blood transfusion

Kleihauer Testing

Clinical Background

Request Reasons: Screening for feto-maternal haemorrhage / trans-placental passage of fetal red cells into the maternal circulation after 20 week gestation. Following a sensitising event in pregnant women, it is possible for fetal red blood cells to pass into the maternal circulation. This is known as a fetomaternal haemorrhage (FMH). FMH can result in the mother being sensitised to produce alloantibodies, the most common is anti-D due to a D positive fetus.

In order to prevent haemolytic disease of the fetus and newborn (HDFN) due to anti-D antibodies, all non-sensitised, D negative pregnant women are offered prophylactic anti-D immunoglobulin. The prophylactic anti-D immunoglobulin coats the baby’s D positive red blood cells found in the maternal blood circulation before they have had time to sensitise the mother’s immune system to produce anti-D alloantibodies. Following a sensitising event further prophylactic anti-D immunoglobulin is required, the quantity depending on the degree of fetal bleed.

The Kleihauer screen (Acid Elution Test) requires the preparation of a blood film. This film is treated so the red cells from the fetus appear deep pink while the maternal red cells appear as ‘ghosts’. The number of fetal and maternal cells is counted then expressed as a calculation of FMH in mLs. A sample that shows a FMH of >2mL in the Kleihauer screen can then be quantified by Flow Cytometry. Flow Cytometry gives a more accurate count by counting thousands of cells (labelled fetal cells, unlabelled maternal cells) so that the size of the FMH can be calculated. This allows the correct dose and further ADDITIONAL anti-D immunoglobulin injections are administered. 1500IU anti-D immunoglobulin dose is sufficient to cover a FMH up to 12mL when administered intramuscularly.

Sample Stability Short term storage: 24 hours at room temperature.
Special requirements Maternal sample for FMH estimation should be taken after a period of 30-45 minutes but prior to 2 hours to allow fetal cells to be distributed within the maternal circulation.
Transport requirements Sample should be transported to Laboratory Reception via hospital air-tube system or hand delivered to maintain storage conditions. Samples must not be subjected to extreme hot or cold conditions prior to testing.
Interpretation Red cells containing adult haemoglobin are completely eluted, leaving “ghost” cells. Cells containing HbF are stained pink. White cells are greyish-purple. All FMH calculated as less than 2mL will be reported as <2 ml. If greater than 2ml is calculated the sample will be referred to Flow Cytometry for quantification. Calculations/Quantifications over 10 ml will be reported to the Haematology clinician on duty.
Factors affecting result False positive results can be obtained if the woman has a high level of HbF. The accuracy and precision of the acid elution procedure is poor. Decisions regarding anti-D dosage in massive fetomaternal haemorrhage should be made when the result has been confirmed by flow cytometry. It is desirable to provide a safety margin when calculating the anti-D dose required. If there is a question regarding the need for additional anti-D Ig, it is preferable to administer another dose to prevent risks of inadequate treatment. Adequate mixing, recommended to ensure an even distribution of fetal cells, may be difficult to achieve after centrifugation, separate samples should be received for maternal blood group and antibody screening and FMH screening, when not possible the FMH testing should be undertaken before the sample is centrifuged.