Kleihauer Test - FAQs

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We usually make up our own fixative - do we have to use that supplied in the kit?
Not necessarily; in fact at one time we used to provide the fixative separately if required. However over time we found that most of the problems associated with the kit were related to poor fixation. For example some alcohol preparations are unstable and will fix the haemoglobin in the adult cells preventing complete elution and resulting in red staining by the counter stain. We find that the stabilised fixative we now supply with the kit eliminates these problems and most of our users prefer to use this than have hassle when making up their own fixative.

At what temperature should blood samples for this test be stored?
If samples must be stored they will be best in the blood-bank refrigerator. If blood is stored at or near freezing haemolysis of the red cells will occur. Higher temperatures (>40°C) may cause fixation of the haemoglobin, haemolysis or crenation of the red cells.

How long can I leave blood samples or films before doing the test?
Blood samples over 4 days old are likely to show problems with improper elution. Much will depend on storage conditions. As with most blood samples, the sooner you make films, or do the test, the better. Once blood films are made it is best to fix them straight away. They may then be left for up to 72 hours if necessary, but no longer as the haemoglobin will denature and be stained by the counterstain. For preference we recommend that fixation, elution and counterstaining be progressed without any lengthy delays.

You recommend using a sink rack and flooding the slides for elution and counterstaining, but we usually use coplan jars for our staining techniques, does this matter?
The use of coplan jars is not recommended because the effectiveness of the eluting reagent is pH dependant. If you elute in a jar the disrupted cells release their haemoglobin into the fluid, which will have a buffering effect and raise the pH of the eluting fluid. This will result in the improper elution of subsequent films and all cells in these films will appear pinkish red.

The tap water in our lab is very acid (or alkaline) will this affect the test?
It is unlikely that very hard or soft tap water will compromise the test results.

Drying of blood films takes a long time, can we speed this up by using a hotplate or hair dryer?
No!  At all stages the blood film must be allowed to air dry: Using a hot plate or hair dryer will heat-fix the haemoglobin resulting in improper elution with all the red cells staining red.

Are there any medical conditions that can give false positive results?
Patients with ß-thalassaemia or sickle cell trait are known to show poor elution of some of their red cells and those with Hereditary Persistence of Fetal Haemoglobin are likely to show between 3 and 20% of their red cells staining pink with the Kleihauer technique. Note: 0.5 - 1% of total adult haemoglobin can be HbF, and depending on how this is distributed over the sample it is possible to see a very occasional adult cell staining pink or red. These cells are often referred to as F-cells or transient cells.

Click here to return to the Guest Medical Kleihauer Test kit.