Sunday, August 12, 2007

Peripheral and Fetal Cord Blood Cultures

In prenatal diagnosis, blood karyotypes are usually done for birth defects, mental retardation, infertility or multiple miscarriages. Stimulated peripheral and fetal cord blood cultures are used to establish the constitutional karyotype of an individual. The procedure is based on the concept that every cell in the body contains the same complement of DNA as any other cell. This is true for most cases; an exception occurs in mosaicism, in which more than one cell line is present in an individual. It is convenient to study chromosomes of peripheral blood lymphocytes as it is easily obtained via venipuncture while fetal cord blood is obtained from the umbilical cord. Blood specimen usually produces long chromosomes and consistently obtains chromosome preparations of good quality.

Lymphocytes in the peripheral blood rarely undergo spontaneous cell division, thus it must be ‘stimulated’ to grow and divide in vitro. Various mitogenic agents are available to stimulate B or T lymphocytes; the most common agent is phytohemagglutinin (PHA), which has a stimulatory effect primarily on T lymphocytes. T lymphocytes account for approximately 85% of the circulating blood lymphocytes and are nearly always present in peripheral blood.

With respect to newborn (< 3 months) and fetuses, a preliminary result is usually available within 3 working days by utilizing a 48hr blood culture, adapted from the 72hr blood culture and harvest procedure. Such method is best utilized for cases of possible aneuploidy ( extra multiple of the haploid number of the chromosomes, eg. Down syndrome, extra 21). The 48hr preliminary result is then followed up with the 72hr result that provides longer chromosomes with higher banding resolution. The difference between 48hr culture and 72 hr culture is, 48hr culture adds EB instead of thymidine, usually being to 72hr cultures.

Culture set-up

Specimens are set up as suspension cultures. To increase the number of metaphase chromosomes, thymidine or methotrexate (MTX) may be added to synchronize the cultures.

After an optimal culture time, the addition of a mitotic inhibitor such as Colcemid® causes the chromosomes to contract and condense, ethidium bromide (EB), a DNA-intercalating agent, may be added to the cultures. When added at G2 in the cell cycle, this interferes with normal condensation of the chromosomes.

The addition of EB during harvesting period results in extended chromosomes and an increase in band resolution. Slide preparations of metaphase chromosomes are obtained following exposure of the culture to a hypotonic solution (Ohnuki’s) to spread the chromosomes apart, followed by a series of fixative (3:1 methanol : acetid acid) steps.

To make slides, 3-6 drops of the cell suspension from a glass pipet along the upper edge of the slide are dropped from height of 10-20cm, depending on the spread, while holding the slide horizontally or vertically to the bench at a 45° angle. Firmly bang the slide on the bench top several times before placing it on a 56°C warming tray. This may help in chromosome spreading. However, do not bang if there is widespread scattering.

Check the spread under the phase contrast microscope before continuing with rest of the slides. Do the appropriate adjustment of the cell density or height to achieve the best spread subsequently. Bake slide at 90°C over for 2 hours, or at 60°C overnight and a further 1hour at 90°C the following morning.

Lau Yi Fang Yvonne


Star team said...

hey yvonne,

Just want to know can you list down a couple of karyotypic disorders commonly found/seen?


J.A.M.M.Y.S said...

Reply to Randall

Some of the common abnormalities seen are:

Autosomal Abnormalities
- Down Syndrome (trisomy 21)
- Edwards Syndrome (trisomy 18)
- Patau Syndrome (trisomy 13)

Sex Chromosome Abnormalities
- Turner Syndrome (45,X)
- Klinefelter Syndrome (47,XXY)

The Lab Freaks said...
This comment has been removed by the author.
The Lab Freaks said...

Hi yvonne,

Sorry to ask so late.. How do you identity different chromosomes in your test? Is it by the different length of the chromosomes or by techniques like Giemsa staining?


J.A.M.M.Y.S said...

Reply to Royston

Yup. The different length of chromosomes does contribute to the identification, but the main difference of each chromosomes are usually distinguish by their distinct banding pattern.

Trypsin digestion helps to form bands at the respective position while Giemsa/Wright's Stain adds colour to it, forming dark and light bands.