Abstract
OBJECTIVES: To calculate the minimum fetal red blood cell concentration required to cause maternal Rh sensitization; validate the use of a flow cytometry protocol below that concentration; preliminarily assess the concentrations of fetal red blood cells in pregnant women before and after uterine aspiration. STUDY DESIGN: Using pre-existing literature, we calculated the lowest concentration of fetal red blood cells found to cause sensitization within adult female circulation. We validated a two-color flow cytometry protocol using fluorescently labeled antibodies to Hemoglobin F (expressed by fetal red blood cells and adult F cells) and Carbonic Anhydrase (expressed in red blood cells during the third trimester and postnatally) by titrating second trimester cord blood into non-pregnant adult blood. We applied this flow cytometry protocol in a prospective cohort study of 42 pregnant women at 5-12 weeks gestational age undergoing uterine aspiration for induced or spontaneous abortion. RESULTS: The calculated threshold for causing Rh sensitization was 250 fetal red blood cells per 10 million total red blood cells. We showed a linear relationship between observed and expected fetal red blood cell fractions in titrated samples. Fetal red blood cell counts were more reliable when samples acquired by flow cytometry contained at least 1 million red blood cells. All 37 subjects with evaluable paired samples demonstrated fetal red blood cell concentrations below the calculated threshold for Rh sensitization both pre- and post-procedure. The fetal RBC concentrations increased from a mean of 4.5 (median 0, range 0-57) fetal RBCS pre- to a mean of 8.6 (median 2, range 0-32) fetal RBCs post- per 10 million total RBCs (p < 0.001). CONCLUSIONS: Flow cytometry was capable of separately quantifying fetal red blood cells and maternal F cells to very dilute concentrations. Fetal red blood cell exposure in the first trimester was well below the calculated threshold for maternal Rh sensitization in our cohort. Larger studies are warranted to confirm our pilot study findings, fill this evidence gap and inform universal guidelines for administering Rh immunoglobulin after first trimester uterine aspiration. IMPLICATIONS: Fetal red blood cell exposure following first trimester uterine aspiration is well below the calculated threshold for maternal Rh sensitization in our cohort.