BB&T Contributing Editor

Recent years have shown a surge of interest in prenatal screening as a method of verifying the wellbeing of a fetus. The objective of ongoing research is to develop a noninvasive method for determining whether a fetus carries a genetic disease in the early weeks of pregnancy. Currently the best method is the integrated screening test which entails a first-trimester ultrasound exam, known as nuchal translucency, in combination with serum tests performed during the first and second trimesters, known as quad and penta marker tests.

The nuchal translucency test measures the fluid that accumulates in the back of the fetus's neck under the skin and the crown rump length, measurements which are associated with the chromosomal disorders of Down syndrome (trisomy 21) and Turner syndrome. The first-trimester serum test is for plasma protein A (PAPP-A) and hCG. The second-trimester serum test is for detecting alpha fetoprotein (AFP), estriol, hCG, and inhibin A. It is important to note that these are screening tests and not definitive diagnostic tests. Patients deemed “at risk“ by these tests are generally referred for genetic counseling and amniocentesis.

Chromosomal analyses performed on amniotic fluid obtained by amniocentesis or harvested placental tissue cells obtained by chorionic villus sampling (CVS) are the diagnostic tests used for determining fetal defects, but they are invasive and costly procedures, and carry a small but significant risk of infection, miscarriage or birth defects. There are about 6 million pregnancies annually in the U.S. and 4.5 million live births, of which 375,000 women have an amniocentesis or CVS procedure.

A growing number of emerging companies are pursuing the development of a noninvasive prenatal diagnostic test on maternal blood for detecting genetic disorders. Obtaining a blood specimen is considered noninvasive. The goal is to have a first-trimester blood test that eliminates the need for amniocentesis or CVS and allows for termination earlier in the pregnancy.

Alternative methods are being developed that seek to enrich and isolate intact fetal cells or nucleic acid fragments from the maternal bloodstream. Throughout pregnancy fetal cells pass the placenta and circulate in the maternal bloodstream. The challenge is to selectively collect a sufficient number of fetal cells or DNA fragments from a maternal blood sample for subsequent molecular diagnostics.

In January 2007, the American College of Obstetrics and Gynecology (ACOG; Washington) released a Practice Bulletin recommending that screening for fetal chromosomal abnormalities be made available for all pregnancies in the U.S.

The importance of conducting early prenatal tests for Down syndrome and other chromosomal abnormalities has taken on greater significance with the increase in the average maternal age of the first birth which is associated with a greater risk of chromosomal disorders. The average age of first-time mothers increased by 3.6 years from 21.4 years in 1970 to 25.0 years in 2006. In 2006, about 1 out of 12 first births were to women aged 35 years and over compared with 1 out of 100 in 1970 (Table 1). The higher risk of Down syndrome and other chromosomal abnormalities with increasing maternal age is shown in Table 2.

Laboratories perform prenatal tests

The U.S. market for prenatal testing is in excess of $1 billion per year. Prenatal screening tests are performed by national clinical testing laboratories, namely LabCorp (Burlington, North Carolina); Quest Diagnostics Nichols Institute (San Juan Capistrano, California), a division of Quest Diagnostics (Madison, New Jersey); and Bio-Reference Laboratories (Elmwood Park, New Jersey). Genzyme Genetics (Westborough, Massachusetts), a business unit of Genzyme (Cambridge, Massachusetts), is the largest clinical testing laboratory that specializes in the prenatal screening test market.

Other clinical testing laboratories that also specialize in this area are reviewed below.

Lenetix (Mineola, New York) is a laboratory that provides an array of genetic screening and diagnostic tests. It has entered into an Institutional Review Board (IRB)-approved screening study of an improved first and second trimester noninvasive fetal diagnostic test to detect Down syndrome. Lenetix's PloidYX maternal blood test is used for the detection of fetal aneuploidies, the presence of extra chromosomes, which cause Down syndrome and other chromosomal abnormalities.

