Application of risk score analysis to low-coverage whole genome sequencing data for the noninvasive detection of trisomy 21, trisomy 18, and trisomy 13.

OBJECTIVES: Clinical performance of a low coverage, low cost, massively parallel sequencing (MPS)-based assay to stratify risk of trisomy 21, 18, and 13 pregnancies was determined. METHODS: The study included 1100 samples with birth outcome or karyotype results, comprising low-risk patients (84.2%) negative for risk indications from maternal age, serum screening, ultrasound, or family history, and high-risk patients (15.8%) with at least one of the aforementioned indications. Cell free DNA (cfDNA) was extracted from maternal plasma. Library preparation incorporated 96 index barcodes to enable sequencing on a HiSeq 2000 or 2500. Risk scores were calculated using chromosomal representation, fetal fraction, and maternal age at the estimated date of delivery. A risk score greater than or equal to 1 in 100 was used to stratify samples as high risk for trisomy 21, trisomy 18, or trisomy 13. RESULTS: Sensitivity and specificity were calculated based on risk group stratification. Trisomy 21, trisomy 18, and trisomy 13 were detected with greater than 99% sensitivity and 99.9% specificity. Fetal sex classification accuracy was 99.3%. CONCLUSIONS: We conclude that simplified MPS can be used to stratify the risk of pregnancies for trisomy 21, trisomy 18, and trisomy 13 and accurately determine fetal sex. © 2015 John Wiley & Sons, Ltd.

Circulating cell free DNA testing: are some test failures informative?

OBJECTIVE: The proportion of circulating cell free DNA derived from the feto-placental unit (fetal fraction or FF) correlates with test success and interpretation reliability. Some fetal disorders are associated with systematically lower FF, sometimes resulting in noninformative results. METHODS: We analyzed results from pregnancies tested in a nested case/control study derived from a cohort of 4664 high-risk pregnancies. Low FF was defined before and after adjusting for maternal weight and gestational age. RESULTS: Compared with euploid pregnancies, the median FF was significantly higher in Down syndrome pregnancies (ratio 1.17) and significantly lower in trisomy 18 and triploid pregnancies (ratios 0.71 and 0.19, respectively). Among 2157 pregnancies tested, 13 (0.6%) had FF <3.0% (all noninformative), including three trisomy 18 and three triploidy fetuses. After adjustment, 16 pregnancies (0.7%) had FF <0.3 multiples of the median (six informative), including one trisomy 18 and three triploidy fetuses. Modeled positive predictive values for low and high-risk populations were 7% and 30%, respectively. CONCLUSION: Among women with noninformative results attributable to low FF, trisomy 18 and/or triploidy risk are sufficiently high to warrant offering additional assessments (e.g. ultrasound). If the testing indication is ultrasound abnormality, amniocentesis and karyotype/microarray should be considered. © 2014 John Wiley & Sons, Ltd.

Circulating cell-free fetal DNA for the detection of RHD status and sex using reflex fetal identifiers.

OBJECTIVE: To determine the sensitivity and specificity of circulating cell-free fetal DNA in determining the fetal RHD status and fetal sex. METHODS: Maternal blood was collected in each trimester of pregnancy from RhD negative nonalloimmunized women. Whole blood was centrifuged, separated into plasma and buffy coat, and frozen at -80°C. DNA analysis was conducted via allele-specific primer extensions for exons 4, 5, and 7 of the RHD gene and for a 37-base pair insertion in exon 4 (RHD pseudogene; psi) three Y-chromosome sequences (SRY, DBY, and TTY2), and an extraction control (TGIFL-like X/Y). RhD serotyping on cord blood and gender assessment of the newborns were entered into a Web-based database. RESULTS: One hundred twenty women were enrolled. The median gestational age at the first venipuncture was 12.4 (range: 10.6-13.9) weeks with 120 samples drawn; 118 samples were drawn at 17.6 (16-20.9) weeks; and 113 samples at 28.7 (27.9-33.9) weeks. Overall accuracy for RHD was 99.1%, 99.1%, and 98.1% for each trimester and was 99.1%, 99.1%, and 100% for fetal sex determination. CONCLUSIONS: Fetal RHD genotyping and sex can be very accurately determined in all three trimesters using circulating cell-free fetal DNA in the maternal circulation.

INTERNATIONAL STANDARDS ON FOOD AND ENVIRONMENTAL RADIOACTIVITY MEASUREMENT FOR RADIOLOGICAL PROTECTION: STATUS AND PERSPECTIVES.

