Kleihauer–Betke Test


Laboratory test used to screen maternal blood samples for the presence of foetal red blood cells. The Kleihauer–Betke Test relies on the fact that foetal RBCs are generally rich in haemoglobin F (HbF) which is resistant to acid.

A maternal blood sample is prepared (smeared) on a glass slide, which is then flooded with acid. Maternal haemoglobin (haemoglobin A in most adults) dissolves away and the HbF remains intact. The slide is then washed, stained, and read.

The foetal RBCs appear bright red, while the maternal RBCs are pale because they have lost their haemoglobin. The percentage of foetal cells (usually from a count of 2000 cells) is used to predict the percentage of foetal red blood cells in the maternal circulation. This value can be used to calculate the total amount of foetal blood in maternal circulation, so that Rhesus D-negative mothers may receive appropriate amounts of prophylactic Rh Immune globulin.


1957 – The method was developed by Kleihauer under the project management of Betke. Dr. Hildegard Braun, second author in the original article, was responsible for the electron microscope preparations. This publication introduced the procedure for detecting HbF cells which today is known as the Kleihauer-Betke technique.

Particular interest was focused on solving the question of how the switch from foetal to adult haemoglobin proceeded within the individual red cell during the perinatal and postnatal period. First of all, a method had to be developed allowing the visualisation of both foetal and adult haemoglobin in individual erythrocytes.

Betke and Kleihauer searched for a method to differentiate Hb A and Hb F in the red cells of a blood smear. In 1930 EG Schenk found that denatured adult haemoglobin is more rapidly digested by pepsin than newborn haemoglobin. So the team ‘digested’ fixed blood smears with pepsin and it worked. Evaluating the best pH for the procedure, Enno Kleihauer ran also a blank of the acid buffer without pepsin, and this produced even better results.

Based on the fact that foetal and adult haemoglobin react differently when digested with trypsin and pepsin, respective procedures were applied for developing the method.

Whereas experiments with trypsin failed, the use of pepsin in an appropriate acid environment (pH approx. 4.3) produced the first successful results. Since the citric acid-phosphate buffer served always as a control, it was readily shown that the ‘digestion’ was possible even without pepsin. Thus, the Elution Method had been established: in an acid buffer solution the adult haemoglobin was ‘washed out’ of the erythrocytes-on the slide these cells appeared as ghosts-whereas the foetal haemoglobin had remained within the cells and, by staining, could be made visible. Applying the elution technique to erythrocytes of newborns and infants it was demonstrated for the first time that the individual red cell precursor is capable of producing both foetal and adult haemoglobin.

Further experiments indicated that the differing reaction of foetal and adult erythrocytes during elution is caused only by haemoglobin properties; the red cell membrane is of no significance for the elution process.

Kleihauer–Betke Test 1957
Abb. la. In Alkohol fixierter Blutausstrich, bei pH 3,5 in Citronensäure-Phosphat-Buffer extrahiert und nach May-Grünwald-Giemsa geftibt. Blut eines jungen Säuglings mit 7,5 % fetalem Hämoglobin. Gut hämoglobingeftilte Zellen neben einzelnen mehr oder weniger schwächer gefärbten. Kleihaur, Braun, Betke. 1957

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the names behind the name

BA MA (Oxon) MBChB (Edin) FACEM FFSEM. Emergency physician, Sir Charles Gairdner Hospital.  Passion for rugby; medical history; medical education; and asynchronous learning #FOAMed evangelist. Co-founder and CTO of Life in the Fast lane | Eponyms | Books | Twitter |

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