Quantification of all fetal nucleated cells in maternal blood between the 18th and 22nd weeks of pregnancy using molecular cytogenetic techniques.

Different types of nucleated fetal cells (trophoblasts, erythroblasts, lymphocytes, and granulocytes) have been recovered in maternal peripheral blood. In spite of many attempts to estimate the number of fetal cells in maternal circulation, there is still much controversy concerning this aspect. The numbers obtained vary widely, ranging from 1 nucleated cell per 104 to 1 per 109 nucleated maternal cells. The purpose of our project was to determine the absolute number of all different types of male fetal nucleated cells per unit volume of peripheral maternal blood. Peripheral blood samples were obtained from 12 normal pregnant women known to carry a male fetus between 18 and 22 weeks of pregnancy. Three milliliters (3 ml) of maternal blood has been processed without any enrichment procedures. Fluorescence in situ hybridization (FISH) and primed in situ labeling (PRINS) were performed, and fetal XY cells were identified (among maternal XX cells) and scored by fluorescent microscopy screening. The total number of male fetal nucleated cells per milliliter of maternal blood was consistent in each woman studied and varied from 2 to 6 cells per milliliter within the group of normal pregnancies. The number of fetal cells in maternal blood, at a given period, is reproducible and can therefore be assessed by cytogenetic methods. This confirms the possibility of developing a non-invasive prenatal diagnosis test for aneuploidies. Furthermore, we demonstrate that it is possible to repeatedly identify an extremely small number of fetal cells among millions of maternal cells.

Creating a new color by omission of 3 end blocking step for simultaneous detection of different chromosomes in multi-PRINS technique.

In the multiple color primed in situ labeling (multi-PRINS) technique, the blocking step using ddNTPs, incorporated by a DNA polymerase, is an important procedure that blocks the free 3' end generated in the previous PRINS reaction, thus avoiding the next PRINS reaction using it as a primer to perform spurious elongation at non-desired sites. However, we found that omission of the blocking step never affected the correct identification of two chromosomes because the signals from the second PRINS reaction site always showed the pure original color. Nevertheless, taking advantage of the color mixing, we successfully used a multi-PRINS technique to create a third color using the two most common forms of labeled dUTP (biotin- and digoxigenin-labeled dUTP) and two fluorochromes (fluorescein and rhodamine) in order simultaneously to detect three chromosomes in the same cell. By arranging the labeling either in bio-dig-bio or in dig-bio-dig order in the sequential PRINS reaction, then detecting with a mixture of avidin-fluorescein/anti-dig-rhodamine or a mixture of anti-dig-fluorescein/avidin-rhodamine, the signals at the centromeres of three different chromosomes displayed perfect yellow, red and green colors, respectively. The entire procedure could be completed in less than 90 min because the blocking step was omitted. We showed that this is a practical and efficient way to carry out multiPRINS so that even more than three chromosome targets could be detected in the same cell.

Optimization of the fluorescence in situ hybridization (FISH) technique for high detection efficiency of very small proportions of target interphase nuclei.

Using commercially available fluorochrome-labeled probes specific for chromosomes X, Y, 13, 18, and 21, we optimized the technical protocols for fluorescence in situ hybridization (FISH) so that the highest sensitivity and specificity were achieved. Also, we compared the optical properties of different types of fluorescent labels in an effort to develop the most efficient FISH protocol, including the determination of which types of labels are the easiest to count accurately. The lymphocytes were purified from blood of normal male and female newborns, normal male and female adults, and a trisomy 21 male adult. Male and female lymphocytes were mixed in five different combinations. For each combination, the male lymphocytes either from newborns or from adults were diluted with female lymphocytes in seven different proportions. For each of these 35 different cell mixtures, 100,000 nuclei were analyzed and scored in a blind fashion. Among the different fluorochrome-labeled probes, the highest sensitivity and specificity were achieved when SpectrumAqua CEP-Y/SpectrumOrange CEP X probe mixture, SpectrumAqua CEP-18, SpectrumOrange LSI-13, and SpectrumOrange LSI-21 were hybridized. The hybridization sensitivity and specificity were higher than 99% for the identification of chromosomes X, Y, 13, and 18, and higher than 98% for the detection of trisomy 21. The proportion of false-positive signals was under 0.005% for XY detection and lower than 0.14% for autosome detection. With these high hybridization sensitivities and specificities, the optimized FISH protocol developed in our laboratory has the potential to detect very rare events, e.g., when the proportion of cells being sought is lower than 0.01%. In other words, our protocol allows the specific detection of one male cell sunken among 10,000 female cells.