Levels of human chorionic gonadotropin (hCG), maternal serum alpha-fetoprotein (MSAFP), and unconjugated estriol have been associated with maternal risk of Down syndrome, a chromosomal abnormality associated with mental retardation, congenital heart defects, and physical anomalies. Measurement of these serum markers has been proposed as a means of identifying pregnant women of all ages who are likely to have a Down syndrome fetus.
All pregnant women should be counseled about the risk of having a Down syndrome fetus. Multiple serum marker testing, in conjunction with adequate counseling, should be offered to pregnant women under age 35 who desire information on their risk of having a Down syndrome fetus. Women found to be at high-risk would be candidates for amniocentesis or chorionic villus sampling (CVS), with karyotyping of the tissue obtained to confirm the diagnosis. Multiple serum markers testing and counseling should also be offered to women age 35 or older who wish to avoid the risks of amniocentesis or CVS but desire information on their risk of having a Down syndrome fetus.
High maternal serum levels of hCG with low levels of MSAFP and/or unconjugated estriol in pregnant women has been associated with an increased risk of carrying a Down syndrome fetus. Measurement of multiple serum markers offers a means of identifying young women who are at high-risk of having a Down syndrome fetus; women found to be at high-risk would be offered confirmatory testing by karyotyping tissue obtained by amniocentesis or CVS. Multiple marker testing may also allow pregnant women at high-risk (such as pregnant women age 35 and older) an alternative means of determining the likelihood of having a Down syndrome fetus if they wish to avoid the risk of fetal harm and death associated with amniocentesis and CVS.
Dimeric inhibin A is now used by some commercial laboratories in combination with the 3 traditional analytes. With a screen-positive rate of 5 % or less, this new 4-analyte combination appears to detect 67 % to 76 % of Down syndrome cases in women younger than 35 years (ACOG, 2001).
Efforts to improve biochemical screening have centered on the investigation of screening in the first trimester and on the search for better markers. Two potential serum markers that can be measured during the first trimester are the free beta subunit of hCG and pregnancy-associated protein A. Serum concentrations of the free beta subunit of hCG are higher than average, and pregnancy-associated protein A concentrations are lower, in the presence of a fetus with Down syndrome. The combination of free beta-hCG, PAPP-A, and maternal age appears to yield detection and false-positive rates comparable to second-trimester serum screening (63 % and 5.5 %, respectively) (ACOG, 2001). Unfortunately, free beta-hCG may not be higher in Down syndrome pregnancies until 12 weeks of gestation, and PAPP-A seems to lose its discrimination value after 13 weeks of gestation, making accurate assessment of gestational age and careful timing of the screening test essential. The American College of Obstetricians and Gynecologists (2001) states, however, that “preliminary data [regarding these analytes] remains controversial and testing is not yet standard of care.” Many other screening analytes, including urinary beta-core and human placental lactogen, are currently being investigated for use in the first and second trimesters to determine whether they alone or in combination, will increase detection to a rate greater than the current 60 %.
Maternal serum screening for Down syndrome in the first trimester, rather than second, is not widespread because: (i) CVS and early amniocentesis are not as widely available as amniocentesis during the second trimester, and they may be less safe; (ii) maternal serum screening for Down syndrome in the first trimester, followed by screening for open neural-tube defects during the second, is likely to be less cost effective than performing all the screening at the same time; (iii) because serum concentrations of pregnancy-associated protein A change rapidly during the first trimester, gestational age needs to be established by ultrasonography in order for the sensitivity of first-trimester screening to be equivalent to second-trimester screening; and (iv) assays for serum pregnancy-associated protein A and the free beta subunit of hCG are not licensed for clinical use in the United States.
The National Institutes of Health sponsored a multi-center prospective study (the First and Second Trimester Evaluation of Aneuploidy Risk or 'FASTER' trial) that compared first- and second-trimester non-invasive methods of screening for fetal aneuploidies with second trimester multiple marker maternal serum screening that is the current standard of care (NICHD, 2001). The results of the FASTER trial are described in CPB 282 - Noninvasive Down Syndrome Screening. First-trimester screening, taken together with maternal age, involves an ultrasound measurement of fetal nuchal translucency thickness at 10 to 14 gestational weeks, as well as serum levels of pregnancy-associated protein A and free beta-hCG. Second-trimester screening is based on the serum “triple screen,” which consists of measurement of levels of AFP, unconjugated estriol (uE3), and hCG, performed at 15 to 18 gestational weeks, taken together with maternal age and serum inhibin-A levels (so called “quad test”).
