Analysis of chromosomes of lymphoblast cells from peripheral blood. Usually
used for confirmation of trisomies or sex chromsome aneuploidy; for other
diagnoses, high resolution studies should be performed.
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. Using tape labels,
label three T-25 flasks with the patient number, type of culture, culture
identification letter, and day of harvest (cultures will be harvested after 3
Pipet 10 ml of complete
RPMI-1640 into each flask. Using a syringe add 0.2 ml Phytohemagglutinin soln.
Using a sterile pipet add 0.5 - 0.7ml of whole blood to each culture. Seal
flasks tightly, mix gently, and incubate at 37 C for 72 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).
and discard all but 0.5 ml of supernatant, (note: the white blood cells 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 g.
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. For baby blood (new born to 6 months old) it is necessary to prefix
the cells. For non-babies go to step I.
G. Slowly add 4 mls of 4%
acetic acid solution to each culture, mix quickly and thoroughly. Centrifuge
H. Centrifuge for 6 minutes at 150 x g. Aspirate and
discard all but 0.5 ml of supernatant, resuspend the cell pellet.
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
J. Centrifuge for 6 minutes at 150 x g.
K. 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.
Repeat steps J,K 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.
IV. Slide Preparation:
A. Use frosted-end slides that have been rinsed with dH2O and stored at
4 C. It may be helpful to warm the slides to room temperature before making
B. Centrifuge the cultures for 6 minutes at 150 x g.
Remove all but 0.2 - 0.4 ml of supernatant. Resuspend the cell
D. Add fixative to dilute the cell suspension, 0.2 - 1 ml may
be required depending on the quantity of cells.
E. Using a pasteur
pipet, place 2 or 3 drops of cell suspension near the frosted end of the
slide. Spread cells by tilting the slide and blowing gently. Wipe dry the back
of the slide and place it on the slide warmer at 38 C for 1- 2 minutes until
the slide is dry. It may be necessary to adjust the temperature of the wet
slides and time on slide warmer to maximize cell spreading.
slides with patient number, culture identification letter, and slide
G. Note the number of slides made from each tube on the tape
label of the tube.
H. If possible, allow slides to age overnight at
room temperature (for rush patients, that may not be possible.) Slides are now
ready for G, Q, C, or NOR banding.
I. After making slides, add a
pipetful of fixative to each tube, cap tightly and store at 4 C for short term
or -20 C for long term.