A. To establish the presence of fragile (fra) sites on chromosomes. Fragile
sites are defined as non-staining gaps of variable width, usually involving both
chromatids, but always occurring at the same chromosome loci and are inherited
in a Mendelian fashion.
B. Fragile sites are best shown by culturing
cells in medium with a folic acid antagonist such as 5-Fluoro-deoxy-uridine
(FUDR) or in media with a DNA synthesis inhibitor such as excess
C. This method is especially useful for establishing the
presence of a fragile site at Xq27 in patients with X-linked mental
II. Culture Procedure:
A. Obtain 2-4 ml of unclotted whole blood aseptically by venipuncture using a
collection tube with sodium heparin. Invert the tube several times to prevent
clotting. Assign accession number to specimen according to procedure in
Laboratory Procedures Manual.
B. Aseptic technique must be used when
setting up cultures, preferably under a laminar flow hood. To maximize the
accuracy of this test, cultures are set up in both folate-deficient medium and
regular medium with a folic acid antagonist. Using tape labels, label three T-25
flasks with the patient number, type of culture (1 flask 0.1 mcM FUDR, 1 flask
0.2 mcM FUDR, 1 flask Excess Thymidine), culture identification letter, and day
of harvest (cultures are harvested after 3 days).
Pipet 10 ml of complete RPMI-1640 media into each
flask. Using a syringe add 0.2 ml of Phytohemagglutinin solution to all three
flasks. For the folic acid antagonist cultures, add 0.1 ml of FUDR to the first
flask and 0.2 ml of FUDR to the second flask. Using a sterile pipet add 0.5 -
0.7 ml of whole blood to each culture. Seal flasks tightly, mix gently, and
incubate cultures at 37 C for 72 hours. Add 0.1 ml Thymidine working solution to
Excess Thymidine culture after 48 hours. Cultures should be gently mixed 2-3
times per day during incubation.
III. Harvest Procedure:
A. 1 1/2 hour before harvest add 65 mcl of colcemid working solution to each
culture flask and mix.
B. Put the 0.075 M KCl hypotonic solution in the
incubator to pre-warm. Prepare fixative of methanol:glacial acetic acid (3:1).
Usually 40 ml of fix will be used per patient.
C. Gently swirl the
flasks. Pour the contents of each flask into a 15 ml screw-cap conical
centrifuge tube; transfer the tape label making sure no label mistakes occur.
Centrifuge the tubes for 6 minutes at 150 x g (900 rpm).
D. Aspirate and
discard all but 0.5 ml of supernatant, (note that the white blood cells will
form a layer on top of the red cells and will be lost if too much supernatant is
removed). Resuspend the cell pellet by mixing by hand, do not use a mechanical
vortexer as this may break cells, this is especially important in step F. Add 5
ml of pre-warmed hypotonic solution and mix. If necessary, use a pasteur pipet
to bubble air through the suspension to completely break up the pellet. Incubate
the tubes at 37 C for 15-30 minutes.
E. Centrifuge for 6 minutes at 150 x
F. Aspirate and discard all but 0.5 ml of supernatant. Mix by hand to
break up the cell pellet. It is important to get the cell pellet completely
resuspended, but cells are fragile and are easily broken.
G. Slowly add
4 ml of fixative (2 pasteur pipetfuls) letting it run down the side of the tube
so that it layers on top of the cell suspension. Cap the tube. Mix rapidly by
hand so that all the cell suspension is fixed evenly; if it is mixed too slowly,
clumps may form. Let sit at room temperature for 15 - 20 minutes.
Centrifuge for 6 minutes at 150 x g.
I. Aspirate and discard all but 0.5
ml of supernatant. Resuspend cell pellet. Add 4 ml of fixative and mix. Let sit
at room temperature 15 - 20 minutes.
J. Repeat steps H,I 1 or 2 times
until the pellet is clean and white. Culture is now ready to make slides. For
best results, slides should be made the same day as the harvest.
See Regular Procedure for Culturing Lymphocytes.
See Regular Procedure for Culturing Lymphocytes, plus:
See Regular Prodedure for Culturing Lymphocytes, plus:
FuDr: Sigma cat # F0503
Thymidine: SIGMA cat# T-9250, 5