Procedure for Culturing and Harvesting

Unstimulated Peripheral Blood Cultures


I. Purpose

This test will occasionally reveal the presence of the Philadelphia chromosome or other anomaly in patients with acute leukemia. Success is contingent on the presence of mitotic immature granulocytes in the peripheral blood. Bone marrow specimens are more desirable, but when not available this method may be substituted. The main cultures are set up with out a mitogen, in addition a regular culture is set up to allow determination of the patient's constituative karyotype.

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, page 7.

B. Aseptic technique must be used when setting up cultures, preferably under a laminar flow hood. Using tape labels, label four T-25 flasks with the patient number, type of culture (1 regular and 3 unstimulated; 24, 48, and 72 hour), culture identification letter, and day of harvest (regular culture will be harvested after 3 days).

example:

Pipet 10 ml of complete RPMI-1640 into each flask. Using a syringe add 0.2 ml Phytohemagglutinin soln to the regular culture, do not add it to the UPB cultures. Using a sterile pipet add 0.5 ml of whole blood to each culture. Seal flasks tightly, mix gently, and incubate at 37 C. Cultures should be gently mixed 2-3 times per day during incubation.

III. Harvest Procedure:

A. 1/2 hour before harvest add 65 mcl of colcemid working solution to 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 in the Sorvall GLC-2B centrifuge).

D. Aspirate 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 immediately.

H. Centrifuge for 6 minutes at 150 x g. Aspirate and discard all but 0.5 ml of supernatant, resuspend the cell pellet.

I. 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.

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.

L. 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 slides.

B. Centrifuge the cultures for 6 minutes at 150 x g.
C. Remove all but 0.2 - 0.4 ml of supernatant. Resuspend the cell pellet.

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 40 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.

F. Label slides with patient number, culture identification letter, and slide number.

G. Allow slides to age overnight at room temperature. Slides are now ready for G, Q, C, or NOR banding.

H. 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.

V. Solutions:

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.

Fixative: 30 ml Methanol and 10 ml Glacial Acetic Acid,
prepared fresh.

4% Acetic acid: 2 ml Glacial Acetic acid in 48 ml dH2O,
prepared fresh.

Hypotonic solution 0.075 M KCl: 2.8 g Potassium chloride dissolved in 500 ml dH2O.

Phytohemagglutinin working solution: Phytohemagglutinin (M form) rehydrated with 10 ml sterile dH2O, store at 4 C.

VI. Reagents:

Colcemid: GIBCO cat# 890-3014. 10 mg bottle.

Fetal calf serum: GIBCO cat# 200-6140. Mycoplasma tested, virus screened, 500 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.