The PloidYX test makes use of methylation differences between the fetal and maternal DNA found in maternal blood in order to achieve selective amplification of fetal cells. Following amplification, a microarray analysis is performed to determine the relative copy number of all the chromosomes. An increase in the relative copy number of chromosomes is indicative of trisomy while a decrease in the number is indicative of monosomy. It is a screening test aimed at lowering the number of women referred to amniocentesis.

NTD Labs (Melville, New York) was acquired in 2006 by PerkinElmer (Waltham, Massachusetts). It does prenatal screening for Down syndrome and has developed the free beta-hCG test used in the first trimester maternal screening test. Its Ultra-Screen protocol uses data from a blood screen (biochemistry) and an ultrasound examination (biophysical) which yields a risk assessment that physicians may present to their patients during the first trimester. The UltraScreen protocol includes tests for free beta-hCG and pregnancy associated plasma protein-A (PAPP-A). NTD Labs also uses the nasal bone assessment protocol as part of its ultrasound screening for Down syndrome.

Ikonisys (New Haven, Connecticut) performs cell-based diagnostic tests for early cancer diagnosis and genetic disorder screening. Its CellOptics technology platform integrates intelligent imaging, microscopy, biology and information for the purpose of single cell detection. The platform includes the Ikoniscope, a robotic digital fluorescence microscope for the detection of FISH (fluorescence in situ hybridization) and antibody signals, eliminating the subjectivity found in manual FISH testing and decreasing turnaround time.

Ikonisys offers fastFISHamnio, an FDA cleared application for prenatal diagnosis of genetic disorders using the Ikoniscope. The test is performed on uncultured amniocytes for the detection of numerical abnormalities of chromosomes 13, 18, 21 X and Y.

In December 2009, Ikonisys entered into a marketing partnership with Enzo Biochem's (New York) clinical lab division for its oncoFISH cervical test. Ikonisys will use its Ikoniscope digital microscopy system for the interpretation of molecular signals. The assay is based on a genetic marker which has been linked to an increased likelihood of developing cervical cancer.

Noninvasive prenatal diagnostic tests

Sequenom's (San Diego) MassARRAY system is a high-performance DNA analysis platform that precisely measures the amount of genetic target material and is able to deliver data from complex biological samples and from genetic target material that is only available in trace amounts. The company has an exclusive license for the development and commercialization of noninvasive prenatal genetic tests for use with the MassARRAY system and other platforms.

Sequenom uses its SEQureDx technology to isolate and analyze circulating fetal nucleic acids in maternal blood and examine the genetic status of the fetus. The first application for using this technology is for the analysis of fetal RhD which was introduced in 2007 through a sublicense to Lenetix. RhD disease can occur when the blood of an expectant mother is incompatible with her unborn child. This incompatibility affects approximately one in one thousand live born infants and can lead to jaundice, anemia, brain damage, heart failure and death. Other potential applications of the technology are for prenatal analysis including noninvasive tests for fetal sex determination and Down syndrome. The SEQureDX technology is based on the research of Dennis Lo, Ph.D., professor of chemical pathology at The Chinese University of Hong Kong, from whom it is licensed.

The company had planned to introduce a noninvasive prenatal maternal blood test for Down syndrome in 2009, but an investigation by independent members of its board of directors found that the company failed to put in place adequate protocols and controls for conducting its studies and its employees did not provide adequate supervision, resulting in inconsistencies and errors. This led to changes in top management and the appointment of new members of its board of directors and the addition of a clinical advisory board member. The company plans to launch its own version of the fetal RhD genotyping test and a fetal sex determination test early this year.

Sequenom operates the Sequenom Center for Molecular Medicine (Grand Rapids, Michigan) which offers a number of prenatal genetic tests based on its platform technologies. In September 2009, it launched the SensiGene cystic fibrosis carrier screening test.

In September 2008, Sequenom reported that it secured an exclusive license from Genomic Nanosystems (Beltsville, Maryland) for use of patents on digital PCR for the noninvasive prenatal diagnosis and analysis on any sample, i.e., fetal cells, plasma, amniocentesis fluid, urine, etc.

