DIRECT AND SHORT-TERM PROCEDURE FOR HARVESTING
BONE MARROW CHROMOSOMES
To identify chromosome anomalies in hematopoietic cells. Used especially for chromosome studies for hematological disorders such as preleukemia and leukemia.
II. Culture Procedure:
A bone marrow aspirate is performed and 0.3 to 2.0 ml of early aspirate is collected in a sodium heparinized syringe. Invert the syringe several times to prevent clotting. If the specimen is clotted, additional heparin may be added when setting up the cultures to dissolve the clot. The specimen should be processed as soon after it is collected as possible. Assign an 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 success of this test, cultures are set up using different supplements to enhance the number and quality of metaphases. Using tape labels, label two centrifuge tubes (for direct harvest; 1 regular, 1 with EtBr) and two T-25 flasks (for short-term culture; 1 regular, 1 with GTC conditioned media) with the patient number, type of culture, culture identification letter, and day and time of harvest (short-term cultures will be harvested after 24 hours.)example:
Direct: Pipet 10 ml hypotonic soln into tube A, add 65 mcl colcemid working solution to the tube. Pipet 10 ml of complete media into tube B, add 45 mcl EtBr and 20 mcl 0.5X colcemid working solution. Add 7 to 9 drops of bone marrow aspirate to each tube; seal tubes tightly, invert to mix and incubate at 37 C. The A culture is incubated for 30 minutes, the B culture is incubated for 60 minutes. Centrifuge for 6 minutes at 150 x g. For A culture go to Harvest Procedure step G. For B culture go to Harvest Procedure step D.
Short-term: Pipet 10 mls of complete RPMI-1640 into each flask, add 1.0 ml of GTC to one flask; add 7 to 9 drops of bone marrow aspirate to each flask. Seal flasks tightly, mix gently, and incubate at 37 C for 24 hours. Cultures should be gently mixed 2-3 times per day during incubation. Note: For lymphomas, set up a 48 hr culture also.
III. Harvest Procedure:
A. 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.
B. Add 45 mcl EtBr working solution and 20 mcl 0.5X colcemid working solution to each flask. Incubate at 37 C for 60 minutes (75 minutes for the 24 and 48 hr cultures.)
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, make sure to remove the fat layer that will be at the top of the supernatent (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 G.
E. Add 10 ml of pre-warmed hypotonic solution colcemid working solution to each tube. Seal tightly and invert to mix. Incubate the tubes at 37 C for 15 minutes.
F. Centrifuge for 6 minutes at 150 x g.
G. 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.
H. 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.
I. Centrifuge for 6 minutes at 150 x g.
J. 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.
K. Repeat steps I,J 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 Making
A. For all CLL, do FISH to test for trisomy 12.
B. For all ALL, be sure to look at non-GCT cultures also.
Colcemid working solution: 10 mg Colcemid in 250 ml dH2O,
sterile filtered, store at 4 C.
Complete RPMI-1640: 100 ml RPMI-1640, 20 ml Fetal calf serum,
1 ml Penicillin/Streptomycin solution,
1 ml L-Glutamine solution, store at 4 C.
Ethidium Bromide working solution: 2 mg/ml Ethidium Bromide in RPMI - 1640, stored in dark.
Fixative: 30 ml Methanol and 10 ml Glacial Acetic Acid,
Hypotonic solution 0.075 M KCl: 2.8 g Potassium chloride dissolved in 500 ml dH2O.
Colcemid: GIBCO cat# 890-3014. 10 mg bottle.
Ethidium Bromide: SIGMA cat# E-8751. 2,7-diamino-10-ethyl-9-phenyl-phenanthridinium, 250 mg bottle, store in dark.
Fetal calf serum: GIBCO cat# 200-6140. Mycoplasma tested, virus screened, 500 ml bottle, store frozen.
Giant Cell Tumor conditioned Medium (GTC): IGEN. Inc cat# 50-0815, 50 ml bottle, store frozen.
L-Glutamine: GIBCO cat#320-5030. L-Glutamine solution 100X
(200 mM), 20 ml bottle, store frozen.
Glacial Acetic acid: AMERICAN SCIENTIFIC PRODUCTS cat# 9508-2. Acetic acid, Glacial ACS (Aldehyde free), 500 ml bottle.
Methanol: AMERICAN SCIENTIFIC PRODUCTS cat# 3016-1. Methyl alcohol anhydrous AR (ACS)(Absolute) Acetone free, 500 ml bottle.
Penicillin/Streptomycin: GIBCO cat# 600-5140. Penicillin/Streptomycin solution,
10,000 units/ml / 10,000 mcg/ml, 20 ml bottle, store frozen.
Potassium Chloride: COLUMBUS CHEMICAL INDUSTRIES, INC. ACS granular, 500 g lots.
RPMI-1640: GIBCO cat# 430-1800 RPMI-1640 powdered form, prepared to instructions, 10 x 1 liter package.
This is a revision of a methodology devised by Rebecca Cornacoff.