Rare‐event analysis methods for detection of fetal red blood cells in maternal blood

Abstract

Methods for analyzing very small subpopulations of cells (“rare-event analysis methods”) are of interest in many areas of biology. In this paper we demonstrate the ability to detect one Rh-positive erythrocyte per 100,000 Rh-negative erythrocytes. This is the level needed to detect human fetal red blood cells in the maternal circulation in the case of fetal-maternal Rh-incompatibility. Erythrocytes stained with fluorescent immunobeads are more than 100 times as bright as the same cells stained by indirect immunofluorescence (or almost 40 times as bright as cells stained by the biotin-avidin technique). More importantly there is greater than a sevenfold increase in signal-to-noise (S/N) ratio, which proves critical in detecting very low frequency („rare event”︁) cell subpopulations. Although equal mixtures of positive and negative cells can easily be detected, positives cannot be easily distinguished from negatives when they are present in relative frequencies of 1:1000 or less as measured by either indirect immunofluorescence or biotin-fluorescent avidin labeling of D antigens. However, use of fluorescent immunobeads and high-speed flow cytometry allows detection of subpopulations of the same cells at relative frequencies of less than 1:100,000. Based on calibrations using free-fluorescent immunobeads, the average Rh-positive cell (when present in a relative frequency of 1:100,000) labeled with 36 ± 19 fluorescent immunobeads. Rh-positive cells of relative frequencies 1: 10,000 and 1: 100,000 can be detected above background (Rh-negative red blood cells treated with the same amount of fluorescent immunobeads). Using defined mixtures of Rh-positive and negative cells, the expected numbers of positive cells were observed over a range of 1:100 to 1:100,000 relative Rh-positive cell frequencies. These methods should prove of general use in applications where original frequency information about rare cell subpopulations is important and would be lost if enrichment procedures were to be used.