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.

The impact of preconception counseling on pregnancy outcomes. The experience of the Maine Diabetes in Pregnancy Program.

OBJECTIVE: To determine if a noncentralized, statewide program could be established to educate health-care providers and women with pregestational diabetes on available strategies to prevent adverse outcomes in pregnancies complicated by diabetes. Characteristics of women who participated in the program and the outcomes of their pregnancies are evaluated. RESEARCH DESIGN AND METHODS: A network of regional providers caring for pregnant women with diabetes was developed. Continuing education sessions were delivered to both providers and women with existing diabetes on the importance of preconception counseling. RESULTS: Maine health-care providers collaborated on the development and adoption of three patient-care guidelines that address preconception counseling, prenatal care, and contraception for women with established diabetes. A total of 185 pregnancies among 160 women with pregestational diabetes reporting estimated delivery dates between 1 January 1987 and 31 December 1990 were identified. Of the total pregnancies, 62 (34%) occurred in women who received preconception counseling: among these 62 pregnancies were one major congenital defect (1.6%) and four fetal or neonatal deaths (6.4%). Among the 123 (66%) pregnancies occurring in women that had not received preconception counseling, 8 (6.5%) infants were born with congenital abnormalities, and 26 (21.1%) fetal or neonatal deaths were documented. CONCLUSIONS: A program promoting preconception counseling can be implemented on a statewide basis by using various health-care providers to deliver the program. Participation in such a program appears to be related to improved pregnancy outcomes among women with pregestational diabetes.

The flow cytometric crossmatch and early renal transplant loss.

Data from this retrospective study indicate that a positive two-color T and/or B cell flow cytometric crossmatch (FCXM) is predictive of early renal allograft loss (less than 2 months) in cadaveric kidney donor recipients who had a negative crossmatch by the antihuman globulin complement-dependent cytotoxicity technique. Among 90 cadaveric kidney donor recipients (67 primary, 23 regrafts), 14 (8 primary, 6 regrafts) lost their renal allografts within 2 months, and 10 of the 14 were FCXM positive and HLA sensitized. The remaining 76 allografts survived beyond 2 months, 12 of which were FCXM-positive. Thus, the FCXM sensitivity rate for detecting early graft loss was 71%, and the specificity rate was 84%. Cadaveric graft-loss rates at 2 months were 33% for primary and 60% for FCXM-positive regrafts in contrast to 7% for primary and 0% for FCXM-negative regrafts. The difference in early graft loss between FCXM-positive and FCXM-negative recipients was statistically significant (P less than 0.0001). Subset analyses of FCXM-positive graft recipients indicate: (1) previous early graft loss contraindicates transplantation of an FXCM-positive regraft (P = 0.03); and (2) panel reactive antibody (PRA) less than or equal to 10% at crossmatch is not associated with early graft loss (P = 0.04). There was no significant difference in 1-year graft survival between primary and regrafts in either FCXM-negative recipients (85% vs. 77%, respectively) or FCXM-positive recipients (67% vs. 40%). All 12 of the FCXM-positive primary and regrafts that survived 2 months continued to function at 2 years. Stepwise logistic regression analysis of 5 independent predictor variables (FCXM status, gender, primary vs. regraft status, PRA level, and HLA mismatched antigens) indicated that the FCXM test was the best predictor of early graft loss. When FCXM results of the 90 cadaveric graft recipients were ranked in three groups, an FCXM channel shift of 29 or greater (third tertile) on a 1024 channel log scale was associated with a 7.0-fold (95% confidence interval 1.9-25.5) increased risk of early graft failure when compared to the first two tertiles. These data indicate that the FCXM offers an additional approach for identifying sensitized patients at risk of early renal allograft loss.

Cigarette smoking-associated changes in blood lipid and lipoprotein levels in the 8- to 19-year-old age group: a meta-analysis.

