Explaining the whole karyotyping technique and procedure

The process of separating and observing chromosomes is known as karyotyping (collectively) includes cell culture, incubation, cell harvesting and slide preparation.

Genetic techniques are of two types; cytogenetics and molecular genetics. Karyotyping, fluorescence in situ hybridization and chromosome microarray like techniques are categorized in cytogenetic techniques while techniques like Polymerase chain reaction, DNA sequencing and restriction digestion can be categorized into the molecular genetics.

The molecular genetic techniques have evolved recently which are capable of examining DNA at the molecular level. On the other side, the cytogenetic techniques can’t be directly related to DNA but we can examine chromosomes and related alterations.

If we differentiate cytogenetics and molecular genetics in terms of technique and process, the cytogenetic techniques are time-consuming, lengthy, laborious and costlier.

This website or blog is for those who want to learn only cytogenetics and karyotyping. Here in the present article, we are explaining the entire procedure of karyotyping and the importance of different steps included in it.

The procedure of karyotyping:

The kartyoping is a combination of technique which can’t be performed using a single technique. Techniques used in the karyotyping are:

Sample collection
Cell culture
Cell harvesting
Microscopy and analysis of results
Preparing a karyogram

This infographics can help you to understand it better: Steps and Procedure of Karyotyping.

Sample collection:

Requirements: syringe and needles, Heparin sample collection tube, cotton, spirit or alcohol.

Blood, bone marrow, amniotic fluid, solid tissue, tumor, chorionic villi, a product of conception are various sample types used for karyotyping. Though the sample collection technique is different from sample to sample.

Blood sample:

Using a sharp needle, approximately 5ml blood is collected from a patient in a heparin tube.

Bone marrow: The bone marrow is present in the hollow part of leg or hand bones. A collector inserts a hollow needle into the bone and collects samples in a tube approximately 5 to 10 ml.

Tumor or solid tissue sample: The sample collector makes a minor incision and surgically removes a small portion of a solid tumor or tissue in a collection tube. The tube usually contains special preservation and transport media for a tissue.

Amniotic fluid of chorionic villi: A fetus sample is collected either by chorionic villi or amniotic fluid- known as amniocentesis.

Here a long sharp is inserted through the abdomen and uterus up to the amniotic sac without hurting a baby.

A few amounts of tissue or fluid is collected carefully for karyotyping analysis.

Once the sample is collected, the sample is transported under 4C temperature. Note that for karyotyping the sample must be sent to the laboratory within 24 hours.

Once the sample is received to the laboratory immediately it is processed for cell culture.

Cell culture:

The cell culture is a technique to grow the metaphase cells under aseptic lab conditions.

Requirements: Cell culture media with lactin, PHA-P, fetal bovine serum, vitamins and other nutrients, colchicine, culture tubes, falcon tubes, pipettes and tips of various sizes, stands.

Instruments: Laminar air flow, incubatory, deep freeze and refrigerator.

Cell harvesting:

Once the cell culture is completed successfully, the samples or culture tubes are removed from the incubator and processed for harvesting.

Cell harvesting is a process in which cells are isolated and cleaned using a special type of physical and chemical treatments.

Requirements: Glacial acetic acid, methanol, falcon tubes, discarder, pipettes, pasteur pipettes, othe glass and plastic wares.

Instruments: Centrifuge, water bath automated spinner or mixture.

Microscopy:

To analyse chromosomes and identify anomalies microscopic analysis is performed.

Requirements: Microscope, giemsa stain, slides and coverslip, pasteur pipettes.

A slide using the Giemsa stain is prepared and observed under the microscope to evaluate chromosomal anomalies.

Preparing a karyograms:

The process of preparing a karyotyping to interpret the results is known as kargogram.

Requirements: Scissor, paper, glue and other related things.

The procedure of karyotyping technique:

The sample is collected as per notified in the sample collection section.

The sample is cultured under strict aseptic conditions using ready to use or pre-prepared culture media. All the steps for culturing are performed in the laminar airflow or class III safety cabinet.

Laminar air flow or class III safety cabinet:

The laminar air flow or the class III safety cabinet is an instrument that provides high end aseptic conditions to culture cells.

One of the major limitations of the cell culture is the contamination, cells get contaminated with other bacteria or cells. Also known as ‘culture hood’, It has a special type of set up to make the air, working place and environment clean and clear for any type of cell culture.

The HEPA filter provides clean air while the UV light present on the top destroys all the viruses and bacteria inside the hood. The process to use the LAF:

Clean the workbench using pure alcohol.
Make it dust free.
Plugin the power supply and switch on the laminar.
Turn on the UV light for 30 or 40 minute to make it free from any microbes.
Turn off the uv light and turn on the main light. Also, turn on the air flow filter.
Open the hood of laminar and place all the chemicals and utilities you required for cell culture. Note that all the utilities should to pre-clined, or autoclaved.

The desired amount of sample as well as other chemicals are inserted into the culture flask or you can use falcon tubes. Close the lid of tubes and place all the culture in the incubator.

Incubate as per the protocol, usually, at 37C temperature for 72 hours.

Incubator:

The incubator is a special type of instrument that maintains the biological conditions for cell culture. It maintains conditions like temperature, humidity and environment for a culture.

Usually for cell culture the CO2 incubator is widely used. That can also provide the CO2 in the culture. It provides constant 37C temperature conditions to grow cells. The process to use it:

Plugin the pin and switch on the machine.
Adjust temperature as per the requirement.
Adjust humidity and CO2 level as per manufacturer’s instructions.
Place the culture tubes and close the lid, put the culture untouched for 72 hours.

Use of other utilities:

Deep freeze:

It is used to store chemicals (some) in -20C temperature

Refrigerator:

It is used to store chemicals, culture, pellets and other materials at 4C temperature.

After that to stop the cell division, a mitotic inhibitor is added at the 70th hour and all the culture tubes are collected for harvesting.

Treat the sample with the hypotonic solution and incubate in the water bath for a few minutes.

Water Bath:

A constant temperature water bath is used to incubate the sample, for 37C temperature, known as a hypotonic treatment. The hypotonic solution is used to swell the cells.

Using the glacial acetic acid and methanol, the fixative is prepared and used to wash the cells.

Perform centrifugation at 25,00 rpm for 15 to 20 minutes, until clear pallets are observed. Everytime use 10ml of fixative to wash the cells and mix gently.

Centrifuge:

Simply put, centrifuge is a special type of instrument used widely in the genetic field to separate biological materials.

Here sample is spinned at higher rpm which settles the larger particles at the bottom of the tube and larger in the supernatant.

After centrifugation, cells settle on the bottom while other debris remains on the top.

Using a sharp pestrue pipette, few drops of culture are placed on the glass slide, allow to dry and stain using the giemsa stain solution.

The slides are observed under a microscope.

Conclusion:

Though there are so many standard versions of karyotyping processes and protocols available, one has to standardized it as per their lab conditions. No such standardized protocol or SOPs are provided by any company or researcher.

However, by using our protocol or procedure you can achieve your karyotype faster, our protocols are used routinely in our labs and are best to investigate chromosomes.