The impact of maternal plasma DNA fetal fraction on next generation sequencing tests for common fetal aneuploidies.

Maternal plasma contains circulating cell-free DNA fragments originating from both the mother and the placenta. The proportion derived from the placenta is known as the fetal fraction. When measured between 10 and 20 gestational weeks, the average fetal fraction in the maternal plasma is 10% to 15% but can range from under 3% to over 30%. Screening performance using next-generation sequencing of circulating cell-free DNA is better with increasing fetal fraction and, generally, samples whose values are less than 3% or 4% are unsuitable. Three examples of the clinical impact of fetal fraction are discussed. First, the distribution of test results for Down syndrome pregnancies improves as fetal fraction increases, and this can be exploited in reporting patient results. Second, the strongest factor associated with fetal fraction is maternal weight; the false negative rate and rate of low fetal fractions are highest for women with high maternal weights. Third, in a mosaic, the degree of mosaicism will impact the performance of the test because it will reduce the effective fetal fraction. By understanding these aspects of the role of fetal fraction in maternal plasma DNA testing for aneuploidy, we can better appreciate the power and the limitations of this impressive new methodology.

Noninvasive prenatal detection of sex chromosomal aneuploidies by sequencing circulating cell-free DNA from maternal plasma.

OBJECTIVE: Whole-genome sequencing of circulating cell free (ccf) DNA from maternal plasma has enabled noninvasive prenatal testing for common autosomal aneuploidies. The purpose of this study was to extend the detection to include common sex chromosome aneuploidies (SCAs): [47,XXX], [45,X], [47,XXY], and [47,XYY] syndromes. METHOD: Massively parallel sequencing was performed on ccf DNA isolated from the plasma of 1564 pregnant women with known fetal karyotype. A classification algorithm for SCA detection was constructed and trained on this cohort. Another study of 411 maternal samples from women with blinded-to-laboratory fetal karyotypes was then performed to determine the accuracy of the classification algorithm. RESULTS: In the training cohort, the new algorithm had a detection rate (DR) of 100% (95%CI: 82.3%, 100%), a false positive rate (FPR) of 0.1% (95%CI: 0%, 0.3%), and nonreportable rate of 6% (95%CI: 4.9%, 7.4%) for SCA determination. The blinded validation yielded similar results: DR of 96.2% (95%CI: 78.4%, 99.8%), FPR of 0.3% (95%CI: 0%, 1.8%), and nonreportable rate of 5% (95%CI: 3.2%, 7.7%) for SCA determination CONCLUSION: Noninvasive prenatal identification of the most common sex chromosome aneuploidies is possible using ccf DNA and massively parallel sequencing with a high DR and a low FPR.

High-throughput massively parallel sequencing for fetal aneuploidy detection from maternal plasma.

BACKGROUND: Circulating cell-free (ccf) fetal DNA comprises 3-20% of all the cell-free DNA present in maternal plasma. Numerous research and clinical studies have described the analysis of ccf DNA using next generation sequencing for the detection of fetal aneuploidies with high sensitivity and specificity. We sought to extend the utility of this approach by assessing semi-automated library preparation, higher sample multiplexing during sequencing, and improved bioinformatic tools to enable a higher throughput, more efficient assay while maintaining or improving clinical performance. METHODS: Whole blood (10mL) was collected from pregnant female donors and plasma separated using centrifugation. Ccf DNA was extracted using column-based methods. Libraries were prepared using an optimized semi-automated library preparation method and sequenced on an Illumina HiSeq2000 sequencer in a 12-plex format. Z-scores were calculated for affected chromosomes using a robust method after normalization and genomic segment filtering. Classification was based upon a standard normal transformed cutoff value of z = 3 for chromosome 21 and z = 3.95 for chromosomes 18 and 13. RESULTS: Two parallel assay development studies using a total of more than 1900 ccf DNA samples were performed to evaluate the technical feasibility of automating library preparation and increasing the sample multiplexing level. These processes were subsequently combined and a study of 1587 samples was completed to verify the stability of the process-optimized assay. Finally, an unblinded clinical evaluation of 1269 euploid and aneuploid samples utilizing this high-throughput assay coupled to improved bioinformatic procedures was performed. We were able to correctly detect all aneuploid cases with extremely low false positive rates of 0.09%, <0.01%, and 0.08% for trisomies 21, 18, and 13, respectively. CONCLUSIONS: These data suggest that the developed laboratory methods in concert with improved bioinformatic approaches enable higher sample throughput while maintaining high classification accuracy.

