Date of Award
Doctor of Philosophy
Dr. Gyula Ficsor
Dr. Ronald J. Flaspohler
Dr. Leonard C. Ginsberg
Dr. Paul Lang
The differential staining of chromosomes with the use of 5-bromo-2'-deoxyuridine (BUdR) was discovered in 1972. Since then, the BUdR technique has been modified and improved. It is used to study cellular kinetics; yet its most extensive use today is focused on studying sister chromatid exchange (SCE). Studies show that the frequency of SCEs increases significantly in the presence of mutagenic and carcinogenic agents. Hence, the BUdR technique (commonly referred to as the SCE technique) has been useful as a diagnostic tool to test for mutagenic anc carcinogenic agents.
Investigators have used differential staining to obtain a baseline incidence of SCEs in human lymphocytes; but their results show a wide variation. This makes the testing of mutagenic and carcinogenic agents problematic. A more basic problem encountered in using the SCE technique is that theyield of differentially stained chromosomes is too low for analytical purposes.
The present study is designed to standardize the differential staining technique so that the problem of low yield can be overcome. This should help tackle the problem of establishing the baseline incidence of SCEs. The three specific objectives of this research, therefore, are to: (a) establish culture conditions that yield a high frequency of metaphase spreads; (b) determine the time of initiation of DNA synthesis; and (c) establish staining conditions that yield an adequate number of differentially stained chromatids for SCE analysis.
These objectives were accomplished as follows. To obtain optimal cell proliferation, Ham's F-10 medium was selected out of five commonly used media on the basis of its high mitotic index (MI) result. The initial lymphocyte concentration was kept at 3.5 x 10('5) per ml. Cell proliferation was measured by determining the amount of tritiated thymidine--('3)(H)-TdR--incorporated into the DNA. Optimal phytohemagglutinin (PHA) concentration was found to range between .36 and .84% (It was discovered that the optimal range of PHA concentration varied from one lot to another). PHA concentration of .48% was used in all further experiments. Optimal FCS concentration was found to be 13%.
Using three optimal conditions, the time of initiation of DNA
synthesis in human lymphocytes was deterined by ('3)(H)-TdR
incorporation as well as a flow cytometer. The first method indicated
a range of 30-40 h, the second indicated a range of 40-50 h. Thus it was found that BUdR should not be added later than 30 h after culture initiation to ensure its incorporation into the DNA.
Using 10 (mu)g/ml of BUdR, clear differentiation of the maximum number of chromosomes was obtained under the following regimen:
Hoechst 33258 50 (mu)g/ml
Soaking time in Hoechst 33258 10 min
Slide warmer (@ 55(DEGREES)C) 10 min
Black light exposure 50 min
Giemsa (3%) staining 15 min.
A procedure for standardizing the differential staining technique in human lymphocytes has been established in this research. It should prove useful in obtaining uniform in vitro culture conditions generally. Moreover, it should help to make SCE analysis a more reliable diagnostic tool for mutagen and carcinogen testing.
Sharma, Kamlesh, "Standardization of the Sister Chromatid Exchange Technique in Human Lumphocytes for Mutagen and Carcinogen Testing" (1981). Dissertations. 2567.