer (haemometer), distilled water, rectified spirit, cotton, lancet.

SAHLI’S HAEMOGLOBINOMETER Apparatus consists of:

·       Comparator box: with color standards on either side with a space for keeping the diluting tube in the middle. The standard is non-fading yellowish brown tinted glass, the color of which is that of acid haematin obtained by treating blood containing 14.5gm of hemoglobin per dl.with 0.1 NHCL and diluting it 100 times

·       Special diluting tube: which is graduated in gm./dl scale on one side and percentage scale on the other side? (1 4.5gm/dI corresponding to 100%).

·       Hemoglobin pipette: This is a capillary pipette with a 20-mm3 marking

·       Glass stirrer

·       A bottle containing 0.1 N  HCL

Procedure:

1.         Take  0.1 N  HCI up to the lowest mark in the diluting tube. Keep the diluting tube in the space provided in the box

2.        Sterilize the fingertip using rectified spirit. Make a quick prick to get moderately large drop of blood.

3.        Suck the blood in the hemoglobin pipette up to the 20mm3 mark without any air bubble.

4.        Wipe off any blood sticking to the tip and sides of the pipette using cotton.

5.        Transfer the blood immediately into the acid taken in the diluting tube.

6.        Rinse the pipette two or three times with the acid and transfer into the diluting tube.

7.             Mix and keep it undisturbed for 10 minutes, so that hemoglobin gets converted to acid haemitin.

8.        After 10 minutes, dilute the contents by adding distilled water drop by drop and mixing the contents after each drop with the stirrer, till the color matches with the color of the standard. Then take the reading both in gram scale and percentage scale by noting the lower meniscus.

Precautions:

1.      There should not be any air bubble or blood clot in the column in the pipette.

2.      Graduations on the diluting tube should not interfere with color matching.

3.     The glass rod should be lifted up before color matching and reading.

4.     Wipe off the excess blood sticking to the sides and tip of the pipette.

5.     Transfer the contents immediately into the diluting tube and note the time.

6.     Take the reading without any delay because on keeping the color will deepen.