Impact of adjusting for the reciprocal relationship between maternal weight and free thyroxine during early pregnancy.

BACKGROUND: Among euthyroid pregnant women in a large clinical trial, free thyroxine (FT4) measurements below the 2.5th centile were associated with a 17 lb higher weight (2.9 kg/m(2)) than in the overall study population. We explore this relationship further. METHODS: Among 9351 women with second trimester thyrotropin (TSH) measurements between 1st and 98th centiles, we examine: (i) the weight/FT4 relationship; (ii) percentages of women in three weight categories at each FT4 decile; (iii) FT4 concentrations in three weight categories at each TSH decile; and (iv) impact of adjusting FT4 for weight--in the reference group and in 190 additional women with elevated TSH measurements. RESULTS: FT4 values decrease steadily as weight increases (p<0.0001 by ANOVA) among women in the reference group (TSH 0.05-3.8 IU/L). TSH follows no consistent pattern with weight. When stratified into weight tertiles, 48% of women at the lowest FT4 decile are heavy; the percentage decreases steadily to 22% at the highest FT4 decile. Median FT4 is lowest in heaviest women regardless of the TSH level. In the reference group, weight adjustment reduces overall variance by 2.9%. Fewer FT4 measurements are at either extreme (below the 5th FT4 centile: 4.8% before adjustment, 4.7% after adjustment; above the 95th FT4 centile: 5.0% and 4.7%, respectively). Adjustment places more light weight women and fewer heavy women below the 5th FT4 centile; the converse above the 95th centile. Between TSH 3.8 and 5 IU/L, the FT4 percentage below the 5th FT4 centile is not elevated (3.8% before adjustment, 3.1% after adjustment). Percentage of FT4 values above the 95th centile, however, is lower (1.5% before adjustment, 0.8% after adjustment). Above TSH 5 IU/L, 25% of women have FT4 values below the 5th FT4 centile; weight adjustment raises this to 30%; no FT4 values remain above the 95th FT4 centile. CONCLUSIONS: During early pregnancy, TSH values are not associated with weight, unlike nonpregnant adults. Lower average FT4 values among heavy women at all TSH deciles partially explain interindividual differences in FT4 reference ranges. The continuous reciprocal relationship between weight and FT4 explains lower FT4 with higher weight. Weight adjustment refines FT4 interpretation.

DNA sequencing of maternal plasma to identify Down syndrome and other trisomies in multiple gestations.

OBJECTIVE: Studies on prenatal testing for Down syndrome (trisomy 21), trisomy 18, and trisomy 13 by massively parallel shotgun sequencing (MPSS) of circulating cell free DNA have been, for the most part, limited to singleton pregnancies. If MPSS testing is offered clinically, it is important to know if these trisomies will also be identified in multiple pregnancies. METHOD: Among a cohort of 4664 high-risk pregnancies, maternal plasma samples were tested from 25 twin pregnancies (17 euploid, five discordant and two concordant for Down syndrome; one discordant for trisomy 13) and two euploid triplet pregnancies [Correction made here after initial online publication.]. Results were corrected for GC content bias. For each target chromosome (21, 18, and 13), z-scores of 3 or higher were considered consistent with trisomy. RESULTS: Seven twin pregnancies with Down syndrome, one with trisomy 13, and all 17 twin euploid pregnancies were correctly classified [detection rate 100%, 95% confidence interval (CI) 59%-100%, false positive rate 0%, 95% CI 0%-19.5%], as were the two triplet euploid pregnancies. CONCLUSION: Although study size is limited, the underlying biology combined with the present data provide evidence that MPSS testing can be reliably used as a secondary screening test for Down syndrome in women with high-risk twin gestations.

DNA sequencing of maternal plasma reliably identifies trisomy 18 and trisomy 13 as well as Down syndrome: an international collaborative study.

