Flow cytometry quantification of feto-maternal hemorrhage following uterine aspiration in the first trimester

Awarded 2017
Complex Family Planning Fellowship Research
Sarah Horvath, MD
University of Pennsylvania

Rh negative women may become sensitized to Rho D when the antigen is present on the fetal red blood cells of an index pregnancy. This sensitization can lead to hemolytic disease of the newborn in subsequent pregnancies.  ~15% of women in the United States are Rh negative and at risk of becoming immunized.  Rh immune globulin is given to Rh negative pregnant women at 28 weeks gestation and again at delivery to prevent this from occurring.  Even before the introduction of prophylaxis, only 9-10% of at-risk pregnancies resulted in iso-immunization.
While there is consensus to give Rh immune globulin in the third trimester, evidence for its necessity earlier in pregnancy is needed to improve management recommendations.  Data showing maternal sensitization following first trimester spontaneous, therapeutic and threatened abortion are lacking.  Thus, all women with vaginal bleeding in the first trimester must undergo blood typing.  Blood bank personnel must then screen all Rh negative samples for pre-existing antibodies, which takes many hours.  Staff in clinics, emergency rooms and labor units must incorporate this extra time for following up results, contacting patients and administering prophylaxis into their work-flow.  Patients must either wait for results or make a separate visit to the clinic.  This additional intervention has a particularly large effect on women receiving abortion by telemedicine or in states with mandated waiting periods.  Currently, international guidelines for Rh immune prophylaxis in the first trimester differ. 
Historically, we have quantified feto-maternal hemorrhage by acid-elution tests such as Kleihauer-Betke (K-B), which have low sensitivity for detecting differences between fetal red blood cells and maternal red blood cells that contain HgbF.  K-B is also prone to human interpretation and difficult to reproduce reliably.  Flow cytometry is more objective, reproducible and sensitive than the K-B.
We aim to test and validate a protocol for flow cytometer detection of 1/100,000 fetal RBCs in a maternal blood sample.  After developing this tool, we will use it to establish a baseline concentration of fetal cells detectable prior to uterine aspiration or vaginal bleeding.  We will then compare concentrations from before and after uterine aspiration in the same cohort of patients.  Results will be correlated to descriptive characteristics and used to refine criteria and hypotheses for future studies.
Our protocol was found to be 94.4% sensitive in repeated laboratory studies, so we used it to evaluate patients in the real world.  In our sample of 42 women with samples taken before and after uterine aspiration, none had enough fetal red blood cells to reach the threshold for sensitization.  We plan to conduct these larger studies using our new technology and hope to provide evidence that will allow new, standardized international guidelines to be developed.  If our results prove true in larger studies, it means that Rh immune globulin may not be necessary before twelve weeks of pregnancy.  

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