Hi everyone, time flies and our sip is ending soon. Anyway, for this month, I was posted to the cytogenetics laboratory which I found it very interesting because this subject is very new to me. In this laboratory, I can analyze my own chromosomes using peripheral blood and fortunately, my chromosomes turned out to be normal. Now, I am going to talk about the procedures to culture peripheral blood for chromosomes studies.

The purpose to culture peripheral blood for chromosomes studies (karyotyping) is to diagnose genetic diseases for e.g. Down’s syndrome, determine the karyotype of families of individuals with chromosome abnormalities, determine carrier status, to confirm structure, numerical abnormalities and/or mosaicism etc. The principle of this test is that the white blood cells (T-lymphocytes) present in the peripheral blood will be exposed to a mitogen (PHA). The function of the mitogen is to stimulate cell division and the cells are arrested at metaphase by the addition of colcemid. The purpose of arresting at metaphase is because at the metaphase phase stage, the cells are at their most contracted stage and the least metabolitically active state and thus chromosomes are analyzed at this stage. Theses metaphases are harvested and spread onto a glass slide for analysis.

The reagents used are:
•Complete RPMI media which contains RPMI, fetal calf serum, penicillin and streptomycin and L-Glutamine
•Complete M199 media which contains M199, fetal calf serum, penicillin and streptomycin, L-Glutamine and sodium heparin
•Sodium heparin which contains sodium heparin and sterile water
•Phytohaemagglutinin (PHA) M Form
•Amethopterin (Methotrexate) (MTX)
•Thymidine

Specimen requirement: 3-5ml of peripheral blood in sterile tube with sodium heparin (anti-coagulant). Collection of blood is done using aseptic techniques.

Method:

•1 tube of complete RPMI medium and 1 tube of complete M199 medium were thawed in water bath at 37ºC.
•The blood specimen was centrifuge at 1200rpm for 10minutes. The volume of blood and condition of blood was recorded.
•The tubes were labeled with the patients’ accession number, patient’s name and date of set-up.
•A sterile transfer pipette was used to pick approximately 0.5-1ml of buffy coat together with some plasma from the blood sample.
•The transfer pipette was inverted and the sample was mixed thoroughly in the bulb. The sample was distributed into the culture tubes and the number of drops added into each tube was noted down.
•0.1ml of PHA was added into each tube.
•The caps were tighten and mixed well by inverting the culture tubes several times.
•The culture tubes were placed in a slant test tube rack to increase the surface area of the cultures for gaseous exchange. The rack was placed in a 37ºC incubator for 48hours.
•The tubes were inverted every morning to resuspend the pellet.
•50µl of MTX 10-5M working solution was added to each tube after 48 hours of incubation. The tubes were mixed well by inverting and were re-incubated at 37ºC for another 18hours.
•50µl of thymidine was added to each tube after 18 hours of incubation with MTX.
•The samples were mixed well and incubated at 37ºC for 4hours.

The function of MTX is to synchronize the cells by stopping the cells in the S phase of cell cycle and collect a large amount of cells which will then go into division together. Thymidine is used to release cells blocked by the MTX.

There are guidelines to determine the amount of blood needed to add to each tube. For neonates less than one year, 4-5 drops was added. For children 1-6years old, 5-6 drops was added. For children 7-13years old, 6-7 drops was added. For males older than 13years old, 7-8 drops was added and for females older than 13years old, 8-12 drops was added.

After blood culturing, the next step will blood harvesting. I will leave the harvesting procedure to the next person who will go to cytogenetics laboratory after me. :)

Rachael
Tg01
0606168C