Detecting Fetomaternal Bleed Using Flow Cytometry

One of the many interesting and unique tests that I learned about upon joining Carter BloodCare was our ability to detect and quantify fetomaternal hemorrhage using flow cytometry. This is very exciting in the pathology world, because it provides an alternative to the traditional Kleihauer-Betke test. I remember, in pathology residency, trying to manually count stained fetal cells on a KB test. In my experience, the KB test is really hard to do!

Calculating the correct volume of fetal blood present in maternal circulation is very important, because that is how we calculate how much anti-D immunoglobulin we need to give. Detecting the % fetal cells by flow cytometry is quite ingenious as it takes advantage of the fact that fetal cells contain hemoglobin F – something which is inherently different about fetal RBCs.

In order to perform the procedure, a maternal sample is collected, and the RBCs are fixed so that the cell structure remains intact. Then, a substance is added that pokes holes in the RBCs’ membrane. It is important to do these steps because we need access inside the red blood cells in order to detect the hemoglobin F. It is also important for the RBCs to maintain their structure because we want to count the number of fetal cells present - and we obviously cannot do that if we destroy them. Once we have fixed, stable RBCs with holes them, we can then add the specialized antibody which detects hemoglobin F. This is a very helpful step as this antibody fluoresces – and our flow cytometer is then able to count how many cells are fluorescing!

One important question I had with this technology is, what if the maternal RBCs have hemoglobin F in them? There are certain conditions which can cause elevated hemoglobin F in adults, and would this cause a false positive result? In fact the answer is “no,” this is unlikely to happen – because fetal RBCs have a higher concentration of hemoglobin F, and so fetal RBCs fluoresce much brighter than adult RBCs with hemoglobin F. Therefore, we simply run positive and negative control samples so that we can make sure we know how bright the fetal cells are fluorescing on each run compared to maternal cells. This is truly a great way to quantify a fetomaternal bleed without having to count cells on a microscope!

References:

1.       Monoclonal antibodies detected to human fetal hemoglobin (HbF). Product Insert, Thermo Fisher Scientific. Effective date Nov 21, 2019.

2.       Fernandes BJ, von Dadelszen P, Fazal I, et al. Flow cytometric assessment of feto-maternal hemorrhage; a comparison with Betke–Kleihauer. Prenat Diagn 2007; 27: 641–643.

3.       Dziegiel MH, Nielsen LK, Berkowicz A. Detecting fetomaternal hemorrhage by flow cytometry. Curr Opin Hematol 2006; 13: 490–495.

Special thanks to Matthew Slaven, MLS (ASCP), our Carter BloodCare flow cytometry specialist, for his help with this blog entry!