In October 1999, the ACOG issued a position statement that first trimester screening is investigational and should not be used in routine clinical practice. The ACOG statement concluded that “[f]irst-trimester screening for fetal chromosome, cardiac, and other abnormalities using the nuchal translucency marker alone or in combination with serum markers appears promising but remains investigational.”
Based on the results of the FASTER trial, which found that first-trimester screening is as good as or better than second-trimester screening, ACOG (2004) stated that first-trimester screening using nuchal translucency, free beta-hCG, and pregnancy-associated plasma protein-A has comparable detection rates and positive screening rates for Down syndrome as second-trimester screening using 4 serum markers (AFP, beta-hCG, uE3, and inhibin-A). The American College of Obstetricians and Gynecologists stated that, although first-trimester screening for Down syndrome and trisomy 18 is an option, it should be offered only if certain criteria can be met.
Laigaard and colleagues (2006) stated that maternal serum A Disintegrin And Metalloprotease 12 (ADAM 12) is reduced, on average, in early first trimester Down and Edwards' syndrome pregnancies; however the extent of reduction declines with gestation. These investigators examined the levels of ADAM 12 at 9 to 12 weeks when the marker might be used concurrently with other established markers. Samples from 16 Down and 2 Edwards' syndrome cases were retrieved from storage and tested together with 313 unaffected singleton pregnancies using a semi-automated time-resolved immuno-fluorometric assay. Results were expressed in multiples of the gestation-specific median (MoM) based on regression. The median in Down syndrome was 0.94 MoM with a 10th to 90th percentile range of 0.22 to 1.63 MoM compared with 1.00 and 0.33 to 2.24 MoM in unaffected controls (p = 0.21, one-side Wilcoxon Rank Sum Test). The 2 Edwards' syndrome cases had values 0.31 and 2.17 MoM. The authors concluded that ADAM12 can not be used concurrently with other markers in the late first trimester. However, it does have the potential to be used earlier in pregnancy either concurrently with other early markers or in a sequential or contingent protocol. The authors stated that more research is needed to reliably predict the performance of either approach. Furthermore, the ACOG practice bulletin on screening for fetal chromosomal abnormalities (2007) does not mention ADAM 12 as a serum marker for screening Down syndrome.
Christiansen et al (2007) examined the potential of ADAM 12 as a second-trimester maternal serum marker of Down syndrome (DS). The concentration of ADAM 12 was determined in gestational week 14 to 19 in 88 DS pregnancies and 341 matched control pregnancies. Medians of normal pregnancies were established by polynomial regression and the distribution of log(10) MoM ADAM 12 values in DS pregnancies and controls determined. Correlations with alpha-fetoprotein (AFP) and free beta-hCG were established and used to model the performance of maternal serum screening with ADAM 12 in combination with other second-trimester serum markers. The ADAM 12 maternal serum concentration was significantly increased with a median MoM of 1.85 and a mean log(10) MoM (SD) of 0.268 (0.2678) compared to a mean log(10) MoM (SD) of 0.013 (0.4318) in controls. ADAM 12 correlated with maternal weight and ethnicity (with the serum concentration increased in Afro-Caribbeans), but neither with maternal age nor gestational age, and only marginally with AFP (r(DS) = 0.078, r(controls) = 0.093) and free beta-hCG (r(DS) = 0.073, r(controls) = 0.144. The increase in detection rate -- for a false positive rate of 5 % -- by adding ADAM 12 to the double test (AFP + free beta-hCG) was 4 %, similar to that of adding unconjugated estriol to the double test. The authors concluded that ADAM 12 is an efficient second-trimester marker for DS. Moreover, they stated that further studies should be conducted to determine whether it may be a useful additional or alternative marker to those currently used in the second-trimester.
