Kleihauer test explained

The Kleihauer test, also called the Kleihauer-Betke or acid elution test, is performed on a blood film made from a sample of the mother's blood. The laboratory treats the film so that fetal red cells stain deep pink while the mother's own red cells appear as "ghosts". The number of fetal and maternal cells is counted and expressed as an estimated fetomaternal haemorrhage in millilitres, according to the Black Country Pathology Services test information.

The test depends on a difference in haemoglobin. Cells containing fetal haemoglobin (HbF) are stained pink after the adult haemoglobin has been washed out, which is how fetal cells can be distinguished from the mother's.

The NHS explains that haemolytic disease of the fetus and newborn (HDFN), sometimes called rhesus disease, can happen when the mother's blood type is D negative and the baby's is D positive. The mother may then have antibodies that break down the baby's red blood cells. Antibodies can develop after contact with D positive blood, for example during a transfusion or a previous pregnancy.

The pathology service explains that following a sensitising event, fetal red cells can pass into the maternal circulation. To prevent anti-D antibodies forming, all non-sensitised D negative pregnant women are offered prophylactic anti-D immunoglobulin, which coats the baby's D positive cells before the mother's immune system reacts. After a sensitising event more anti-D is needed, and the amount depends on the size of the fetal bleed. NICE recommends routine antenatal anti-D prophylaxis as a treatment option for all RhD negative pregnant women who are not known to be sensitised.

The Kleihauer test is the way of sizing that bleed. It is not a test of whether the baby is well; it is a measure used to choose the right anti-D dose.

It is an ordinary blood test from a vein. The laboratory information describes some practical requirements: the maternal sample for FMH estimation should be taken after 30 to 45 minutes but before 2 hours, to allow fetal cells to distribute within the mother's circulation. Short-term storage is 24 hours at room temperature, and the sample must not be exposed to extreme heat or cold before testing.

Adequate mixing is needed to ensure an even distribution of fetal cells, and the laboratory prefers a separate sample from the one used for blood group and antibody screening, which is why you may be asked for more than one tube.

Reporting follows a stepped approach. A calculated bleed below 2 mL is reported as less than 2 mL. If more than 2 mL is calculated, the sample is sent for flow cytometry, which labels fetal and unlabelled maternal cells and counts thousands of them to give a more accurate size. Results over 10 mL are reported to the haematology clinician on duty.

The result is then converted into an anti-D dose. The pathology service states that 1500 IU of anti-D immunoglobulin is sufficient to cover an FMH of up to 12 mL when given by intramuscular injection. It also recommends a safety margin when calculating the dose, and says that if there is doubt about whether more anti-D is needed it is preferable to give another dose to avoid inadequate treatment.

The laboratory information is frank that the accuracy and precision of the acid elution procedure is poor. False positives can be obtained if the woman has a high level of fetal haemoglobin, which is why more accurate flow cytometry is used to confirm larger bleeds before decisions are made about massive FMH.

Because of these limits the test is interpreted by the transfusion laboratory and obstetric or haematology team together, rather than as a number to act on in isolation.

If you are RhD negative and have been told you need a Kleihauer test, it can help to ask your midwife or obstetrician why it has been requested, when the sample needs to be taken, when results are expected, and what dose of anti-D is likely to follow. The NHS says that if there is a risk of HDFN during pregnancy you will usually be offered injections to stop you developing antibodies, so the test is one step in a prevention pathway.

Any bleeding, abdominal injury or reduced baby movements in pregnancy should be reported to your maternity unit promptly regardless of your blood group. This page is general information, not individual advice.