In this meta-analysis it was demonstrated that, when compared with nonsmokers of similar age, smokers in the 8- to 19-year-old age group have significantly higher serum levels of triglyceride (+11.8%), very-low-density lipoprotein (VLDL)-cholesterol (+12.4%) and low-density lipoprotein (LDL)-cholesterol (+4.1%) and significantly lower serum levels of high-density lipoprotein (HDL)-cholesterol (-8.5%) and total cholesterol (-3.7%). All of these smoking-associated changes are in the same direction as those found in adults, with the exception of total cholesterol levels, which are significantly increased in adult smokers. The extent to which mean triglyceride, LDL-cholesterol, and HDL-cholesterol levels are shifted is significantly greater in the 8- to 19-year-old smokers than in adult smokers. The changes in mean total cholesterol levels among smokers in both age groups represent only the net shifts in the lipoprotein fractions and are therefore likely to be a less sensitive indicator of the possible lipid-related excess coronary artery disease risk in smokers.

Tay-Sachs disease in persons of French-Canadian heritage in northern New England.

This study sought to determine whether persons of French-Canadian heritage in northern New England are at high risk for the lethal infantile form of Tay-Sachs disease. In order to accomplish this, death records and laboratory diagnostic records were surveyed to ascertain Tay-Sachs deaths in a cohort of 372,000 live births between 1977-1986. The proportion of the total population with French-Canadian or Jewish heritage was determined from census and birth records, and the ethnic background of Tay-Sachs cases was determined from the corresponding birth records. In 1,860 births, both parents were of Ashkenazi Jewish heritage. One of those children was diagnosed with Tay-Sachs disease. In 41,000 births, both parents were of French-Canadian heritage, and in an additional 93,000 births, one parent was of French-Canadian heritage. No cases of Tay-Sachs disease were identified in the offspring of those individuals. Approximately 14 cases (95% confidence interval 8-20) would be expected, if the gene frequency approximated that reported for individuals of Ashkenazi Jewish heritage. Based on the results of this study, routine testing for Tay-Sachs disease heterozygosity is not indicated for persons of French-Canadian heritage in northern New England. This conclusion may not necessarily be valid for persons of French-Canadian heritage residing in other states. Further studies of Tay-Sachs disease mutations and prevalence among persons of French-Canadian heritage will be important to determine possible regional variations in gene frequencies.

Can low birth weight after elevated maternal serum alpha-fetoprotein be explained by maternal weight?

Low birth weight infants are delivered with increased frequency in women who have elevated maternal serum alpha-fetoprotein values in the second trimester. Maternal serum alpha-fetoprotein elevations, however, are found more often in lighter-weight women, a group known to have lower-weight infants regardless of maternal serum alpha-fetoprotein levels. To clarify the association between elevated maternal serum alpha-fetoprotein levels and low birth weight independent of maternal weight, we applied a weight correction formula to maternal serum alpha-fetoprotein values from 9507 singleton viable pregnancies without major fetal malformations. Before adjusting for weight, 486 of the women (5.1%) had maternal serum alpha-fetoprotein values of 2.0 or more multiples of the median. The weight adjustment process removed 100 lighter-weight women from this category, added 58 heavier women, and led to an equivalent proportion of women in the various weight categories who were classified as having maternal serum alpha-fetoprotein values of 2.0 or more multiples of the median. Of the 388 low birth weight pregnancies (2500 g or less), 50 initially had maternal serum alpha-fetoprotein values of 2.0 or more multiples of the median; after weight adjustment, seven lighter-weight women were removed, four heavier women were added, the median birth weight fell from 2217 to 1956 g, and a threefold increase in risk was found for low birth weight outcome regardless of weight class. Maternal serum alpha-fetoprotein elevations predict increased risk for low birth weight outcome independent of maternal weight.

Self-rated physical activity level during the second trimester and pregnancy outcome.

To examine the relationship between level of physical activity during pregnancy and subsequent pregnancy outcome, we asked women enrolling for maternal serum alpha-fetoprotein (MSAFP) screening in Maine during the years 1984-1988 to rate their usual physical activity level as light, moderate, or vigorous. Overall, 23,091 women were approached and 21,342 (92%) responded. Ten percent rated their physical activity level as light, 84% as moderate, and 6% as vigorous. Those who identified their physical activity level as vigorous were, on average, slightly older and more educated, and weighted less than women in the other two categories. However, there were no significant differences in the rates of low birth weight or fetal or neonatal death.

Second-trimester maternal serum alpha-fetoprotein, unconjugated estriol, and hCG levels in pregnancies with ventral wall defects.