Wright and Burton (2009) stated that cell-free fetal nucleic acids (cffNA) can be detected in the maternal circulation during pregnancy, potentially offering an excellent method for early non-invasive prenatal diagnosis (NIPD) of the genetic status of a fetus. Using molecular techniques, fetal DNA and RNA can be detected from 5 weeks gestation and are rapidly cleared from the circulation following birth. These investigators searched PubMed systematically using keywords free fetal DNA and NIPD. Reference lists from relevant papers were also searched to ensure comprehensive coverage of the area. Cell-free fetal DNA comprises only 3 % to 6 % of the total circulating cell-free DNA, thus diagnoses are primarily limited to those caused by paternally inherited sequences as well as conditions that can be inferred by the unique gene expression patterns in the fetus and placenta. Broadly, the potential applications of this technology fall into 2 categories: (i) high genetic risk families with inheritable monogenic diseases, including sex determination in cases at risk of X-linked diseases and detection of specific paternally inherited single gene disorders; and (ii) routine antenatal care offered to all pregnant women, including prenatal screening/diagnosis for aneuploidy, particularly DS, and diagnosis of Rhesus factor status in RhD negative women. Already sex determination and Rhesus factor diagnosis are nearing translation into clinical practice for high-risk individuals. The authors concluded that the analysis of cffNA may allow NIPD for a variety of genetic conditions and may in future form part of national antenatal screening programs for DS and other common genetic disorders.
The American College of Obstetricians and Gynecologists (2012) has stated that noninvasive prenatal testing that uses cell free fetal DNA from the plasma of pregnant women offers tremendous potential as a screening tool for fetal aneuploidy. The ACOG Committee Opinion has concluded that measurement of cell-free DNA may be considered for the following indications: maternal age 35 years or older at delivery; fetal ultrasonographic findings predicting an increased risk of fetal aneuploidy; history of a prior pregnancy with an aneuploidy; positive screening test for an aneuploidy, including first trimester, sequential, or integrated screen, or a positive quadruple screen; parental balanced robertsonian translocation with increased risk for fetal trisomy 13 or trisomy 21.The ACOG Committee Opinion stated that cell free fetal DNA testing should be an informed patient choice after pretest counseling and should not be part of routine prenatal laboratory assessment. The ACOG Committee Opinion stated that cell free fetal DNA testing should not be offered to
low-risk women or women with multiple gestations because it has not been sufficiently evaluated in these groups. A negative cell free fetal DNA test result does not ensure an unaffected pregnancy. The Committee Opinion states that a patient with a positive test result should be referred for genetic counseling and should be offered invasive prenatal diagnosis for confirmation of test results.
Assessments of the literature on cell-free DNA for detection of fetal aneuploidy are currently underway by the BlueCross BlueShield Association Technology Evaluation Center and the California Technology Assessment Forum.
Currently available cell-free DNA tests are laboratory developed tests, and there is no requirement for premarket approval by the U.S. Food and Drug Administration. Such laboratory developed tests are regulated by the Centers for Medicare & Medicaid Services as part of the Clinical Laboratory Improvement Amendments of 1988 (CLIA). However, CLIA regulations are restricted to certifying internal procedures and qualifications of laboratories rather than the safety and efficacy of laboratory developed tests specifically. CLIA regulations of genetic tests are designed to ensure procedural compliance at laboratory level and do not extend to validation of specific tests.
Ehrich and colleagues (2011) evaluated a multi-plexed massively parallel shotgun sequencing assay for non-invasive trisomy 21 detection using circulating cell-free fetal DNA. Sample multi-plexing and cost-optimized reagents were evaluated as improvements to a non-invasive fetal trisomy 21 detection assay. A total of 480 plasma samples from high-risk pregnant women were employed. In all, 480 prospectively collected samples were obtained from third-party storage site; 13 of these were removed due to insufficient quantity or quality. Eighteen samples failed pre-specified assay quality control parameters. In all, 449 samples remained: 39 trisomy 21 samples were correctly classified; 1 sample was mis-classified as trisomy 21. The overall classification showed 100 % sensitivity (95 % confidence interval [CI]: 89 to 100 %) and 99.7 % specificity (95 % CI: 98.5 to 99.9 %). The authors concluded that extending the scope of previous reports, this study demonstrated that plasma DNA sequencing is a viable method for non-invasive detection of fetal trisomy 21 and warrants clinical validation in a larger multi-center study.
Sehnert, et al. (2011) reported on a cross sectional study of the use of cell free DNA to detect fetal aneuploidy. Blood samples from 119 adult pregnant women underwent massively parallel DNA sequencing. Fifty-three sequenced samples came from women with an abnormal fetal karyotype. To minimize the intra- and interrun sequencing variation, the investigators developed an optimized algorithm by using normalized chromosome values (NCVs) from the sequencing data on a training set of 71 samples with 26 abnormal karyotypes. The classification process was then evaluated on an independent test set of 48 samples with 27 abnormal karyotypes. The authors reported that sequencing of the independent test set led to 100% correct classification of T21 (13 of 13) and T18 (8 of 8) samples. The authors noted that other chromosomal abnormalities were also identified.