In October 2008, Sequenom licensed from Xenomics (New York) exclusive rights to patented technology for prenatal diagnostic products using transrenal fetal nucleic acids found in maternal urine. Xenomics' technology utilizes fragmented DNA materials which cross the kidney barrier (transrenal DNA) and are available in urine. The company is targeting the use of its technology for infectious disease detection and monitoring of cancer, prenatal genetic testing and monitoring organ/cell transplants. In October 2009, Xenomics said that it had filed a lawsuit alleging that Sequenom “fraudulently misrepresented to Xenomics the status of its testing for Down syndrome.“

Qiagen (Venlo, the Netherlands/Germantown, Maryland) markets more than 500 sample and assay products used for the isolation of DNA, RNA and proteins from biological samples. Its products are sold to molecular diagnostic laboratories, academic research markets, pharmaceutical and biotechnology companies, and to customers in applied testing markets such as forensics, animal or food testing and pharmaceutical process control.

Qiagen's QIAamp circulating nucleic acid kit is used for the isolation and purification of all types and sizes of nucleic acids. The kit is projected to facilitate corresponding biomarker research. Qiagen is collaborating with Sequenom for the development of noninvasive prenatal diagnostic tests.

Artemis Health (San Carlos, California) uses proprietary microfluidic technology to separate intact fetal cells from hydrodynamically smaller maternal red blood cells and platelets, yielding a fetal cell enriched sample. The advantage of this technique over other sized-based sorting methods (e.g., filters) is that it is inherently a non-clogging and gentle procedure. This technology is being developed in conjunction with Diana Bianchi, MD, professor of pediatrics and obstetrics and gynecology at Tufts University School of Medicine (Boston).

Artemis is developing prenatal diagnostic tests by quantifying fetal DNA fragments found in maternal circulation and for accurately predicting chromosomal and genetic disorders. The company has a co-exclusive worldwide license from Stanford University (Palo Alto, California) to develop cell-free fetal DNA prenatal diagnostic tests based on the research of Stephen Quake, PhD, professor of bioengineering. The test is being used to develop a noninvasive prenatal diagnostic test solely for the detection of fetal aneuploidy, a clinical concern for pregnant women and their physicians.

Quake also is pursuing in a separate program under license from Fluidigm (South San Francisco, California), a company which he co-founded, the development of a noninvasive prenatal diagnostic test using a technique that counts chromosomes in trace amounts of fetal DNA in the maternal bloodstream for detecting fetal genetic characteristics. It uses a combination of digital polymerase chain reaction and high-throughput screening.

BioCep (Safed, Israel) has developed technology for immuno-magnetic separation of cells from diverse biological sources. The company's proprietary Cell Enrichment Process (CEP) allows effective separation of rare cell populations. BioCep's initial focus is to create a system for separating and isolating fetal nucleated red blood cells from maternal blood in the first trimester of pregnancy to be used for genetic diagnosis. Other potential applications for the technology are for improving the purity of stem cell transplants and the isolation of cancerous cells from blood for monitoring cancer progression.

Parsortix (Philadelphia) is a particle separation company. Its physical separation device uses a patented separation process to selectively capture particles of different sizes and types suspended in fluid media. It is collaborating with the Ben Franklin Technology Partners of southeastern Pennsylvania. The initial application of its technology is for the noninvasive separation of intact fetal cells from maternal blood. Discussions are under way with several potential strategic partners for the fetal cell application. Other application areas that are being investigated include infectious diseases, oncology and stem cell capture.

FCMB (Vejle, Denmark) has identified specific markers that recognize intact fetal cells from maternal blood samples for prenatal genetic analysis. The company has created a cDNA library originating from fetal cells for revealing expression patterns different from maternal blood cells. FCMB uses magnetic separation technology for isolating cells.

Ravgen (Columbia, Maryland) is developing a noninvasive prenatal diagnostic test based on single nucleotide polymorphisms (SNPs). The test entails the use of formaldehyde to enrich fetal DNA in maternal blood, SNPs to distinguish fetal DNA from maternal DNA, and calculating the ratio of heterozygous SNPs to determine the chromosome copy number.