Hemoglobin

Hemoglobin 

Principle

Blood is mixed with an acid solution so that hemoglobin is converted to brown-colored acid hematin.

This is then diluted with water till the brown color matches that of the brown glass standard. The hemoglobin value is read directly from the scale.

Equipment

(1) Sahli’s hemoglobinometer: This consists of Sahli’s graduated hemoglobin tube (marked in grams and percent) and a comparator with a brown glass standard.

(2) Sahli’s pipette or hemoglobin pipette (marked at 20 μl or 0.02 ml).

(3) Small glass rod (stirrer).

(4) Dropping pipette.

Reagents

(1) N/10 hydrochloric acid

(2) Distilled water

Specimen: EDTA-anticoagulated venous blood or blood obtained by skin puncture.

Method

(1) Place N/10 hydrochloric acid into Sahli’s graduated hemoglobin tube up to the mark of 2 grams.

(2) Take blood sample in Sahli’s pipette exactly up to 20 μl mark. Blood adhering to the exterior of the pipette is wiped away using absorbent paper or gauze.

(3) Add blood sample to the acid solution, mix with a glass stirrer, and allow to stand for 10 minutes.

(4) Add distilled water drop by drop till the color of the solution matches that of the brown glass standard.

(5) Take the reading of the lower meniscus from the graduated tube in grams.

Note:

(1) Although the graduated tube is marked in both grams and percent figures, result should always be reported in grams. This is because (a) no single hemoglobin value can be considered as 100% since it varies with the age and sex of the individual and altitude, and

(b) hemoglobinometers of different manufacturers have different values as 100%, so that same sample of blood will yield different results on different instruments.

(2) Disadvantages of Sahli’method:

• About 95% color of acid hematin is attained at the end of 10 minutes. For maximum color development, much longer time (1 hour) is required.

• Perfect matching with the brown glass standard is not possible.

• Carboxyhemoglobin, methemo-globin, and sulfhemoglobin are not converted to acid hematin. HbF is also not converted to acid hematin and therefore this method is not suitable in small infants.

• Development of color is slow and acid hematin solution is not stable.

• Source of light (daylight or artificial) will influence the visual comparison of colors.

• Personal error in matching brown glass standard with test solution is 10%.

• Color of brown glass standard fades with time.