Palomaki, et al. (2011) reported on a nested case-control study of the use of massively parallel DNA sequencing to detect fetal aneuploidy. The investigators used blood samples that were collected in a prospective, blinded study from 4664 pregnancies at high risk for Down syndrome by maternal age, family history, or positive screening test. Fetal karyotyping results from amniocentesis or chorionic villus sampling were compared to cell free DNA sequencing in 212 Down syndrome and 1484 matched euploid pregnancies. 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.
In a subsequent report, Palomaki, et al. (2012) selected 62 pregnancies with trisomy 18 and 12 with trisomy 13 from the cohort of 4,664 pregnancies along with matched euploid controls (including 212 additional Down syndrome and matched controls already reported in Palomaki, et al., 2011), and their samples tested by massively parallel DNA sequencing. Among the 99.1% of samples interpreted (1,971/1,988), observed trisomy 18 detection rates was 100% (59/59), with a false positive rate of 0.28%. Observed trisomy 13 detection rate was 91.7% (11/12) with a false-positive rates of 0.97%l however, this estimate was based upon only 12 cases. Among the 17 samples without an interpretation, three were trisomy 18. The authors stated that, 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.
Bianchi, et al. (2012) reported on a nested case-control study of the use of massively parallel DNA sequencing to detect fetal aneuploidy. The investigators used blood samples that were collected in a prospective, blinded study from 2,882 high-risk women scheduled to undergo amniocentesis or chorionic villus sampling procedures at 60 U.S. sites. An independent biostatistician selected from these blood samples all singleton pregnancies with any abnormal karyotype, and for comparison, selected a balanced number of randomly selected pregnancies with euploid karyotypes. Chromosome classifications were made for each sample by massively parallel sequencing and compared with fetal karyotype determined by amniocentesis or chorionic villus sampling. The authors had 532 samples, 221 of which had abnormal karyotypes. The authors reported that 89 of 89 trisomy 21 cases were classified correctly (sensitivity 100%, 95% confidence interval [CI] 95.9 to 100), 35 of 36 trisomy 18 cases were classified correctly (sensitivity 97.2%, 95% CI 85.5 to -99.9), 11 of 14 trisomy 13 cases (sensitivity 78.6%, 95% CI 49.2 to 99.9), and 15 of 16 monosomy X cases (sensitivity 93.8%, 95% CI 69.8 to 99.8). The authors reported that there were no false-positive results for autosomal aneuploidies (100% specificity, 95% CI more than 98.5 to 100). In addition, fetuses with mosaicism for trisomy 21 (3/3), trisomy 18 (1/1), and monosomy X (2/7), three cases of translocation trisomy, two cases of other autosomal trisomies (20 and 16), and other sex chromosome aneuploidies (XXX, XXY, and XYY) were classified correctly. Because this was a nested case control study, and therefore it did not reflect true population prevalence of the fetal aneuploidies, positive and negative predictive values cannot be calculated. Specificities were estimated based upon a relatively small number of controls; further studies involving a larger number of unaffected controls would estimate the specificity with greater precision.
Regarding the evidence for cell-free DNA tests for fetal aneuploidy, Allyse, et al. (2012) has commented that companies offering such tests have limited themselves to publishing the results of clinical and analytic validation studies. However, more contextual, but no less important, issues of consistent and systematic validation, timing, risk and scope of cell-free DNA testing still need to be resolved. The authors stated that, at present, however, cell-free DNA tests have not achieved sufficient specificity and sensitivity to replace existing invasive tests as a diagnostic tool. The authors stated that demonstrated detection rates with cell-free DNA show a significant improvement over existing noninvasive integrated screening regimes. Nevertheless, they do not match the near-perfect diagnostic capabilities of invasive tests. Furthermore, existing clinical validation trials have taken place only in high-risk populations. The authors stated that it is unclear whether acceptable positive and negative predictive values can be attained in lower risk populations.