OBJECTIVE: To determine if second-trimester maternal serum concentrations of unconjugated estriol (E3) and hCG are altered in pregnancies associated with fetal gastroschisis or omphalocele. METHODS: Concentrations of alpha-fetoprotein (AFP), unconjugated E3, and hCG were measured in a case-control study involving 23 cases of gastroschisis, 17 cases of omphalocele, and 200 matched unaffected pregnancies. RESULTS: As reported previously, median AFP levels were significantly higher in pregnancies with gastroschisis and omphalocele compared to unaffected pregnancies (9.42 and 4.18 multiples of the unaffected population median [MoM], respectively). The median hCG values were not significantly different for the two defects (1.10 and 1.13 MoM, respectively). Six of the cases of omphalocele were associated with other anomalies, but exclusion of these cases from the analysis did not alter the conclusions. CONCLUSIONS: Unconjugated E3 and hCG measurements are not useful in screening for, or distinguishing between, open ventral wall defects. Alpha-fetoprotein measurements alone will detect nearly all cases of gastroschisis and most cases of omphalocele.

Second-trimester maternal serum alpha-fetoprotein levels in pregnancies associated with gastroschisis and omphalocele.

This population-based study analyzes maternal serum alpha-fetoprotein (MSAFP) distributions for 20 cases of gastroschisis and 13 cases of omphalocele occurring in singleton pregnancies from among 72,782 second-trimester pregnancies in Maine and Rhode Island screened consecutively between January 1, 1979 and February 28, 1987. Median values (and ranges) for the two lesions were 4.1 multiples of the median (0.5-29.8) for omphalocele and 7.0 multiples of the median (3.6-13.5) for gastroschisis. The MSAFP distributions for the two conditions were both log-Gaussian, and the log standard deviation was smaller for gastroschisis than for omphalocele. The MSAFP screening sensitivity was greater for gastroschisis than for omphalocele at any given cutoff, and the overall sensitivity of this screening process for detecting open ventral wall defects will differ, therefore, depending upon the relative proportion of gastroschisis and omphalocele cases that occur in the screened population.

Prospective multicenter study of second-trimester nuchal skinfold thickness in unaffected and Down syndrome pregnancies.

OBJECTIVE: To determine the distribution of nuchal skinfold thickness in normal and Down syndrome pregnancies and to evaluate the use of this sonographic measurement as a screening test for fetal Down syndrome. METHODS: A prospective, multicenter, population-based study was performed by experienced obstetric sonographers on 1382 women with sonographically normal fetuses undergoing second-trimester amniocentesis for the indication of advanced maternal age. A standard, well-defined sonographic image was obtained at all collaborating centers. The distributions of nuchal skinfold thickness were compared between euploid and Down syndrome fetuses. RESULTS: There were 1346 chromosomally normal pregnancies, 13 fetuses with Down syndrome (1:106), and 23 other chromosome abnormalities. Seventeen fetuses had measurements of 6 mm or greater, and one of these had Down syndrome. The median nuchal skinfold thickness in Down syndrome was 3.2 mm and in euploid fetuses was 3.1 mm. By the Mann-Whitney rank-sum test, there was no statistically significant difference in nuchal skinfold between the euploid and Down syndrome fetuses (P = .5). Overall, using a nuchal skinfold thickness of 6 mm or greater as a screening test, the detection rate for Down syndrome was one of 13 (8%), the false-positive rate was 16 of 1382 (1.2%), the positive predictive value was one of 17 (6%), and the probability of detecting Down syndrome was 6.5%. CONCLUSION: In this investigation, excess nuchal skinfold thickness was a poor and unreliable screening test for Down syndrome.

Cigarette smoking and levels of maternal serum alpha-fetoprotein, unconjugated estriol, and hCG: impact on Down syndrome screening.

