Hemoglobin Determination Methods in the Philippines

This article is intended to address the common violation of clinical laboratories with regards to haemoglobin determination.  Majority of the non-hospital based freestanding clinical laboratories specially primary categories do not even perform the test but a result is being released by multiplying a factor (0.333) to the hematocrit of the patient.  Poor patients, they are paying for the test but without knowing that it is actually not being done by the medical technologist involved.  Such thing is very unprofessional and with all the notices and warnings issued by the regulatory officers, this practice still exist.

The approved standard for quantitative blood  haemoglobin determination in the Philippines is by using the hemiglobincyanide (HiCN) or commonly known as the cyanmethemoglobin method.  To perform this method, blood is mixed with Drabkin's solution, a solution that contains ferricyanide and cyanide. The ferricyanide oxidizes the iron in the hemoglobin, thereby changing hemoglobin to methemoglobin. Methemoglobin then unites with the cyanide to form cyanmethemoglobin.  All forms of hemoglobin likely to occur in circulatory blood, with the exception of sulfhemoglobin, are determined. Cyanmethemoglobin produces a color which is measured in a colorimeter, spectrophotometer, or automated instrument.  The color is suitable for measurement in filter as well as in narrow-band spectrophotometers, because its absorption band at a wavelength of 540 nanometers is broad and relatively flat.  The color relates to the concentration of hemoglobin in the blood.  Standards prepared from either crystalline hemoglobin or washed erythrocytes and stored in a brown glass container and in sterile condition are stable for at least nine months (change 2%).

Since very few primary category clinical laboratory are equipped with hemoglobinometer  or a photometer, the regulatory officers consider manual methods which include the Haden-Hausse and Sahli-Hellige methods. In both methods, blood is mixed with dilute hydrochloric acid. This process hemolyzes the red cells, disrupting the integrity of the red cells' membrane and causing the release of hemoglobin, which, in turn, is converted to a brownish-colored solution of acid hematin. The acid hematin solution is then compared with a color standard.

This methods of determination of haemoglobin as haeminchloride (acid haematin) though not recommended because of their unreliability, are sufficient for routine examination provided they are properly done.

Below is the procedure for the Sahli-Hellige method.

Materials Required:

• ·         Distilled water

• ·         Sahli-helligehemoglobinometerkit containing:

o   Small bottle of dilute (approx. 0.1N) hydrochloric acid. Prepare this solution by adding 1 ml of concentrated HCl to 99 ml of distilled water. POUR ACID INTO WATER. Replenish this peri-odically–it must be of proper strength.

o   Graduated tube, with a scale on two sides. On one side is the percentage scale, and on the opposite side is the gram scale. The percentage scale reads from 0 to 170. The gram scale reads from 0 to 24.

• ·          Pipette, marked at the 20 mm 3 level

• ·         Stirring rod

• ·         Color comparator, with a window in the side. On the right and left sides of this opening is the color standard for comparison. The center has an open slot to hold the graduated tube.  

Procedure 

1. With a medicine dropper, place 5 drops of the 0.lN HCl in the bottom of the graduated tube. Place the tube in the color comparator.
2. Using well-mixed venous blood or fingertip blood, fill the pipette to the 20 mm mark.
3. Wipe blood from the outside of the pipette. Transfer blood to the Sahli tube. Note the time.
4. Aspirate distilled water into the pipette two or three times and transfer these washings to the tube.
5. Shake until the blood is well mixed and the tube is a uniform color.
6. Add distilled water, drop by drop, each time mixing the solution with the stirring rod. Keep adding water and mixing until the color of the solution matches the standards on either side. Remove the stirring rod from the tube each time before com-paring. Natural light makes more accurate readings possible.
7. Five minutes after time noted, read the result from the scale on the tube by noting the graduation mark at the lower edge of the meniscus. Read and report both scales.