AMNIOTIC FLUID

Amniotic fluid (AF) contains fetal cells that can be grown in culture and also can be used for screening genetic anomalies at DNA level by PCR study. Approximately, 20-30 ml AF is obtained in a sterile container (tissue culture grade) by amniocentesis at 16-20 weeks gestational age, under USG guidance. The AF should be clear, without any RBCs. First 2 ml of AF should be discarded to avoid maternal cell contamination. AF obtained after this is centrifuged to concentrate the cells. These cells are then diluted in a specific culture medium and the cell suspension is incubated in a flask at 37°C. for 10-12 days. Here, the cells will settle down and grow. As the cells divide, they form colonies where all the cells are descended from the original cell that settled down. Usually, sufficient colonies can be obtained after 10-14 days, with actively dividing cells ready for harvest.

The same Amniotic fluid sample is used for FISH study. The AF cells are processed by using trypsin and treated with hypotonic solution. The cells are then fixed and the cell nuclei is studied using specific FISH probes (13, 18, 21, X & Y) on fluorescence microscope with specialized filters and imaging software.

For DNA study, the sediment, containing the amniotic cells obtained after centrifugation of the amniotic fluid, is used to isolate the DNA using standard protocol. The necessary DNA is then used for requested molecular genetic studies. (thalassaemia etc.)
 

CVS is done for prenatal cytogenetic and molecular genetic studies. The CVS is collected at 8-12 weeks of gestational age. Under USG guidance, small fragments of placental tissue (Chorionic villi) are collected in sterile tissue culture media with few drops of antibiotic (strepto-penicillin). The Chorionic tissue is isolated from blood clots and maternal tissue under inverted microscope. The clean villi are then treated with collagenase, and incubated in three different flasks containing tissue culture medium in 5% CO2 incubator. The remaining tissue is processed in a different way to obtain short culture metaphases. The cells in the flasks adhere to the surface and proliferate rapidly. The growing cells are harvested between 10-15 days to obtain metaphases. Minimum of 25 well spread metaphases from each flask are studied.

The same CVS is used for FISH study. The CV cells are processed by using trypsin and treated with hypotonic solution. The cells are then fixed, and the cell nuclei is studied using specific FISH probes (13, 18, 21, X & Y) on fluorescence microscope with specialized filters and imaging software.

The DNA is isolated from chorionic cells using standard protocol. The necessary DNA is then used for requested molecular genetic studies. (thalassaemia etc.)
 

Bone marrow sample is the sample of choice for cytogenetic investigations in cases of leukemia and other proliferative disorders. The BM is studied for chromosome changes present in the blast cells in human leukemia. The BM is processed by two different ways. As per the blast and total number of cells present, one part of the BM is directly harvested to obtain metaphases. The other portion is incubated in a specialized medium in a CO₂ incubator for 24 hours to obtain quality metaphases. In some cases of leukemia, the BM specimen is incubated for 48-72 hours. The BM is harvested by routine protocol to obtain quality metaphases. Usually 20 well spread metaphases are studied for analysis. In some cases of hypoplastic bone marrow, or a very few blasts, where the dividing cells are not present, the metaphases studied may be less in number, or the repeat sample is requested.

FISH is also studied using BM samples in variety of leukemias. The BM sample is directly processed using hypotonic solution and fixed in fixative. Specific probes are used for hybridization, and the interphase cells are observed under the fluorescence microscope with specialized imaging software. The FISH thus studied, is helpful in evaluating minimal residual disease (MRD) after therapy or transplantation. Some of the chromosome translocations cannot be detected by routine cytogenetic studies, hence FISH, using specific probes, can be applied on the interphase cells to study such anomalies.
 

Fragile X testing is a technique where cells are grown in a special media in order to detect a heritable fragile site on the X chromosome. Expression of this fragile site is associated with a syndrome of X-linked mental retardation, Martin-Bell syndrome.

The blood cells are cultured using two different techniques. The first uses routine growth media plus excess thymidine. The second uses routine growth media that is supplemented with Fluoro-deoxy-uridine (FudR). FudR is a folic acid antagonist, which inhibits the enzyme dihydrofolate reductase from reducing folate and dihydrofolate to tetrahydrofolate. Folic acid is required for thymidine incorporation. In patients with fragile X syndrome, this pathway is disrupted, causing the expression of a fragile site in the long arm of the X chromosome.

Because fragile X segregates in families, it is important to test males with mental retardation for the presence of fragile X, since other family members may be at risk for having affected offspring.
 

High resolution banding is a technique for obtaining longer, less condensed chromosomes, where more sub-bands can be visualized.

After a 72 hour growth period, an intercalating agent, Ethidium Bromide, is added to the blood cultures. The molecules of Ethidium Bromide get inserted between the base pairs of the DNA in the chromosome, and inhibit condensation of the chromosomes during the mitotic cycle. When colcemid is added to block the cells during metaphase, the chromosomes are still in an uncondensed stage similar to late prophase.

Approximately 800 bands can be identified in a haploid karyotype as a result of this technique, instead of 400 bands. Subtle loss or gain of genetic material, which can be missed otherwise, is easily detected by this technique. The main purpose of prometaphase technique is that it gives us a better chromosome resolution, resulting in easy, early and reliable detection of minor abnormalities.
 

In cases of leukemias, where the BM aspiration is not possible, and the total number of blasts is more than 40% in peripheral blood, the sample can be used for cytogenetic study. The procedure is same as BM technique. The peripheral blood is processed without adding mitogen so that only the spontaneously dividing cells will give the metaphases for analysis. The failure rate to obtain quality metaphases is almost 30%, hence unstimulated peripheral blood for leukemia is only processed in case of exception.

FISH using peripheral blood is the most convenient method for specific anomalies. The FISH study does not require dividing cells, hence can easily be studied on interphase cells for analysis. The procedure is same as for BM FISH studies.

Peripheral blood can also be used for DNA study by PCR. The procedure remains same as BM.
 

The peripheral blood sample is stimulated by phytohemagglutinin and incubated for 72 hours. The mitogenic inhibitor (colcemid) is added 2 hours before harvesting to get analyzable metaphase.
 

The DNA can be isolated from different specimens e.g. peripheral blood, buccal cells, sputum etc. The cells are lysed with lysis buffer and nucleic acids are separated from the cell debris by centrifugation. The DNA is now precipitated with repeated washes of alcohol.

The isolated DNA is analyzed qualitatively by 0.8% agarose gel electrophoresis and quantitatively by spectrophotometry.
 

PCR is a technique for amplification and enzymatic conversion of genetic material (DNA & RNA). The isolated nucleic acid is amplified using two specific oligonucleotides (Primers) in the presence of specific thermostable enzyme, Taq DNA polymerase with repeated cycles of denaturation, renaturation and extension on thermal cycler.

The amplified DNA is again analyzed qualitatively by 0.8% agarose gel electrophoresis and quantitatively by spectrophotometry.

The reaction and time may vary according to the tests done.

SIGNIFICANCE OF GENETIC STUDY

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