Cytogenetics

For the past 4 weeks, I was attached to Cytogenetics Laboratory. In this entry, I will discuss about peripheral blood culture for chromosome studies.

Peripheral Blood Culture

Specimen Requirement

• 3 - 5ml of peripheral blood in sterile tube wuth sodium heparin as anticoagulant.
• Sterile techniques must be observed when collecting blood.
• The sample is sent to cytogenetics lab as soon as possible.

Clinical Significance

1. T-lymphocytes present in peripheral blood are exposed to a mitogen (e.g. PHA) to stimulate cell divison. They are then arrested at metaphase with the addition of colcemid. Chromosomes for analysis are obtained upon harvesting.
2. Clinical uses of constitutional karyotype obtained from blood cultures include:

• Genetics diagnosis/genetic counselling
• Determine the constitutional karyotype of families of individuals with chromosome abnormalities
• Determine carrier status
• Comparison of lymphocyte karyotypes with those of other tissue e.g. skin, AF or CV
• Confirmation of structural and numerical abnormalities or mosaisim
• Determine the constitutional karyotypes of patient with haematologic malignancies or solid tumors
• Aid in the diagnosis of chromosome breakage syndromes e.g. Fanconi Anaemia
• To provide material for metaphase FISH

Method

1. Thaw 1 tube of complete Rpml medium and 1 tube of complete M199 medium in water bath at 37ºC.
2. Add 0.1ml PHA into each tube.
3. Centrifuge blood specimen at 1200rpm for 10 minutes. Record volume of blood received and note down whether there is haemolysis, jaundice, clot etc. in worksheet.
4. With a sterile transfer pipette, gently pick up »1ml of buffy coat together with some plasma from the blood.
5. Invert transfer pipette and mix the sample thoroughly in the bulb. Distribute the sample into the culture tubes.
6. Tighten caps and mix well by inverting the culture tubes several times.
7. Place the culture tubes in a slant test-tube rack. The slant is at approximately 45º angle and the purpose is to increase the surface area of the culture for gaseous exchange. Leave the rack in a 37ºC incubator for 48 hrs.
8. Invert the tubes every morning to re-suspend pellet.
9. 48 hrs after culture set-up, add 50μl of MTX x 10-5 m working solution into each tube. Mix well by inverting the tubes and re-incubate at 37ºC for another 18 hrs.
10. After 18 hrs, add 50μl of thymidine to each tube.
11. Mix well and incubate at 37ºC for 4 hrs.
12. Proceed to harvesting.

Note: MTX is to block cells in the S (DNA synthesis) phase of cell cycle. Thymidine is to promote longer and better G-banded chromosomes in routine blood cultures.

Harvesting of Peripheral Blood Cultures

Harvest Process

1. Centrifuge tubes at 1200 rpm for 10 minutes. Aspirate supernatant.
2. Loosen pellet gently. Add approximately 8-10ml warmed hypotonic solution into each tube; invert tubes 2-3 times to ensure the suspension is well mixed.
3. At the beginning of handling the first tube, start a timer on 5 minutes counting down for every batch of »12 tubes. Leave the tubes at room temperature for the whole period of 5 minutes.
4. At the end of the 5 minutes, transfer tubes unto a covered centrifuge buckets and spin tubes at 1500 rpm for 6 minutes.
5. Aspirate and discard supernatant.
6. Repeat step 2 and 3.
7. At the end of the 5 minutes, add 1 pipetteful of fixative and invert tubes 2-3 times to mix well.
8. Repeat step 4 and 5.
9. Re-suspend cell pellet by flicking tubes until no pellet clumps are visible.
10. Add another 5 pipetteful of fixative. Invert tubes 2-3 times to mix well.
11. Stand the tubes at room temperature for at least 15 minutes. Aspirate and discard supernatant.
12. Centrifuge the tubes at 1000 rpm for 10 minutes.
13. Re-suspend pellet in 4 pipetteful of fixative. And repeat step 12. check that the suspension is clear after centrifugation, if not repeat this step again especially for clumpy and jelly-like pellets.
14. Re-suspend pellet in 3 pipetteful of fixative and store in refrigerator at 4ºC until ready to make slides. Proceed to slide making.
    

After slide making and staining, the slide is ready for chromosome analysis.
That’s all for this entry. Enjoy reading. Feel free to ask questions. =)

Doreen (tg 01)

p.s. for the interview question, the amniocytes are not lysed. Fixation can strengthen cell membranes, improve chromosome morphology, and rid the sample of debris such as lysed red cells. Hence, after fixation, the cells are ready for slide making and staining.