Bone marrow culture set-up

Hi everyone, I’m Justin here again. Today I’m going to talk about bone marrow culture set-up. This step involves the initiation of cultures by inoculating cells into the media. The specimen is bone marrow aspirate in sodium heparin anti-coagulant (the green top). Other types of anti-coagulants should not be used as they will affect cell viability. The purpose is to get viable cells that are dividing.

The specimen is centrifuged at 1500rpm for 10 minutes and the buffy layer is obtained. The buffy layer is the layer in between the red cells and the plasma portion. The amount of buffy layer to inoculate depends on its thickness and the WBC count. It is very important to inoculate the correct amount of cells. Adding too much cells will cause nutrients to be depleted fast, resulting in cell death too early or stopping of mitotic activity. Adding too little cells will result in not enough metaphases for analysis. As a guide from the technical procedures manual, if the layer is more than 1 mm thick, use ½-1 drop. If it is about 1 mm thick, use 2 to 3 drops.

The duration of culture depends on the suspected diagnosis. The purpose of using 2 durations is because certain abnormal clones can be found immediately while others need more time to grow. For most cases, setting a 24 and 48 hour culture will be appropriate to yield the abnormality present. Only abnormal clones show the specific cytogenetic abnormality.

For conditions such as AML (acute myeloid leukemia); CML (chronic myeloid leukemia); (MPD) myeloproliferative diseases eg, polycythemia vera, a 24 and 48 hour culture will be set up.

For cases of chronic lymphocytic leukemia (CLL), mitogens are added to stimulate specific clones as clones are dividing very slowly. TPA (Tetradecanoylphorbol acetate) and PHA (phytohemagglutinin) are growth factors specific for B-cells and T-cells respectively. For new cases of CLL the 2 cultures are 72 hours with TPA and PHA so as to determine the specific cause, whether the CLL is due to B-cells or T-cells. For follow up cases, a 48 hour culture is set up and another culture is the 72 hour with the specific mitogen.

For cases of Multiple myeloma (MM), a direct harvest is done and a 72 hour culture with Il-6 is set. The direct harvest is done so as to get abnormal clones characteristic of MM present that are aggressively dividing. They will die off fast, thus the need for the direct harvest. The 72 hour culture is set up and because other clones divide very slowly. Il 6 is a cytokine which would help abnormal clones proliferate.

The culture media used is the RPMI 1640 complete culture media. The media is supplemented with L-glutamine, antibiotics, and fetal bovine serum. L-glutamine is an essential amino acid. Fetal bovine serum provides proteins and growth factors. Antibiotics such as penicillin and streptomycin inhibit the growth of bacteria in the culture.

Cultures are incubated for their respective durations in a 37ºC incubator in 5% CO2. The CO2 allows the pH of cultures to be in equilibrium. The temperature of incubation at 37 ºC is the physiological body temperature which is optimal for growth of human tissues.

That’s all for now. Take care everyone ! All the best for MP and the remaining SIP !

Ng Tze Yang Justin
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