PURPOSE: To determine whether maternal plasma cell-free DNA sequencing can effectively identify trisomy 18 and 13. METHODS: Sixty-two pregnancies with trisomy 18 and 12 with trisomy 13 were selected from a cohort of 4,664 pregnancies along with matched euploid controls (including 212 additional Down syndrome and matched controls already reported), and their samples tested using a laboratory-developed, next-generation sequencing test. Interpretation of the results for chromosome 18 and 13 included adjustment for CG content bias. RESULTS: Among the 99.1% of samples interpreted (1,971/1,988), observed trisomy 18 and 13 detection rates were 100% (59/59) and 91.7% (11/12) at false-positive rates of 0.28% and 0.97%, respectively. Among the 17 samples without an interpretation, three were trisomy 18. If z-score cutoffs for trisomy 18 and 13 were raised slightly, the overall false-positive rates for the three aneuploidies could be as low as 0.1% (2/1,688) at an overall detection rate of 98.9% (280/283) for common aneuploidies. An independent academic laboratory confirmed performance in a subset. CONCLUSION: Among high-risk pregnancies, sequencing circulating cell-free DNA detects nearly all cases of Down syndrome, trisomy 18, and trisomy 13, at a low false-positive rate. This can potentially reduce invasive diagnostic procedures and related fetal losses by 95%. Evidence supports clinical testing for these aneuploidies.

Feasibility of using plasma rather than serum in first and second trimester multiple marker Down's syndrome screening.

OBJECTIVE: To compare maternal plasma with serum for measuring markers currently used in first and second trimester screening for Down's syndrome. SETTING: A laboratory-based investigation of two sample types in assays used in prenatal screening for Down's syndrome. METHODS: A paired data-set included both plasma and serum from 101 pregnant women. A nested case/control data-set included only plasma samples from 34 first and 23 second trimester Down's syndrome pregnancies, each matched with six euploid controls. Analyte levels were measured and converted to multiples of the median (MoM). RESULTS: In the paired data-set, each of the five analytes (alphafetoprotein, unconjugated estriol, human chorionic gonadotropin, inhibin-A and pregnancy-associated plasma protein A) in serum and plasma was highly correlated (r > 0.970) and after conversion to MoM, the resulting distributions were equivalent (P > 0.7). In the matched data-set, plasma-based median MoM levels in cases were consistent with the published serum counterparts for all markers. CONCLUSIONS: This study provides strong evidence that current serum-based prenatal screening can be performed equally well using plasma samples. This may prove useful, especially if secondary screening using a DNA-based test requires maternal plasma.

Prenatal screening for trisomy 21: recent advances and guidelines.

The performance of prenatal screening tests for the identification of trisomy 21 (Down syndrome) has markedly improved since the 1970s and early 1980s when maternal age was the sole mode of screening the general pregnant population. With the discovery of second trimester serum markers in the 1980s and 1990s and implementation of double, triple, and quad marker testing; the discovery of first trimester serum and ultrasound markers in the 1990s and implementation of the combined test; and the development of the integrated test and sequential screening strategies over the past decade, the performance of screening has improved to a detection rate of 90%­95% at a false positive rate of 2%­5%. In this review, I will describe the advances in prenatal screening for trisomy 21, present current screening strategies, and discuss guidelines published by professional societies and regulatory bodies, with a focus on current prenatal screening practice in the USA.

DNA sequencing of maternal plasma to detect Down syndrome: an international clinical validation study.

PURPOSE: Prenatal screening for Down syndrome has improved, but the number of resulting invasive diagnostic procedures remains problematic. Measurement of circulating cell-free DNA in maternal plasma might offer improvement. METHODS: A blinded, nested case-control study was designed within a cohort of 4664 pregnancies at high risk for Down syndrome. Fetal karyotyping was compared with an internally validated, laboratory-developed test based on next-generation sequencing in 212 Down syndrome and 1484 matched euploid pregnancies. None had been previously tested. Primary testing occurred at a CLIA-certified commercial laboratory, with cross validation by a CLIA-certified university laboratory. RESULTS: Down syndrome detection rate was 98.6% (209/212), the false-positive rate was 0.20% (3/1471), and the testing failed in 13 pregnancies (0.8%); all were euploid. Before unblinding, the primary testing laboratory also reported multiple alternative interpretations. Adjusting chromosome 21 counts for guanine cytosine base content had the largest impact on improving performance. CONCLUSION: When applied to high-risk pregnancies, measuring maternal plasma DNA detects nearly all cases of Down syndrome at a very low false-positive rate. This method can substantially reduce the need for invasive diagnostic procedures and attendant procedure-related fetal losses. Although implementation issues need to be addressed, the evidence supports introducing this testing on a clinical basis.

The relationship between PTH and 25-hydroxy vitamin D early in pregnancy.

