Sahli’s Acid Hematin Method for the Estimation of Hemoglobin

When blood is mixed with an acid solution, the hemoglobin converts into the brown-colored acid hematin. The acid hematin is then diluted with distilled water till the color of the acid hematin matches that of the brown glass standard. The hemoglobin is estimated by reading the value directly from the scale.

Principle

When blood is mixed with an acid solution, the hemoglobin converts into the brown-colored acid hematin. The acid hematin is then diluted with distilled water till the color of the acid hematin matches that of the brown glass standard. The hemoglobin is estimated by reading the value directly from the scale.

Equipment

1. Sahli’s hemoglobinometer: This equipment consists of a comparator with a brown glass standard and Sahli’s graduated hemoglobin tube which is marked in percent and gram.
2. Hemoglobin pipette or Sahli’s pipette (marked at 0.02 ml or 20 μl).
3. Stirrer (a small glass rod).
4. Dropping pipette (dropper).

Reagents

1. N/10 hydrochloric acid
2. Distilled water

Specimen

Blood is obtained directly by skin puncture or EDTA-anticoagulated venous blood.

Method

1. The N/10 hydrochloric acid is placed into Sahli’s graduated tube up to mark 2 grams.
2. With the help of Sahli’s pipette, take blood sample exactly up to 20 μl mark. Blood adhering to the outer part of the pipette is wiped away with the help of tissue paper (absorbent paper) or cotton (gauze piece).
3. Add blood sample to the N/10 hydrochloric acid solution which is placed into Sahli’s graduated tube, mix the mixture with the help of a glass stirrer, and allow the tube to stand for 10 minutes.
4. Add distilled water drop by drop into the mixture placed in Sahli’s graduated tube, till the color of the solution matches that of the brown glass standard.
5. Take the reading of the lower meniscus from the Sahli’s graduated tube in grams.

Note:

1. All the Sahli’s graduated tube is marked in both percent and grams figures, this is because (a) different manufacturers of hemoglobinometers have different values as 100%, so that blood sample will yield different results on different instruments and (b) no single hemoglobin is can be evaluated as 100% since it is different according to the sex and age of the individual and altitude.
2. Disadvantages of Sahli’s method:
• It is impossible to match the color perfectly of the mixture into the Sahli’s graduated tube with the brown glass standard.
• Minimum 1 hour is required for the maximum color development of acid hematin because 95% color of acid hematin is attained at the end of 10 minutes.
• Sulfhemoglobin, methemoglobin, and carboxyhemoglobin cannot be converted into acid hematin. Fetal hemoglobin is also not converted to acid hematin and therefore this technique is not appropriate for in small infants.
• The acid hematin solution is not firm and stable, and the color development is slow.
• Lights may affect the visual comparison of color.
• The color of the brown glass standard dims with time.
• Personal error in matching the color of the mixture in Sahli’s graduated tube with the brown glass standard is 10%.