Flow cytometry based micronucleus assays and kits
View Patent ↗The invention is a method for analyzing immature reticulocytes for the presence of micronuclei. The method includes reticulocyte enrichment, fluorescent labeling, micronuclei staining, and analysis using single-laser flow cytometry. The invention also includes kits containing reagents to use in the method.
1. A method for analyzing immature reticulocytes for the presence of micronuclei comprising the steps of:
a) immuno-magnetically isolating reticulocytes from a human blood cell sample by contacting the sample with first reticulocyte surface protein—selective first antibody coated, flow cytometry-compatible magnetic beads, under conditions wherein the first antibody selectively binds the reticulocytes, applying a magnetic field to the sample to magnetically segregate antibody-bound reticulocytes, and washing away unsegregated material;
b) labeling magnetic bead-bound reticulocytes of step a) with a fluorescently labeled second reticulocyte surface protein—selective second antibody;
c) permeabilizing the magnetic bead-bound reticulocytes of step b) to an RNase and a DNA-staining fluorescent dye, and then treating the reticulocytes with the RNase to prevent RNA-staining from interfering with analysis of the micronuclei and staining micronuclei of the reticulocytes with the DNA-staining fluorescent dye, wherein the dye and the fluorescently-labeled second antibody have overlapping absorption spectra, and distinguishable emission spectra; and
d) analyzing the labeled magnetic bead-bound reticulocytes of step c) for the presence of stained micronuclei using single-laser flow cytometry;
wherein the first and second reticulocyte surface proteins are independently selected from the group consisting of transferrin receptor (CD71), glycophorin A (CD235a), thrombospondin (TSP) receptor (glycoprotein IV; CD36), and integrin α4β1(very late activation antigen-4(VLA- 4 ), and CD49d/CD29).
2. The method of claim 1 wherein the beads have a diameter of 1 μm.
3. The method of claim 1 wherein the first reticulocyte surface protein is selected from the group consisting of CD36, CD71, and VLA-4.
4. The method of claim 1 wherein the first reticulocyte surface protein is CD71.
5. The method of claim 1 wherein the beads are BIOMAG Anti-CD71 beads (BIOMAG is a trademark of Polysciences, Warrington, Pa.).
6. The method of claim 1 wherein the beads are polynucleotide-free.
7. The method of claim 1 wherein the sample is whole peripheral blood.
8. The method of claim 1 wherein the sample is obtained by centrifuging whole blood and discarding plasma and buffy coat layers from the sample.
9. The method of claim 1 wherein the first reticulocyte surface protein is selected from the group consisting of CD71, CD36, and CD49d/CD29; and the second reticulocyte surface protein is selected from the group consisting of CD71, CD235a, CD36, and CD49d/CD29.
10. The method of claim 1 wherein the second reticulocyte surface protein is CD71.
11. The method of claim 1 wherein the second antibody is labeled with fluorescein isothiocyanate (FITC).
12. The method of claim 1 wherein the DNA staining dye is 7-aminoactinomycin D.
13. The method of claim 1 wherein in the analyzing step, an argon ion laser is used.
14. The method of claim 1 wherein: the sample is obtained by centrifuging whole blood and discarding plasma and buffy coat layers from the sample, the first and second surface proteins are both CD71, the beads are polynucleotide-free and have a diameter of 1 μm, the second antibody is labeled with FITC, the DNA staining dye is 7-aminoactinomycin D, and an argon ion laser is used in the analyzing step.