Simultaneous immunophenotyping and chromosomal FISH.
(A) Primitive ε-positive fetal erythroblast at 10 weeks' gestation stained with AMCA. Cell morphology is well preserved. No DAPI has been used as counterstain because accumulation of AMCA around the nucleus acts as an excellent counterstain. (B-D) Fetal whole blood at 9 weeks' gestation. (B) Use of the Spectrumaqua channel allows the visualization of all heme-containing cells that autofluoresce. One NRBC is positive for ε and the other is negative (arrow); (C) the same group of cells viewed with the red and green filters switched on to show X and Y signals; and (D) the same group of cells viewed with the Spectrumaqua filter off. (E, F) Representative ε-positive and ε-negative cells as seen in a representative mixing experiment of male fetal erythroblasts in never-pregnant adult female nucleated cells in a ratio of 1:10−5. (E) ε-Globin–positive male fetal erythroblast. (F) Nearby ε-globin–negative female nucleated cell. The autofluorescence of the cell through the green channel was deliberately potentiated to demonstrate the outline of the female leukocyte. (G) Mixing experiment of ε-globin–positive male fetal erythroblast with K562 cells cultured in the absence of hemin. One of the X chromosome signals in both neighboring K562 cells are beyond the focal plane captured. (H) Mixing experiment of first trimester, ε-positive, male fetal erythroblasts with female adult bone marrow–derived erythroblasts. Fetal ε-positive NRBC stained with AMCA is clearly distinguished from the AMCA-negative cell. Two X signals are visible in the female erythroblast with demonstrable autofluorescence of heme through red. The Y probe is easily visualized in the male erythroblast, but the X signal is only barely visible at the nuclear periphery in this focal plane. All fluorescence images represent a single optical plane.