Radiological protection is a matter of concern for members of the public and thus national authorities are more likely to trust the quality of radioactivity data provided by accredited laboratories using common standards. Normative approach based on international standards aims to ensure the accuracy or validity of the test result through calibrations and measurements traceable to the International System of Units. This approach guarantees that radioactivity test results on the same types of samples are comparable over time and space as well as between different testing laboratories. Today, testing laboratories involved in radioactivity measurement have a set of more than 150 international standards to help them perform their work. Most of them are published by the International Standardization Organization (ISO) and the International Electrotechnical Commission (IEC). This paper reviews the most essential ISO standards that give guidance to testing laboratories at different stages from sampling planning to the transmission of the test report to their customers, summarizes recent activities and achievements and present the perspectives on new standards under development by the ISO Working Groups dealing with radioactivity measurement in connection with radiological protection.

Anti-inflammatory drugs in experimental atherosclerosis. Part 5. Influence of cortisone acetate on short-term and long-term cholesterol fluxes in atherosclerotic aorta.

Previous studies in this series have shown that cortisone and other anti-inflammatory drugs inhibit atherosclerotic plaque development in cholesterol-fed rabbits. The present study was designed to investigate the influence of cortisone on the processes of cholesterol influx and efflux in the aorta wall. Forty-seven New Zealand white rabbits were fed a 1% cholesterol diet for periods up to 12 weeks. In addition 23 of the animals were fed 5 mg cortisone acetate daily. At 0, 5 and 12 weeks, groups were fed a tracer dose of [3H]cholesterol. Plasma cholesterol specific radioactivity was measured at intervals during the next 10 days. Total aorta cholesterol and its specific activity were measured by killing groups of animals at 2 days and 10 days. Atherosclerotic plaque intensity at 12 weeks, measured planimetrically, averaged 68 +/- 12% in controls vs. only 6 +/- 3% in cortisone-treated animals. During the period between 5 and 12 weeks, net cholesterol accumulation by chemical analysis averaged only 11 micrograms/g aorta/day in cortisone-treated animals vs. 117 micrograms/g in controls. [3H]cholesterol influx (measured during a brief 2-day exposure) at 5 weeks averaged 206 and 199 micrograms/g tissue/day and at 12 weeks 586 micrograms and 281 micrograms/day in control and cortisone-treated animals, respectively. Measured over a longer 10-day period, however, the apparent influx of [3H]cholesterol was much less in cortisone-treated animals, averaging only 53 micrograms/day compared to 269 micrograms/day in controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Parental decision following prenatal diagnosis of fetal chromosome abnormality.

In the event of prenatal diagnosis of fetal chromosome abnormality, parents must choose between continuation and termination of the pregnancy. To determine whether parents are capable of understanding differences in severity among aneuploidy syndromes, we examined the outcome chosen for all pregnancies in which a fetal chromosome disorder was diagnosed at Northwestern Memorial Hospital between January, 1977 and June, 1986. Among amniocentesis cases, 88% with autosomal aneuploidy were terminated, but only 41% with sex chromosome abnormalities and none with de novo structural rearrangements were terminated. Among a smaller group of chorionic villus sampling cases, all with abnormal results were terminated. Similar patterns of parental behavior were noted in other prenatal diagnosis units. We conclude that parents do distinguish among, and respond specifically to, fetal chromosome disorders of differing severity, at least in the second trimester of pregnancy. However, parents appear more inclined to terminate all pregnancies with chromosome abnormalities when the diagnosis has been made in the first trimester.

HLA sharing and fertility in Hutterite couples: evidence for prenatal selection against compatible fetuses.

Antigenic differences between mother and fetus (i.e., blood group incompatibilities) were traditionally considered deleterious for viviparous reproduction. Recently, evidence has accumulated suggesting that maternal response to paternally derived fetal antigens, paradoxically, may facilitate maintenance of pregnancy. Thus, fetuses whose paternally derived antigens do not differ from maternal antigens (i.e., histocompatible pregnancies) may be at a selective disadvantage during pregnancy. Parents sharing histocompatibility antigens (i.e., HLA) may produce compatible fetuses and show overall reduced fertility. Indeed, increased HLA sharing has been reported in some couples experiencing repetitive spontaneous abortion. However, the effects of HLA sharing in couples not selected because of previous pregnancy losses have not been assessed. To elucidate the reproductive effects of maternal-fetal histocompatibility, we initiated prospective population-based studies of parental HLA sharing and reproductive outcome in the Hutterites, a population isolate that lives communally and proscribes contraception. The relationship between HLA-A, -B, and -DR sharing and reproductive outcome was examined in 111 Hutterite couples. Intervals from marriage to each birth were no longer among couples sharing antigens; differences were significant at the second birth and remained significant through the sixth birth (P less than .05). When the effects of sharing at individual loci were examined, HLA-DR was the only individual locus that was a significant predictor of birth interval length (P = .025). Completed family sizes were 6.5 and 9.0 among couples sharing and not sharing HLA-DR, respectively (P = .082, 2-tailed). However, recognized fetal loss rates did not differ among couples sharing and not sharing antigens.(ABSTRACT TRUNCATED AT 250 WORDS)

Noninvasive first-trimester screening for fetal aneuploidy.

