Cytogenetics

Hi everyone, I’m Justin here and I’m posted to cytogenetics lab for 20 weeks. The things I learnt are quite interesting as most of the stuff is not taught at school. For week 1 and 2, I’ve been observing procedures; reading documents; and practicing how to pair and arrange chromosomes. This week, I started performing some procedures.

Cytogenetics is the study of chromosome number, morphology, and the abnormalities that can be correlated to diseases. The lab I’m in is classified into 3 areas. Namely, Prenatal, which handles amniotic fluid and chorionic vili for diagnosis of chromosomal disorders ; Bone marrow, which handles bone marrow, bone core, and peripheral blood samples for haematological disorders; and FISH(fluorescence in situ hybridization), which uses specific molecular probes linked to fluorescent dyes to detect conditions eg. deletion syndromes. I’m in the bone marrow area, and will talk about the workflow for producing a karyotype, with some bone marrow specific procedures.

Sample: Bone Marrow/ Bone Core

When the sample arrives and test requisition has been performed, the first part of the process is the culture set up. The samples will be centrifuged and the buffy layer is obtained. 2 cultures will be set up. The type of cultures set up (direct harvest; 24-hr; 48-hr; 72 hr with mitogens) depends on the suspected diagnosis of the patient. The reason is that certain abnormal cell clones can be detected immediately but others need a longer time to grow, hence the need for 2 cultures and disease specific durations. The addition of mitogens (growth factors) allows the stimulation of specific clones. For example, in suspected cases of CLL (chronic lymphocytic leukemia), the 2 cultures set up would be 72-hr with TPA and PHA. TPA and PHA are growth factors specific for B-cells and T-cells respectively. This is so that we can determine the exact cause of the CLL (whether it’s due to B or T cells). For direct harvest, the harvesting process is done immediately (see next step), while for 24, 48, and 72 hour cultures are prepared, by inoculating the buffy coat into complete RPMI 1640 media (used by my lab, contains nutrients for bone marrow cell culture), and incubating them for the respective durations in a 37ºC CO2 incubator. I’ll go into more specific details of culture durations and procedures in subsequent posts.

The next part is the harvesting, which are the processing steps so as to obtain metaphases for chromosomal analysis. The first step is the mitotic arrest. Cell cycle is arrested at metaphase using a reagent known as colcemid. Colcemid inhibits spindle fiber formation by depolymerising tubulin, thus preventing the progress of the cell cycle. The next step is hypotonic treatment. A hypotonic solution (potassium chloride is used in my lab) causes water to enter the cell. This allows cells to swell and allows the chromosomes to spread out for easier analysis. In my lab the colcemid and potassium chloride is incorporated into 1 solution-“direct-harvest media”. The third step is fixation. The fixative used is a modified Carnoy’s fixative (without the chlorofoam?), which consist of methanol and acetic acid in the ratio of 3:1. The purpose of fixation is to harden cells and chromosomes, making them resistant to changes, and allowing for staining. The fixative also destroys debris (of cell clots etc), thus allowing a clearer suspension for easier analysis.

After harvesting, fixed cells are dropped onto slides so that they can be analysed. The slides must be grease and dirt free as other particles may interfere with analysis. The slides are then baked in an oven at 90ºC for 2 hours. The purpose of baking as that it gives better contrast for staining and allows the chromosomes to be stained. For staining, the most common staining method for routine analysis is G banding by trypsin using Giemsa and Wright’s stain (GTG banding). This staining allows chromosomes to be individually identified. The enzyme trypsin allows the staining dyes to enter the chromosomes.

After staining, the slide is sent for analysis. 20 metaphases will be analysed. The use of 20 metaphases is to ensure that an extra chromosome in one metaphase is not due to some random error (eg. overspreading because hypotonic treatment too long). A minimum of 5 metaphases must be karyotyped for analysis. Karyotyping refers to the arranging and pairing of chromosomes in order (1-22, XX or XY) Karyotyping can be done through computer programs or manually, in which the photo of the metaphase is taken and chromosomes cut out by hand (I do that as part of training for identifying chromosomes).

In terms of clinical significance, the use of bone marrow specimens allows for the diagnosis and prognosis of haematological disorders. This is because consistent chromosomal abnormalities will be associated with a particular disease. For example, in patients with Chronic Myelogenous Leukemia (CML), the 9-22 translocation will be observed (t(9;22)(q34;q11)) in abnormal cells. For prognostic information, a change in karyotype will show it. In patients with CML, if an extra chromosome 8; chromosome 19 or Philadelphia chromosome is observed, it indicates progression to the blast crisis of CML, whereby the prognosis is poor and the survival rate for patients is about 2-5 months (refer to MBIO lecture notes topic 5).

That’s all for now. I’ll go into more specific details of each stage in subsequent posts. Sorry for the long post. Enjoy your SIP !

Ng Tze Yang Justin
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