Kleihauer-Betke Test

The Kleihauer-Betke test detects and counts foetal red cells in maternal blood. A maternal blood film is exposed to acid buffer (about pH 3.2), which elutes haemoglobin A from adult red cells and leaves them pale "ghosts". Foetal haemoglobin resists elution, so foetal cells stain pink red.

The proportion of foetal cells among at least 2000 maternal cells estimates the volume of foetomaternal haemorrhage, which determines the dose of anti-D immunoglobulin needed to prevent sensitisation of a Rh D-negative mother. It also identifies a massive foetomaternal haemorrhage as a cause of foetal anaemia or death.

Flow cytometry with anti-haemoglobin F or anti-D is more precise and reproducible, and is preferred where available. The acid elution test remains in use where flow cytometry is not.

Small foetomaternal haemorrhages occur in most pregnancies and become more frequent with advancing gestation. A bleed large enough to need additional anti-D (about 30 mL or more of foetal blood) complicates roughly 1 in 300 deliveries. It occurs more often after abruption, caesarean section, manual removal of the placenta, trauma and external cephalic version, but it often occurs with no risk factor.

Indications.

• Rh D-negative women after delivery of a Rh D-positive or unknown group infant, in services that quantify routinely.
• Rh D-negative women after a sensitising event after 20 weeks (antepartum haemorrhage, abdominal trauma, external cephalic version, stillbirth, invasive procedure), when a large bleed is suspected.
• Suspected massive foetomaternal haemorrhage in any mother, including Rh D-positive women: reduced foetal movements, sinusoidal cardiotocograph, unexplained foetal anaemia, hydrops, unexplained stillbirth, or neonatal anaemia with a negative direct antiglobulin test.
• Confirmation that an additional dose of anti-D has cleared foetal cells.

Specimen. Maternal EDTA blood, taken before anti-D is given. Allow time for mixing, usually 30 to 60 minutes after a sensitising event, and after delivery of the placenta at birth. Local practice varies. Test promptly or store at 4 °C for a short period.

Not required. Routine testing before 20 weeks (a fixed dose is given), and when the foetus or infant is Rh D-negative.

Method. A thin film is made from maternal blood. Positive control (cord or adult blood containing foetal cells) and negative control films are run in parallel. The slide is fixed, eluted in acid buffer, rinsed and stained with erythrosin or eosin. At least 2000 cells are counted by two scientists, and the counts are compared. If they disagree widely, the count is repeated.

Calculation. The estimated volume of foetal blood is the percentage of foetal cells multiplied by about 50 mL, assuming a maternal blood volume of 5000 mL. Local correction factors for cell volume are applied by some laboratories. The result is an estimate with wide error.

Anti-D dosing. About 100 to 125 IU of anti-D neutralises 1 mL of foetal red cells (about 2 mL foetal whole blood), with the exact ratio set by the product label. The usual post-delivery dose of 500 IU covers up to about 4 mL of foetal red cells.

A larger bleed needs an additional dose calculated from the estimated volume, given intramuscularly in divided doses or intravenously where the product label permits, as soon as possible and ideally within 72 hours. Later administration, up to about 10 days, still gives some protection.

Result categories.

• No or few foetal cells: usual dose of anti-D is adequate.
• Estimated foetal red cells over about 4 mL: additional anti-D is needed, and the test is repeated at about 48 hours to confirm clearance.
• Estimated foetal blood over 30 mL (about 0.6% foetal cells): large bleed. Foetal anaemia, hydrops or death follows large or chronic bleeds, and counts may exceed several percent.

Causes of a positive result. Physiological transfer, delivery, caesarean section, manual placental removal, placental abruption, placenta praevia, trauma, amniocentesis, chorionic villus sampling, external cephalic version, intrauterine death, twin pregnancy, and spontaneous massive foetomaternal haemorrhage.

Causes of a false positive. Maternal cells that contain foetal haemoglobin stain pink, including hereditary persistence of foetal haemoglobin, beta thalassaemia, sickle cell disease and, modestly, normal pregnancy. These cells stain uniformly pale pink and variably, and flow cytometry separates them from true foetal cells.

Causes of a false negative or underestimate.

• ABO-incompatible foetal cells are cleared quickly by maternal anti-A or anti-B.
• Foetal cells with low haemoglobin F content (preterm infants).
• Poor film quality, inadequate counting and delay between the event and sampling.

Critical findings needing immediate action.

• A large bleed in a Rh D-negative mother needs additional anti-D without delay.
• Suspected massive foetomaternal haemorrhage with foetal compromise needs urgent obstetric review. Foetal anaemia is assessed by middle cerebral artery Doppler, and delivery or intrauterine transfusion may be needed.

Pitfalls.

• Manual counting is imprecise, and estimates can differ by two fold between observers.
• A positive screen is best confirmed by flow cytometry where available.
• Maternal haemoglobinopathy limits interpretation.
• The test does not apply if the foetus is Rh D-negative.

Special groups.

• Rh D-positive women: tested only when massive foetomaternal haemorrhage is suspected, never for anti-D dosing.
• Haemoglobinopathy carriers: use flow cytometry with anti-D when the foetus is Rh D-positive.
• Twin pregnancy: sample after delivery of both infants.
• Recent transfusion or intrauterine transfusion: interpretation is unreliable.

Follow up tests. Flow cytometric quantification of foetal cells, maternal blood group and antibody screen, repeat Kleihauer-Betke test or flow cytometry about 48 hours after additional anti-D, foetal RHD genotyping from maternal plasma, middle cerebral artery Doppler, neonatal haemoglobin, direct antiglobulin test, reticulocyte count and bilirubin.