OBJECTIVE: Measure serum PTH and 25(OH)D in a cross-sectional sample of pregnant women at 11th through 13th weeks' gestation to examine vitamin D status and consider implications. DESIGN: Observational: we retrieved residual sera stored at -20 °C after routine first trimester Down's syndrome screening, distributed over 12 months. PATIENTS: 430 African American women and 586 Caucasian women. MEASUREMENTS: PTH and 25-hydroxy vitamin D [25(OH)D] immunoassays. RESULTS: PTH medians were: 1·33 pmol/l (African American women); 1·20 pmol/l (Caucasian women) (t = 0·43, P = 0·7). Concentrations were highest in winter and decreased significantly in spring, fall, and summer. There was a direct PTH/weight relationship in Caucasian (t = 3·12, P < 0·002), but not African American women (t = 1·34, P = 0·18). Median 25(OH)D concentrations were 47·5 nmol/l (African American women) and 65 nmol/l (Caucasian women) (t = 13·7, P < 0·001). Concentrations were lowest in winter and rose significantly in spring, fall, and summer. Reciprocal 25(OH)D/weight relationships existed for both racial groups (t =-4·31 P < 0·001; t = 4·54, P < 0·001, respectively). Among 68 Caucasian women who smoked, median PTH and 25(OH)D concentrations were somewhat lower (P = ns). In separate regression models with PTH and 25(OH)D [dependent variables] and season, weight and smoking [independent variables], the only qualifying interactive term was in the Caucasian PTH model (season*1/weight). A regression model applied to adjusted scatter plots of PTH vs 25(OH)D indicated a weak relationship. CONCLUSIONS: The PTH/25(OH)D relationship is weaker during early pregnancy than in non-pregnant adults, making it unreliable for estimating vitamin D sufficiency. A suitable reference point for sufficiency might be the maternal 25(OH)D level considered sufficient for adequate transfer to neonates.

Impact of smoking on maternal serum markers and prenatal screening in the first and second trimesters.

OBJECTIVES: To examine the effects of smoking on first and second trimester screening markers and to determine the overall impact of these effects on Down syndrome and trisomy 18 risks in first trimester combined, second trimester quadruple and integrated tests. METHODS: Examination of screening records at Women and Infants Hospital during 2006-2008. First trimester pregnancy-associated plasma protein-A (PAPP-A), beta-human chorionic gonadotrophin (hCG) and nuchal translucency and second trimester alpha-fetoprotein (AFP), unconjugated estriol (uE3), hCG and inhibin A (inhA) multiple of the median (MoM) values were extracted from the database along with risk results, smoking status and relevant demographic information. RESULTS: Smoking led to significantly reduced median levels of first trimester PAPP-A (0.89 MoM) and hCG (0.80 MoM), reduced second trimester uE3 (0.96 MoM) and hCG (0.84 MoM), and increased AFP (1.03 MoM) and inhA (1.39 MoM). After accounting for the differences in age between groups, smokers had higher Down syndrome screen positive rates for the second trimester quadruple test, but not for first trimester combined or integrated tests. Screen positive rates for trisomy 18 were markedly increased in smokers relative to age-matched non-smokers when using first trimester combined or integrated tests. CONCLUSION: Smoking leads to increased screen positive rates, especially for trisomy 18 using combined or integrated tests.

Thyroperoxidase and thyroglobulin antibodies in early pregnancy and placental abruption.

OBJECTIVE: To estimate the relationship between thyroid antibodies and placental abruption. METHODS: This cohort study assesses thyroperoxidase and thyroglobulin antibodies in relation to placental abruption among 10,062 women with singleton viable pregnancies (from the First and Second Trimester Risk of Aneuploidy [FaSTER] trial). A thyroperoxidase antibody cutoff of 50 international units/mL is used for comparison with published data from another cohort. RESULTS: Women with elevated thyroperoxidase antibody levels in the first and second trimesters have a higher rate of placental abruption than antibody-negative women. This relationship is less strong in the first trimester (1.51% compared with 0.83%; odds ratio [OR], 1.83; 95% confidence interval [CI], 0.99-3.37) than in the second trimester (1.78% compared with 0.82%; OR, 2.20; 95% CI, 1.21-3.99). A similar, but weaker, relationship is present for thyroglobulin antibodies. Sixty-four of 782 thyroperoxidase antibody-positive pregnancies without abruption become negative by the second trimester; one pregnancy with abruption becomes antibody-positive. Odds ratios for pregnancies with both thyroperoxidase and thyroglobulin antibody elevations are also higher (first trimester: OR, 2.10; 95% CI, 0.91-4.86; second trimester: OR, 2.73; 95% CI, 1.17-6.33). CONCLUSION: The present data confirm an association between thyroid antibody elevations and placental abruption described in a recent report. These findings, however, do not provide support for recommending routine testing for thyroid antibodies during pregnancy. LEVEL OF EVIDENCE: II.

