3 Important Photometric Techniques for Measuring Haemoglobin

Before the introduction of novel laboratory test procedures, haemoglobin was measured photometrically or estimated using a visual comparative technique. Haemoglobin values are expressed in grams per lire (g/l) or grams per decilitre (g/dl). However, photometric techniques for measuring haemoglobin now use electronic means to achieve a more precise diagnostic result in the laboratory. 

Photometric Techniques

Photometric techniques for measuring haemoglobin: In photometric techniques, the absorbance of haemoglgin in a blood sample is measured electronically using a filter colorimeter or a direct read-out haemoglobin meter. Ways of measuring haemoglobin photometrically include:

Haemiglobincyanide (cyanmethaemoglobin) technique using a filter colorimeter or direct read-out meter: This technique is ICSH recommended because stable haemiglobincyandie (HiCN) standards are available to calibrate instruments. It has howver, several disadvantages when used in tropical countries.

Using a direct read-out meter such as the Developing Health Technology (DHT) Haemoglobinometer: which is a haemoglobin meter with specifications and technique suitable for use in tropical counties.

Oxyhaemoglobin (HbO₂) technique using a filter colorimeter: While this is a reliable and inexpensive method of measuring haemoglobin (HbO₂) there is no stable HbO₂ reference standard solution. A control haemolysate or a whole blood of know haemoglobin value (previously measures by the HiCN method) is required to calibrate a colorimeter.

Note: Most photometric techniques require dilution of the blood. A non-dilution photometric technique is more precise because blood is not measures into a diluting fluid. An example of a non-dilution method is the HaemoCue system. Although not affordable by most district laboratories, it is briefly described at the end of photometric techniques because it is used in some situations.

5 Sources of Error When Measuring Haemoglobin 

The following are the most important and commonest errors that can lead to unreliable test results when measuring haemoglobin photometrically:

Not measuring the correct volume of blood due to poor technique or using a wet or chipped pipette.

When using anticogulated venous blood, not mixing the sample sufficiently.

Not ensuring that the optical surfaces of a cuvette are clean and dry and there are no air bubbles in the solution.

Techniques to Prevent Cuvette-Related Errors

There are important steps to take to prevent cuvette-related errors in adopting the photometric techniques in measuring heamoglobin. These techniques adopted include: 

Hold a clean cuvette only by its frosted (matt) or ridged sides. When transferring a solution to a cuvette, allow the fluid to run down the inside wall of the cuvette. This will help to avoid air bubbles in the solution. Do not fill a cuvette more than three quarters full.

Using a tissue or soft clean cloth, wipe clean the clear optical surface of the cuvette carefully insert the cuvette in the colorimeter or haemoglobin meter (optical surface facing the light source).

Note: Ensure a solution is at room temperature before reading its absorbance otherwise condensation will form on the outside of the cuvette which will give an incorrect reading.

Not protecting a colorimeter or haemoglobin meter from direct sunlight and not checking the performance of an instrument or maintaining it as instructed by the filter colorimeter is using a glass filter which is not clean.

Not checking a diluting fluid such as Drabkin’s for signs of deterioration as explained in the HiCN technique.