GENOTOXICITY ASSAY WITH CYTOPLASM BOUNDARY LABELING
A method of detecting chromosomal damage in a subject is described that includes the steps of stimulating proliferation of the lymphocytes of a blood sample from a subject by contacting them with a labeled mitogen; growing the stimulated lymphocytes in cell culture; contacting the lymphocytes with a mitotic arrest agent to arrest the lymphocytes as binucleate lymphocytes; staining the lymphocytes with a DNA binding dye; detecting labeled mitogen to define the cytoplasm boundary of binucleated lymphocytes; counting the micronuclei in one or more binucleated lymphocytes to obtain an observed micronuclei value; and determining the level of chromosomal damage in the subject by comparing the observed micronuclei value with a control micronuclei value. The method can be used to conduct biological dosimetry on subjects who are suspected of having been exposed to radiation.
1 . A method of detecting chromosomal damage in a subject, comprising the steps of:
a) stimulating proliferation of the lymphocytes of a blood sample from a subject by contacting them with a labeled mitogen;
b) growing the stimulated lymphocytes in cell culture;
c) contacting the lymphocytes with a mitotic arrest agent to arrest the lymphocytes as binucleate lymphocytes;
d) staining the lymphocytes with a DNA binding dye;
e) detecting labeled mitogen to define the cytoplasm boundary of binucleated lymphocytes;
f) counting the micronuclei in one or more binucleated lymphocytes to obtain an observed micronuclei value; and
g) determining the level of chromosomal damage in the subject by comparing the observed micronuclei value with a control micronuclei value.
2 . The method of claim 1 , further comprising the step of obtaining a blood sample from the subject.
3 . The method of claim 1 , further comprising the step of isolating lymphocytes from the blood sample before contacting them with a labeled mitogen.
4 . The method of claim 1 , wherein the mitogen is concanavalin A.
5 . The method of claim 1 , wherein the mitogen is labeled with a fluorescent compound.
6 . The method of claim 1 , wherein the mitotic arrest agent is cytochalasin D.
7 . The method of claim 1 , wherein the DNA binding dye is 4′,6-diamidino-2-phenylindole (DAPI).
8 . The method of claim 1 , wherein the subject is suspected of having been exposed to a hazardous level of radiation.
9 . The method of claim 1 , wherein the subject is human.
10 . The method of claim 1 , further comprising the step of fixing the lymphocytes to a substrate before staining the lymphocytes.
11 . The method of claim 9 , wherein the substrate is a glass microscope slide.
12 . The method of claim 1 , further comprising the step of detecting the presence of a nucleoplasmic bridge in one or more binucleated lymphocytes.
13 . The method of claim 1 , wherein blood samples from a plurality of subjects are evaluated using the method.
14 . The method of claim 1 , wherein one or more of the steps are automated.
15 . The method of claim 1 , wherein the step of counting the micronuclei in one or more binucleated lymphocytes uses an algorithm for automated image analysis.
16 . The method of claim 1 , wherein the method of detecting chromosomal damage in a subject further comprises translocation analysis of lymphocytes from a blood sample from the subject.