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Screening for and quantification of foetal-maternal haemorrhage by flow cytometry

Pictogramme horloge Elodie Rabut Pictogramme horloge March 2015

Foetal-maternal haemorrhage (FMH) is defined as the passage of foetal red blood cells into the mother’s circulatory system, either during pregnancy or at the time of delivery.

Notable risk factors for FMH include abdominal injury, obstetric intervention, abortion, delivery and foetal death in utero. Even very slight FMH exposes the mother to a risk of Rhesus alloimmunisation, and also the risk of alloimmunisation to other blood-group systems. Massive FMH can cause severe foetal anaemia and hypovolaemic shock that can lead to foetal death.

Screening for FMH is therefore indicated in cases of abdominal trauma, obstetric complications and when there is a reduction in the perception of foetal movement.

If FMH is detected in a woman who is Rh- (D-), preventative treatment against Rhesus alloimmunisation must be undertaken by injection of anti-D gammaglobulins (e.g. Rhophylac®), ideally in the 72 hours following the FMH (preferably by IV route).

In such cases, the dosage of Rhophylac® is adjusted to the size of the FMH: 200 µg for up to 12 mL foetal blood, then 200 µg for every further 20 mL foetal blood (reference 1).

The screening and quantification of FMH are traditionally carried out by the Kleihauer–Betke cytochemical test first described in 1957. This test is based on the resistance of foetal red blood cells to acid: after elution with acid, the foetal red blood cells are counted microscopically out of a total of approximately 1000–2000 visualised red blood cells. This manual technique requires expertise and is poorly reproducible, as the difference between foetal red blood cells and adult red blood cells rich in haemoglobin F (HbF) is particularly difficult. The precision of the method is mediocre (CV: 30–40%), and a systematic overestimation of the extent of the FMH has been shown with this technique (reference 2).

In flow cytometry, the use of a specific anti-HbF reagent linked to the fluorophore FITC allows good distinction between foetal and adult red blood cells. Acquisition is carried out in a few minutes with 100,000 cells, and the data analysis is conducted by defining the zone of foetal cells using an internal control of high positive quality. The detection threshold of the technique used at Biomnis is 0.06% foetal red blood cells, equivalent to a volume of 3 mL foetal blood.

Screening for FMH by flow cytometry is well-correlated with the reference cytochemical technique, but it is more precise and more reproducible. The use of flow cytometry enables the systematic overestimation observed with the Kleihauer test, which could lead to excessive injection of anti-D gammaglobulins, to be avoided. This is because the anti-HbF reagent used in flow cytometry allows good distinction between foetal red blood cells and adult red blood cells rich in HbF, which makes the interpretation of the results simpler in women with a moderately raised level of HbF (which frequently occurs in pregnancy and in mothers suffering from haemoglobin disorders).

More information

  1. Prévention de l’allo-immunisation RHD foeto-maternelle
    Recommandations du Collège National des Gynécologues Obstétriciens Français, 2005.
  2. Evaluation of FMH QuikQuant for the Detection and Quantification of Fetomaternal Hemorrhage
    Pastoret C, Le Priol J, Fest T, Roussel M. Cytometry Part B 2013; 84B: 37-43.

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