Adjustment of maternal serum alpha-fetoprotein levels in women with pregestational diabetes.

OBJECTIVE: Decreased second trimester levels of maternal serum alpha-fetoprotein (MSAFP) have been reported in women with pregestational diabetes leading some laboratories to use a correction factor. The aim of this study is to determine if MSAFP levels in pregnant women with diabetes managed on oral antidiabetic agents is lower than non-diabetic controls and require adjustment similar to those on insulin. STUDY DESIGN: We performed a nested case/control study of an existing dataset using women with pregestational diabetes who had routine MSAFP values available. RESULTS: Before adjusting the MSAFP value for weight, both the diabetic patients who used insulin (n = 68) and those who used oral antidiabetic agents (n = 37) showed a non-significant trend toward a lower multiples of the median (MoM) as compared with controls (n = 244). After converting the raw MSAFP values to race-adjusted MoM and adjusting for weight, the median MSAFP MoM for women taking insulin (1.01) versus those on oral antidiabetic agents (1.00) were essentially the same. Furthermore, both of the diabetic groups were virtually identical to non-diabetic controls. CONCLUSIONS: In our study, women with pregestational diabetes managed on either insulin or oral antidiabetic agents had weight-adjusted MSAFP MoM levels equivalent to those in control pregnancies and did not require a correction factor.

Four years' experience with an interlaboratory comparison program involving first-trimester markers of Down syndrome.

CONTEXT: We initiated a voluntary, self-funded interlaboratory comparison program in the fall of 2005 because no proficiency testing program was available to laboratories in North America offering first-trimester, combined serum and ultrasound, Down syndrome screening. Objectives.-To evaluate the first 4 years of the interlaboratory comparison program against stated goals, to identify areas of concern, and to create new initiatives as indicated. DESIGN: Five serum samples are distributed 3 times a year to be tested for pregnancy-associated plasma protein A, human chorionic gonadotropin or its ß subunit, and dimeric inhibin-A; participants convert these results into multiples of the median. Patient histories include nuchal translucency information that enables the calculation of the risk of Down syndrome. Also included are educational components linked to interlaboratory comparison program results. Assessment of integrated (first- and second-trimester markers) risks is accomplished by having participants combine interlaboratory comparison program results with their results from a second-trimester proficiency testing program administered by the College of American Pathologists. RESULTS: The precision profile for pregnancy-associated plasma protein A shows decreasing coefficients of variation with increasing pregnancy-associated plasma protein A concentrations and multiples of the median (25% to 11% and 30% to 15%, respectively). In contrast, coefficients of variation are a relatively constant 12% throughout the entire range of human chorionic gonadotropin results. On a logarithmic scale, the median coefficient of variation of the risk of Down syndrome is 9%. CONCLUSIONS: Participants in the interlaboratory comparison program reliably measure analytes, compute multiples of the median, and calculate consistent Down syndrome risks. Assays for the measurement of pregnancy-associated plasma protein A are not standardized and are less precise than those for human chorionic gonadotropin. Participants calculate reliable median equations given sonographer-specific sets of paired crown-rump length and nuchal translucency measurements.

Examination of the pregnancy-associated plasma protein-A assay on the Beckman Coulter Access(®) platform: suitability for use in first trimester Down's syndrome screening.

OBJECTIVE: To explore the clinical validity of the new Access(®) pregnancy-associated plasma protein-A (PAPP-A) assay for Down's syndrome screening. SETTING: Academic hospital. METHOD: Residual serum samples (n = 416) received for routine first trimester Down's syndrome screening (10-13 weeks) were retrieved from freezer storage and tested using two PAPP-A immunoassay methods. The new Access(®) assay was specifically compared, on a subset of these samples (n = 194), with an assay proven to give acceptable Down's syndrome screening performance, the PerkinElmer (PE) AutoDELFIA method. Access(®) PAPP-A levels were examined in relation to gestational age and maternal weight, and were compared with the PE method by regression and Bland-Altman analyses. RESULTS: Access(®) PAPP-A assay results were highly correlated with the PE AutoDELFIA method (r = 0.97). PAPP-A levels obtained using the Access(®) assay increased with advancing gestational age and were inversely related to maternal weight, as expected. The distribution of multiples of the median (MoM) values fit a log Gaussian distribution and the standard deviation of the log MoM (0.2331) matched published estimates. PAPP-A MoM levels in Down's syndrome pregnancies (n = 6, median 0.30) showed the expected reduction. CONCLUSION: Using an appropriate gestational age-specific median equation, the Access(®) PAPP-A assay is expected to perform acceptably in first trimester Down's syndrome screening.