In addition to its noninvasive nature, another commonly espoused feature of non-invasive tests using cell-free DNA is its potential to detect fetal DNA beginning at 10 weeks of gestation (Allyse, et al., 2012). Some women may want cell-free DNA testing to enable them to terminate a non-viable pregnancy as early as possible to avoid physical and emotional discomfort. However, a majority of pregnancies with trisomy 13, 18 and 21 spontaneously abort during the first trimester. Conducting early testing to recognize a trisomic pregnancy could require women to make wrenching decisions about termination and generate considerable guilt and stress that might have been avoided had the fetus spontaneously aborted. In addition to the psychosocial effects, this process would also entail spending considerable medical resources on prenatal care for non-viable pregnancies (Allyse, et al., 2012).
The UK National Health Service Fetal Anomaly Screening Programme has stated that cell-free DNA testing "is very much in the early stages of development" and more research is needed to make sure cell-free DNA is a better test than those currently offered to women wanting information on the health of their baby (Delbarre, 2012). As such, non-invasive prenatal diagnosis using cell-free DNA to test for genetic conditions is unlikely to be available on the UK National Health Service for at least five years. The UK National Health Service would not currently consider using these tests to replace any of the tests currently offered as part of the Fetal Anomaly Ultrasound and Down’s syndrome Screening Programme.
Koster and colleagues (2009) examined if placental protein 13 (PP13) could be an additional marker in first trimester screening for aneuploidies. These researchers assessed differences in multiples of the gestation-specific normal median (MoMs), PP13 concentrations were measured in serum samples from DS, trisomy 18 and 13 affected pregnancies and euploid singleton pregnancies (4 for each case matched for duration of storage, maternal weight and age). The PP13 MoM in DS cases (n = 153) was 0.91 [not statistically significant from controls (n = 853); p = 0.06; Wilcoxon rank sum test, 2-tail]. Placental protein 13 MoMs were decreased in trisomy 18 (n = 38- median MoM 0.64; p < 0.0001) and trisomy 13 cases (n = 23-median MoM 0.46; p < 0.0001). There was a slight upward trend in MoM values of the DS cases with gestational weeks. The PP13 MoM was significantly correlated with the pregnancy associated plasma protein-A MoM and the free beta-subunit of hCG (fbeta-hCG) MoM. The authors concluded that PP13 does not seem to be a good marker for DS.
Koster and co-workers (2010) ascertained the distributions of pregnancy-associated plasma protein A (PAPP-A), fbeta-hCG, ADAM12 and PP13 in first trimester twin pregnancies. Serum marker concentrations were measured in monochorionic and dichorionic twin pregnancies and singleton controls to study differences in MoMs. Median PAPP-A and fbeta-hCG MoMs were 2.03 and 1.87 for monochorionic twins (n = 116) and 2.18 and 1.89 for dichorionic twins (n = 650). Furthermore, ADAM12 and PP13 MoMs were 1.66 and 1.56 for monochorionic twins (n = 51) and 1.64 and 1.53 for dichorionic twins (n = 249). No statistically significant differences between monochorionic and dichorionic twin pregnancies were found. Correlations between markers in these pregnancies did not differ from singletons. The authors concluded that for first-trimester screening, different parameters for monochorionic and dichorionic twin pregnancies is not necessary. Furthermore, if ADAM12 and PP13 will be adopted as screening markers, the presented median MoM values, standard deviations and correlation coefficients for twin pregnancies may contribute to a proper twin risk estimation.
In a case control study, Torring and colleagues (2010) examined if ADAM12-S is a useful serum marker for fetal trisomy 21 using the mixture model. These researchers measured ADAM12-S by KRYPTOR ADAM12-S immunoassay in maternal serum from gestational weeks 8 to 11 in 46 samples of fetal trisomy 21 and in 645 controls. Comparison of sensitivity and specificity of first trimester screening for fetal trisomy 21 with or without ADAM12-S was included in the risk assessment using the mixture model. The concentration of ADAM12-S increased from week 8 to 11 and was negatively correlated with maternal weight. Log MoM ADAM12-S was positively correlated with log MoM PAPP-A (r = 0.39, p < 0.001), and with log MoM free beta hCG (r = 0.21, p < 0.001). The median ADAM12-S MoM in cases of fetal trisomy 21 in gestational week 8 was 0.66 increasing to about 0.9 MoM in weeks 9 and 10. The use of ADAM12-S along with biochemical markers from the combined test (PAPP-A, free beta-hCG) with or without nuchal translucency measurement did not affect the detection rate or false positive rate of fetal aneuploidy as compared to routine screening using PAPP-A and free beta-hCG with or without nuchal translucency. The authors concluded that these findings showed moderately decreased levels of ADAM12-S in cases of fetal aneuploidy in gestational weeks 8 to 11. However, including ADAM12-S in the routine risk does not improve the performance of first trimester screening for fetal trisomy 21.