We reviewed all studies concerning noninvasive first trimester screening for fetal aneuploidy obtained from a MEDLINE search through June 1996 with additional sources identified through cross-referencing. Three screening and diagnostic modalities are of potential application in noninvasive first trimester testing for fetal aneuploidy: ultrasound, maternal biochemical markers, and analysis of fetal cells retrieved from maternal sources. Sensitivities of the sonographic finding of nuchal translucency thickness in combination with maternal age for trisomy 21, performed between 10 and 14 weeks of gestation in experienced hands, and maternal biochemical markers independently may be as high as 86 percent and 60 percent, respectively. Sensitivity, specificity, and predictive values of these diagnostic modalities alone, in combination with each other, or in conjunction with other predisposing factors such as maternal age, in large low risk populations have not currently been established. Analysis of fetal cells retrieved from maternal sources, although more complex, may offer definitive noninvasive prenatal diagnosis yet is not currently available in clinical practice. We conclude that noninvasive first trimester screening for fetal aneuploidy modalities including sonographic examination for nuchal translucency thickness and maternal biochemical markers, is feasible. Clinical feasibility; and all-encompassing clinical management paradigms of these and other early noninvasive first trimester screening methods for fetal aneuploidy, are not yet available.

Reproductive genetics for couples older than 40 years of age.

The counseling process, which involves genetic screening and prenatal diagnosis, is presented. Also addressed are the recent advances in assisted reproductive technologies, which may prove to be particularly useful for these patients.

Screening for the aneuploid fetus.

Advances in ultrasound technology have dramatically improved the detection of fetal defects. Although only an invasive test can provide a diagnosis, the incorporation of sonography into current biochemically based screening programs should significantly improve the detection of a host of other physically based fetal abnormalities. This article provides an overview and discussion of the prenatal sonographic features that may suggest the presence of a significant chromosomal abnormality.

Unexplained positive/elevated maternal serum alpha-fetoprotein associated with placenta increta. A case report.

Maternal serum alpha-fetoprotein (MSAFP) is a regularly utilized antenatal screening test for the identification of pregnancies at increased risk for a variety of genetic and nongenetic abnormalities. Complete mid-trimester evaluation of the patient with a positive screening test may fail to reveal an etiology for a positive MSAFP value. This case report concerns an unexplained positive/elevated MSAFP screening test for a patient found at delivery to have abnormal placentation.

Informed consent for maternal serum alpha-fetoprotein screening in an inner city population: how informed is it?

OBJECTIVE: To determine if women who received information from a provider and viewed a videotape about maternal serum alpha-fetoprotein (MSAFP) screening understood enough to sign informed consent. DESIGN: A prospective qualitative design using tape recorded interviews of women who were provided information regarding MSAFP testing from a provider and from viewing a videotape. PARTICIPANTS: Fifty-three inner city pregnant women (58% Hispanic, 39% African-American, 3% white). RESULTS: Two women answered all questions correctly; no one answered all questions incorrectly. Sixty-two percent correctly answered "What is MSAFP?" Sixteen percent thought "something has to be taken from my belly" for the test. Fifty-nine percent understood that children with spina bifida could have difficulty walking or urinary problems. Seventy-two percent thought their infant would be healthy in all respects if the test was negative. Only 45% could describe the follow-up to a positive test. Eighty percent planned to have the test. Many misconceptions were apparent, and for some knowledge items, as many as 80% of the women answered incorrectly. CONCLUSIONS: Obtaining truly informed consent for a complex test is not a simple process. Participants met a few, but not all, of the criteria for informed consent. Women understood that the test was voluntary, but their comprehension of the meaning and implication of a positive test results was deficient. Despite this, they signed the informed consent document. The larger question of just how much comprehension is required to consider a woman "informed" has not been answered.

Molecular analysis and chorionic villus sampling (CVS): opportunities for rapid prenatal diagnosis in the military.