OBJECTIVE: To investigate the association between maternal cigarette smoking and serum levels of alpha-fetoprotein (AFP), unconjugated estriol (uE3), and hCG and to determine whether it is appropriate to take smoking status into account when screening for fetal Down syndrome. METHODS: Smoking information was obtained from 23,668 pregnant women at the time of routine screening for fetal Down syndrome. Serum levels of AFP, uE3, and hCG were stratified by smoking status and compared. Individual risks for fetal Down syndrome were analyzed both before and after adjusting the hCG levels for the effect of smoking. The prevalence of Down syndrome in the study population was calculated for both smokers and non-smokers. RESULTS: The average AFP, uE3, and hCG levels in women who smoked cigarettes were 3% greater (95% confidence interval [CI] 2-4%), 3% less (95% CI 2-4%), and 23% less (95% CI 22-24%), respectively, compared with levels in non-smokers. At comparable maternal ages, women who smoked cigarettes were identified as being at high risk for fetal Down syndrome 40% less often than non-smokers (4.0 versus 7.1% at a risk level of 1:250). Adjusting hCG levels for smoking status reduced the overall false-positive rate from 6.4 to 6.1% (using a term risk cutoff level of 1:250). The predicted effect on detection was small (a 0.7% increase). The prevalence of Down syndrome was lower among smokers, but the difference was not statistically significant (odds ratio 0.51, 95% CI 0.15-1.5). CONCLUSIONS: Adjusting the serum markers used for Down syndrome screening for the effect of maternal smoking has a small effect on overall screening performance. Given the uncertainty over whether there is a lower birth prevalence among women who smoke cigarettes, such adjustment is not currently justified.

The relationship between serum levels of lipoprotein(a) and proteins associated with the acute phase response.

The association of serum lipoprotein(a) (Lp(a)) with inflammation was investigated in a primarily rheumatologic study group (n = 570; 202 males and 368 females) by studying the relationship between serum levels of Lp(a) and a panel of acute phase proteins (C-reactive protein (CRP), alpha 1-antitrypsin (AAT), alpha 1-acid glycoprotein (AGP), haptoglobin (HPT), complement components 3 and 4 (C3, C4), prealbumin (PAL), albumin (ALB) and transferrin (TRF)). Lp(a) data were adjusted for age and sex, but not clinical condition as no significant differences in Lp(a) levels were observed, using analysis of variance, among the 15 diagnostic categories in the study group. Univariate analyses revealed significant positive associations between Lp(a) levels and levels of C4, AGP, C3 and HPT. Multivariate analysis revealed that C4 and AGP (in descending order of significance) were significant independent predictors of Lp(a) concentration, together accounting for 2.9% of the variability in Lp(a) concentration in the present study group. The data indicate that confounding effects of an acute phase response should be considered in epidemiologic studies, if a high prevalence of inflammation is suspected.

Effect of parity on human chorionic gonadotrophin levels and Down's syndrome screening.

OBJECTIVE: To investigate further the association between parity and human chorionic gonadotrophin (hCG) levels during the second trimester and determine whether prenatal screening for Down's syndrome might be made more efficient by adjusting for this variable. METHODS: Measurements of hCG were analysed in relation to parity from a cohort of 16,675 singleton pregnancies with viable outcomes, and from 108 singleton pregnancies affected by Down's syndrome. Cigarette smoking during pregnancy and maternal age were also analysed in relation to hCG levels. RESULTS: For both unaffected pregnancies and pregnancies with Down's syndrome the median hCG levels were 7% lower in parous than in nulliparous women. A dose-response relation was also documented for the unaffected pregnancies. When para 0, para 1, and para 2 women were studied separately, hCG levels rose steadily as age advanced. Adjusting hCG levels for parity led to a 0.1% reduction in variance of the distribution of hCG measurements in unaffected pregnancies and to a negligible increase in Down's syndrome screening efficiency. CONCLUSION: The effect of parity is not sufficiently great to warrant routine adjustment of hCG levels as part of prenatal screening for Down's syndrome.

Can reliable Down's syndrome detection rates be determined from prenatal screening intervention trials?

OBJECTIVES - To develop a standardised approach for analysing Down's syndrome screening performance in clinical practice and to apply it to published intervention trials in order to estimate detection and false positive rates more accurately. METHODS - Peer reviewed intervention trials, grouped by specific combination of analytes, were reanalysed. Revised detection rates were calculated for each study, taking into account both the high spontaneous loss during the last half of pregnancy and the possible under ascertainment of Down's syndrome live births not detected by screening. Collective screening performance was estimated, when possible, using a published methodology based on fitting receiver-operator characteristic curves. RESULTS - Sixteen trials were analysed; 11 using three, and five using two, analytes. Collective screening performance for the triple analyte trials was Down's syndrome detection rates of 57, 64, and 69% at amniocentesis referral rates of 3, 5, and 7% respectively. Four of the five studies involving two analytes performed less well, individually, when compared with the overall performance of the three analyte studies. It was not possible to estimate collective performance for the two analyte studies because there were too few. CONCLUSIONS - Accurate Down's syndrome detection rates are difficult to obtain in intervention trials owing to two potential biases, both of which tend to produce overestimates of the true rates. These sources of bias need to be taken into account when analysing and reporting Down's syndrome intervention trials. The methodology presented here offers the opportunity to achieve a more reliable, standardised estimate of both individual and collective intervention trial screening performance.