Cowans et al (2010) examined the stability of ADAM-12 with time and at different temperatures. Maternal serum and whole blood pools were stored at 30 degrees C, room temperature and refrigerator temperature or subjected to repeated freeze-thaw cycles. ADAM-12 was measured at set time points using an automated DELFIA research assay. Using a 10 % change in concentration as a limit of stability, ADAM-12 is stable in serum for less than 15 hrs at 30 degrees C, less than 20 hrs at room temperature and for 51 hrs at refrigerator temperature. ADAM-12 levels are not altered following 3 -20 degrees C to room temperature freeze-thaw cycles. The stability of ADAM-12 in whole blood appears similar to that in serum. The authors concluded that these findings suggested that ADAM-12 may be unstable under many routine laboratory conditions, and the marker's instability may also be partly responsible for the discrepancies in the literature.
Li et al (2010) compared the difference in maternal serum anti-Mullerian hormone (AMH) level between DS pregnancies and unaffected pregnancies, and evaluated its performance as a screening marker for DS pregnancy. A total of 145 pregnancies affected by fetal DS and 290 unaffected controls matched with maternal age and gestational age were selected, and their archived first or second trimester serum retrieved for AMH assay. There was no significant difference in maternal serum AMH level between pregnancies affected and unaffected by fetal DS. First trimester serum samples had higher AMH level compared to second trimester samples. The authors concluded that maternal serum AMH level, as a marker of ovarian age, is not superior to chronological age in predicting DS pregnancies. They stated that despite the cross-sectional nature of the study, the variation of maternal serum AMH concentration with gestational age warrants further investigation.
Wright et al (2010) provided estimates and confidence intervals for the performance (detection and false-positive rates) of screening for Down's syndrome using repeated measures of biochemical markers from first and second trimester maternal serum samples taken from the same woman. Stored serum on Down's syndrome cases and controls was used to provide independent test data for the assessment of screening performance of published risk algorithms and for the development and testing of new risk assessment algorithms. A total of 78 women with pregnancy affected by Down's syndrome and 390 matched unaffected controls, with maternal blood samples obtained at 11 to 13 and 15 to 18 weeks' gestation, and women who received integrated prenatal screening at North York General Hospital at 2 time intervals: between December 1, 1999 and October 31, 2003, and between October 1, 2006 and November 23, 2007 were include in this analysis. Repeated measurements (first and second trimester) of maternal serum levels of hCG, uE3 and PAPP-A together with AFP in the second trimester were carried out. Main outcome measures were detection and false-positive rates for screening with a threshold risk of 1 in 200 at term, and the detection rate achieved for a false-positive rate of 2 %. Published distributional models for Down's syndrome were inconsistent with the test data. When these test data were classified using these models, screening performance deteriorated substantially through the addition of repeated measures. This contradicts the very optimistic results obtained from predictive modeling of performance. Simplified distributional assumptions showed some evidence of benefit from the use of repeated measures of PAPP-A but not for repeated measures of uE3 or hCG. Each of the 2 test data sets was used to create new parameter estimates against which screening test performance was assessed using the other data set. The results were equivocal but there was evidence suggesting improvement in screening performance through the use of repeated measures of PAPP-A when the first trimester sample was collected before 13 weeks' gestation. A Bayesian analysis of the combined data from the 2 test data sets showed that adding a second trimester repeated measurement of PAPP-A to the base test increased detection rates and reduced false-positive rates. The benefit decreased with increasing gestational age at the time of the first sample. There was no evidence of any benefit from repeated measures of hCG or uE3. The authors concluded that If realized, a reduction of 1 % in false-positive rate with no loss in detection rate would give important benefits in terms of health service provision and the large number of invasive tests avoided. The Bayesian analysis, which shows evidence of benefit, is based on strong distributional assumptions and should not be regarded as confirmatory. The evidence of potential benefit suggests the need for a prospective study of repeated measurements of PAPP-A with samples from early in the first trimester. A formal clinical effectiveness and cost-effectiveness analysis should be undertaken. This study has shown that the established modeling methodology for assessing screening performance may be optimistically biased and should be interpreted with caution.