Prior to recent advances in the field of molecular biology, prenatal diagnosis of hemoglobinopathies was accomplished by direct analysis of the gene product(s) in samples obtained by fetoscopy or, more recently, percutaneous umbilical blood sampling. The use of the polymerase chain reaction enables quicker diagnosis than with indirect or cumbersome Southern blot techniques. This case, reporting a couple at risk for fetal sickle cell disease, is a model for rapid prenatal diagnosis and demonstrates the capabilities which exist in the military health care referral system. Within 24 hours of a couple's presentation for consultation and accomplishment of chorionic villus sampling (CVS), preliminary results of both cytogenetic and sickle cell status of an at-risk fetus were determined. Final analysis was accomplished by 48 hours. The combination of early referral, early sampling by CVS, and application of advances in DNA laboratory technologies offers tremendous improvements for care heretofore unavailable as a complete "package" in the military. The implications and limitations for prenatal testing for single gene disorders within the military medical health care system will be discussed.

Maternal serum alpha-fetoprotein (MSAFP) screening: the Keesler experience.

Maternal serum alpha-fetoprotein (MSAFP) screening is rapidly becoming a standard of practice in all regions of the United States and Canada. This paper will review the initial results of MSAFP screening in 761 patients at USAF Medical Center Keesler, and offer recommendations for Department of Defense hospitals offering such testing.

Management of immune thrombocytopenia purpura at a military medical center.

OBJECTIVE: To investigate whether fetal platelets in immune thrombocytopenia purpura (ITP) may be predicted by antepartum assessment. METHODS: A prospective analysis was conducted of 28 pregnant women with ITP out of 8,056 deliveries. Of the 28 patients, 13 were evaluted by fetal scalp sampling and 11 were evaluated by percutaneous umbilical blood sampling (PUBS). RESULTS: The mean fetal and maternal platelet counts prior to delivery were 146,000 and 176,000, respectively. The mean fetal and maternal platelet counts after delivery were 245,000 and 149,000, respectively. Fetal cord platelet counts could not be predicted by maternal platelet count, the presence/absence of maternal direct/indirect anti-platelet antibodies, steroid therapy, or history of splenectomy. PUBS for fetal platelet assessment correlated well with fetal postdelivery counts. CONCLUSIONS: Patients with ITP rarely exhibit complications. No antepartum characteristic enhances reliable prediction of neonatal outcome. Method of delivery should be based on obstetric guidelines.

Placental echolucencies: does their presence influence outcome following genetic amniocentesis?

Women undergoing genetic amniocentesis procedures were evaluated for the presence of ultrasonographic echolucencies within the placenta. Non-calcified sonolucencies < 4 to 5 cm were classified as subchorionic hematomas (SH). Of the 1,000 pregnancies evaluated, 153 (15%) pregnancies manifested SH prior to amniocentesis (study group). The indications for referral were similar in the study and control groups. There were 13 (1.3%) losses: 3 and 10 in the study and control groups, respectively, with no statistical difference in fetal losses between the groups: RR 1.52 (95% confidence interval 0.56-4.14; p = 0.32). Placental sonolucencies < 4 to 5 cm in diameter appear to be incidental ultrasound findings not associated with increased fetal loss following genetic amniocentesis. Complications following invasive prenatal diagnosis cannot be attributed to the presence of these common ultrasound findings.

ISO standards on test methods for water radioactivity monitoring.

Water is vital to humans and each of us needs at least 1.5L of safe water a day to drink. Beginning as long ago as 1958 the World Health Organization (WHO) has published guidelines to help ensure water is safe to drink. Focused from the start on monitoring radionuclides in water, and continually cooperating with WHO, the International Standardization Organization (ISO) has been publishing standards on radioactivity test methods since 1978. As reliable, comparable and 'fit for purpose' results are an essential requirement for any public health decision based on radioactivity measurements, international standards of tested and validated radionuclide test methods are an important tool for production of such measurements. This paper presents the ISO standards already published that could be used as normative references by testing laboratories in charge of radioactivity monitoring of drinking water as well as those currently under drafting and the prospect of standardized fast test methods in response to a nuclear accident.

International standardisation work on the measurement of radon in air and water.

Radon is considered to be the main source of human exposure to natural radiation. As stated by the World Health Organization, the exposure due to the inhalation of indoor radon is much greater than the one via the ingestion of water as radon degasses from water during handling. In response to these concerns about the universal presence of radon, environmental assessment studies are regularly commissioned to assess the radon exposure of public and workers. The credibility of such studies relies on the quality and reliability of radon analysis as well as on the sample representativeness of the radiological situation. The standard-setting approach, based on consensus, seemed to lend itself to a settlement of technical aspects of potential comparison. At present, two Working Groups of the International Standardization Organization are focussing on drafting standards on radon and its decay products measurement in air and water. These standards, which aim for a set of rigorous metrology practices, will be useful for persons in charge of the initial characterisation of a site with respect to natural radioactivity as well as to those performing the routine surveillance of specific sites.