High Resolution Procedure for Culturing & Harvesting Lymphocyte Chromosomes


I. Purpose

Analysis of chromosomes of lymphoblast cells from peripheral blood to detect small structural rearrangements, duplications, or deletions that may result in an abnormal phenotype or increased fetal wastage.

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. Since not all patients will work adequately with the high-resolution cultures, one culture should be set up as a regular culture for back-up. Using tape labels, label one T-25 flasks for using EtBr during culture with the patient number, type of culture, culture identification (start with R), and day of harvest (cultures will be harvested after 3 days).

example:

Label one flask for using Fudr during culture.
example:

Label one flask for using both Fudr and EtBr during culture (start with M):
example:

Pipet 10 ml of complete RPMI-1640 into each flask. Using a syringe add 0.2 ml Phytohemagglutinin soln. To the two Fudr cultures, add 0.1 ml stock Fudr. Using a sterile pipet add 0.5- 0.7 ml 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. 2 hours before harvest add 65 mcl Ethidium Bromide working solution to the EtBr cultures, 30 minutes before harvest add 65 mcl colcemid working solution to each high-resolution culture.

B. 1 1/2 hour before harvest add 65 mcl of colcemid working solution to the Fudr culture flask.

C. 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.

D. 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).

E. 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 G. 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.

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. For baby blood (new born to 6 months old) it is necessary to prefix the cells. For non-babies go to step J.

H. Slowly add 4 mls of 4% acetic acid solution to each culture, mix quickly and thoroughly. Centrifuge immediately.

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

J. 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.

K. Centrifuge for 6 minutes at 150 x g.

L. 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.

M. Repeat steps K,L 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:

See Regular Procedure for Culturing Lymphocytes.

V. Solutions:

See Regular Procedure for Culturing Lymphocytes

VI. Reagents:

See Regular Prodedure for Culturing Lymphocytes