Moise (2012) reviewed the evidence for the use of cell-free fetal DNA to determine the fetal RHD gene. The feasibility of using cell-free fetal DNA circulating in maternal serum to determine fetal RHD gene and guide administration of prophylaxis has been shown in several studies (Rouillac-Le Sciellour et al, 2004; Finning et al, 2008; Muller et al, 2008; Van der School et a., 2006; Clausen et al, 2012). In the largest of these studies (n = 2,312 Rh(D)-negative women), fetal RHD gene detection sensitivity was 99.9 % at 25 weeks of gestation using an automated system that targeted 2 RHD exons (Clausen et al, 2012). Six fetuses were falsely identified as RHD-positive and 74 results were inconclusive due to methodologic issues or variant D types; all of these women received antenatal Rh(D) prophylaxis. Prophylaxis was unnecessary and avoided in 862 true-negative cases (37.3 %), unnecessary antenatal prophylaxis was administered to 39 women who had a positive or inconclusive result antenatally but delivered a Rh(D)-negative newborn (1.7 %). In 2 pregnancies (0.087 %), a Rh(D)-positive fetus was not detected antenatally so antenatal prophylaxis was not given; however, the women received post-natal prophylaxis.
An assessment by the Swedish Council on Technology Assessment in Health Care (SBU, 2011) found that there is moderately strong scientific evidence that fetal RHD determination by non-invasive fetal diagnostic tests has a sensitivity and specificity of nearly 99 %. The assessment stated that these results are largely based on studies of RhD-negative pregnant women who are not RhD-immunized. The report stated that those studies that also included pregnant women who have been immunized against RhD showed similar results. The report concluded that screening for fetal blood group using non-invasive fetal diagnostic tests, in combination with specific prenatal preventive measures (targeted RhD prophylaxis), could result in fewer RhD-negative pregnant women developing antibodies to RhD. The report concluded that the organizational and health economic consequences of introducing this type of screening have not been established.
In some European countries, fetal RHD gene determination is performed clinically in Rh(D)-negative women and administration of antenatal anti-D is avoided in the case of a RHD-negative fetus. An opinion by the Royal College of Obstetricians and Gynaecologists (Chitty and Crolla, 2009) stated that obstetricians have used non-invasive prenatal diagnosis to guide management of women who are RhD-negative and at risk of hemolytic disease of the newborn for years and guidelines should be revised to reflect this change in practice.
The American College of Obstetricians and Gynecologists (2012) has no recommendation for use of fetal cell-free DNA in preventing RHD alloimmunization.
Moise and Argoti (2012) evaluated the application of new technologies to the management of the red cell alloimmunized pregnancy. These investigators searched 3 computerized databases for studies that described treatment or prevention of alloimmunization in pregnancy (MEDLINE, Embase, and the Cochrane Central Register of Controlled Trials [1990 to July 2012]). The text words and MeSH included Rhesus alloimmunization, Rhesus isoimmunization, Rhesus prophylaxis, Rhesus disease, red cell alloimmunization, red cell isoimmunization, and intrauterine transfusion. Of the 2,264 studies initially identified, 246 were chosen after limiting the review to those articles published in English and cross-referencing to eliminate duplication. Both authors independently reviewed the articles to eliminate publications involving less than 6 patients. Special emphasis was given to publications that have appeared since 2008. Quantitative polymerase chain reaction can be used instead of serology to more accurately determine the paternal RHD zygosity. In the case of unknown or a heterozygous paternal RHD genotype, new DNA techniques now make it possible to diagnose the fetal blood type through cell-free fetal DNA in maternal plasma. Serial Doppler assessment of the peak systolic velocity in the middle cerebral artery is now the standard to detect fetal anemia and determine the need for the first intrauterine transfusion. Assessment of the peak systolic velocity in the middle cerebral artery can be used to time the second transfusion, but its use to decide when to perform subsequent procedures awaits further study. New data suggested normal neurologic outcome in 94 % of cases after intrauterine transfusion, although severe hydrops fetalis may be associated with a higher risk of impairment. Recombinant Rh immune globulin is on the horizon. The authors stated that cell-free fetal DNA for fetal RHD genotyping may be used in the future to decide which patients should receive antenatal Rh immune globulin.