Thyroperoxidase and thyroglobulin antibodies in early pregnancy and preterm delivery.

OBJECTIVE: To further evaluate the relationship between thyroid antibodies and preterm births. METHODS: This is a prospective study of pregnancy outcome and demographic data combined with retrospective measurement of thyroperoxidase and thyroglobulin antibodies. Sera were obtained at 11-13 and 15-18 weeks of gestation from 10,062 women with singleton viable pregnancies (a subset from the First- and Second-Trimester Risk of Aneuploidy [FaSTER] trial). RESULTS: Women with elevated levels of thyroperoxidase, thyroglobulin antibodies, or both in the first trimester have a higher rate of preterm delivery before 37 weeks of gestation than antibody-negative women (7.5% compared with 6.4%, odds ratio [OR] 1.18; 95% confidence interval [CI] 0.95-1.46). This is also the case for very preterm delivery before 32 weeks of gestation (1.2% compared with 0.7%, OR 1.70; 95% CI 0.98-2.94). Preterm premature rupture of membranes is also increased (2.0% compared with 1.2%, OR 1.67; 95% CI 1.05-2.44). These associations are less strong for second-trimester antibody measurements. CONCLUSION: The present data do not confirm strong associations between thyroid antibody elevations and preterm birth found in three of five previously published reports. Preterm premature rupture of membranes appears to contribute to the thyroid antibody-associated early deliveries, possibly as a result of inflammation. LEVEL OF EVIDENCE: II.

Early onset preeclampsia and second trimester serum markers.

OBJECTIVE: To examine serum markers measured in the second trimester to identify women who subsequently develop preeclampsia. METHODS: Clinically defined preeclampsia was confirmed in 45 women who had provided a serum sample as part of Down syndrome screening. Preeclampsia was categorized as mild or severe, as well as early (<32 weeks) or late onset. Each case was matched with five controls based on gestational age and date of serum collection. Stored sera were retrieved and tested for inhibin A, soluble vascular endothelial growth factor receptor 1 (sVEGF R1), placental growth factor (PlGF), and endoglin. Results were converted to multiples of the median (MoM) and compared in case and control pregnancies. Univariate analysis was used to identify the strongest markers, which were then used in a multivariate model. RESULTS: Inhibin A, PlGF, and endoglin were consistently associated with preeclampsia, especially for early onset disease. A multivariate model using the three markers could identify 50% of the pregnancies with early onset preeclampsia with a 2% false positive rate. CONCLUSION: The levels of inhibin A, PlGF, and endoglin in the second trimester can be combined using a predictive model to provide individualized risk estimates for early onset preeclampsia.

Technical standards and guidelines: prenatal screening for Down syndrome that includes first-trimester biochemistry and/or ultrasound measurements.

This statement is intended to augment the current general ACMG Standards and Guidelines for Clinical Genetics Laboratories and to address guidelines specific to first-trimester screening for Down syndrome. The aim is to provide the laboratory the necessary information to ensure accurate and reliable Down syndrome screening results given a screening protocol (e.g., combined first trimester and integrated testing). Information about various test combinations and their expected performance are provided, but other issues such as availability of reagents, patient interest in early test results, access to open neural tube defect screening, and availability of chorionic villus sampling are all contextual factors in deciding which screening protocol(s) will be selected by individual health care providers. Individual laboratories are responsible for meeting the quality assurance standards described by the Clinical Laboratory Improvement Act, the College of American Pathologists, and other regulatory agencies, with respect to appropriate sample documentation, assay validation, general proficiency, and quality control measures. These guidelines address first-trimester screening that includes ultrasound measurement and interpretation of nuchal translucency thickness and protocols that combine markers from both the first and second trimesters. Laboratories can use their professional judgment to make modification or additions.