Table 2 Common techniques used for diagnosis of Down’s syndrome along with its advantages and disadvantages

1

Cytogenetics analysis

Giemsa banding (G-banding) is performed on fetal cells at metaphase stage on amniocytes (grown in vitro) or CVS.

• Suitable for low income countries where physician can be presumed to have acquired a high level of diagnostic skill in the absence of laboratory services.

• Time consuming.

• Resolution of special importance for the detection of structural. abnormalities may be quite low as the spontaneous dividing cells are more condensed than those obtained after cell culture in vitro.

• In CVS, occurrence of confined placental mosaicism and occurrence of aberrant cells that do not represent the status of fetus.

• Chances of giving a false positive and false negative result.

2

FISH(Fluorescence in situ hybridization)

FISH involves hybridization of selected chromosome specific DNA sequences that have been labeled with fluorescent dye to chromosome preparation. The fluorescently labeled sequences stick to corresponding DNA of chromosome and can be visualized under microscope.

• As it uses smaller probes thus the signals appears to be more distinct as dots.

• Sometimes diffused signals are obtained because it uses chromosome at interphase stage which appears less condense than those of metaphase.

• It uses higher number of interphase nuclei for analysis, so the problem of any suspected mosaicism is resolved.

• Time consuming since it involves preparation of slides, fluorescent microscopy and spot counting (~30min per sample is expected).

• Maternal and fetal XX is not distinguished by FISH.

3

QF-PCR (Quantitative fluorescent-polymerase chain reaction)

Involves amplification and detection of STR using fluorescently labeled primers. The product is thus visualized and quantified as peaks areas of respective length using an automated DNA sequencer with Gene Scan software.

• Highly reliable and reproducible.

• Poses a challenge in the case of mosaicism.

• Chances of getting false negative and false positive cases are rare.

• While testing sex chromosome abnormalities samples from normal XX female may show homozygous QF-PCR pattern indistinguishable from those produced by sample with single X as in Turner syndrome.

• Maternal contamination is easily detected.

• Faster approach as it can give the diagnosis within 24 hours.

4

Paralogous sequence quantification (PSQ)

A PCR based method for detection of targeted chromosome number abnormalities, based on the use of paralogous genes. Paralogous sequences have high degree of sequence identity but accumulate nucleotide substitution in a locus specific manner. These differences are called as paralogous sequence mismatches which can be quantified using pyrosequencing.

• The first generation design of test requires 10 separate PCR reaction per sample, which significantly reduces the sample throughput and increases the probability of handling errors.

• Expensive when compared to others.

• It can handle 30–40 samples in a day and report result in less than 48 hours.

5.

MLPA (multiplex probe ligation assay)

MLPA is based on hybridization and PCR method. Divided into 4 phases: DNA denaturation, hybridization of probe to the complementary target sequence, probe ligation and PCR amplification of ligated probe. These amplified products are analysed through capillary electropheresis.

• Very short time for diagnosis (2–4 days).

• Unable to exclude low level placental and true mosaicism.

• Relatively low costs

6.

NGS (Next Generation Sequencing)

Clonally amplified DNA templates are sequenced in a massively parallel. It provides a digital quantitative information, in that each sequence read is a countable “sequence tag” representing an individual clonal DNA template or a single DNA molecule.

• The current time for sample processing, sequencing, and data interpretation in experienced hands is 5 to 8 days.

• The cost of sequencing is approximately $700 –$1000 per sample.

• Complex data analysis.