Kleihauer–Betke test

Principle

• Fetal haemoglobin (HbF) resists acid elution. When a fixed maternal blood film is exposed to acid buffer, adult Hb is eluted from red cells, leaving pale ghosts, while fetal cells retain HbF and stain darkly.

Specimen

• Maternal EDTA blood collected after delivery (ideally within about 1–2 h) or after a sensitising event, before anti-D is given.
• A screening test (e.g. rosette test) may be done first; positives are quantified.

Procedure

• Make thin films; fix in 80% ethanol for about 5 min.
• Immerse in citrate-phosphate buffer at pH about 3.2–3.3, 37 °C, for about 5 min (time and pH per local validated method).
• Rinse, stain with erythrosine or eosin, counterstain with haematoxylin.
• Include positive (cord and adult blood mixture) and negative controls.
• Count fetal cells among at least 2000 maternal cells, ideally using an ocular grid.

Results

• Fetal cells: bright pink-red, refractile. Adult cells: pale ghost cells.
• Fetal cell % = fetal cells ÷ total cells counted × 100.
• Fetomaternal haemorrhage (mL fetal whole blood) = fetal cell % × 50 (assuming maternal blood volume of 5000 mL).
• One 300 µg dose of RhIG covers about 30 mL fetal whole blood (15 mL red cells). Vials = FMH ÷ 30; round up if the decimal is 0.5 or more, down if less than 0.5, then add one vial.

Quality control and pitfalls

• False positives from maternal cells with raised HbF (HPFH, beta-thalassaemia trait, sickle cell disease, pregnancy-related F cells), usually as intermediate staining.
• Critical control of pH, temperature and timing; wrong conditions cause under- or over-elution.
• Poor precision with small bleeds; flow cytometry with anti-HbF is more accurate.
• ABO-incompatible fetal cells may be cleared rapidly, underestimating the bleed.

Clinical use

• Quantifies fetomaternal haemorrhage to calculate RhIG dose in D-negative mothers; investigates stillbirth or fetal anaemia.