Hereditary haemochromatosis and hepatocellular carcinoma in males: a strategy for estimating the potential for primary prevention.

OBJECTIVES: Homozygosity for the C282Y mutation of the HFE gene is the main cause of iron overload in hereditary haemochromatosis. This study calculated the number of hepatocellular carcinoma cases among a cohort of white males that could be attributed to C282Y homozygosity. A better understanding of the extent of potentially preventable mortality arising from this cancer might help with decision making about the feasibility of population screening. METHODS: We combined information from published life tables, age-specific cancer rates and DNA studies of archived liver biopsy specimens to calculate the number of cases of hepatocellular carcinoma that might occur during the lifetime of a cohort of 1,000,000 men, including a subgroup of 5000 C282Y homozygotes. RESULTS: Hepatocellular carcinoma was estimated to occur in 2673 men in the cohort (1:374); 267 of these cases were in the subgroup of 5000 C282Y homozygotes (1:17). If these 267 cases were prevented, the remaining lifetime risk among all males would be 1:416. The relative risk for this cancer in C282Y homozygotes is 23. CONCLUSIONS: There continues to be uncertainty about the efficacy of screening for haemochromatosis. Hepatocellular carcinoma is the most readily quantifiable serious health problem attributable to this source. Further confirmatory DNA (C282Y) studies would be helpful in larger, unbiased sets of archived biopsy specimens, as a way to confirm the present estimate. Any strategy designed to prevent attributable liver cancer is likely to prevent other serious problems from haemochromatasis as well.

Population screening for haemochromatosis: expectations based on a study of relatives of symptomatic probands.

OBJECTIVES: The frequency of symptomatic haemochromatosis in the general population and the potential efficacy of population screening is uncertain. Data from family members of clinically diagnosed index cases were used to estimate the frequency of the haemochromatosis genotype, the proportion of homozygous individuals with clinical manifestations, and the efficacy of transferrin saturation and serum ferritin measurements as screening tests. SETTING: English language studies from Europe, North America, and Australia. METHODS: Haemochromatosis zygosity was classified only by HLA haplotyping, the most reliable available method. All subsequent analyses were based on family members classified in this way. RESULTS: An estimated 53 individuals per 10,000 are homozygous for haemochromatosis. Overall, 67% of male and 41% of female family members display at least one clinical manifestation; for men, the frequency increases with age. Transferrin saturation levels are 70% or above in an estimated 72% of homozygous men, along with three per 1000 heterozygous or unaffected men. Transferrin saturation levels are 60% or above in an estimated 67% of homozygous women, along with six per 1000 heterozygous or unaffected women. Serum ferritin levels, but not transferrin saturation levels, are associated with clinical manifestations. CONCLUSIONS: This information can be used to compare expected versus actual screening performance for intervention trials aimed at detecting haemochromatosis in the general population.

Population screening for haemochromatosis: a unifying analysis of published intervention trials.

OBJECTIVES: To examine the efficacy of population screening for haemochromatosis by analysing the screening performance of seven intervention trials, and to compare this with the expected performance derived from family studies. SETTING: Seven population intervention trials carried out between 1983 and 1995 in Australia, Scandinavia, Iceland, and the United State. METHODS: Seven of 23 English language trials identified were suitable for the meta-analysis. Transferrin saturation and serum ferritin measurements derived from family studies were used to predict detection and false positive rates for each trial. RESULTS: The seven trials used various screening and diagnostic criteria. A total of 18,396 men and 12,254 women were screened. Because some cases were not detected by screening, and some screen positive individuals did not complete diagnostic testing, the prevalence of homozygous individuals was underestimated in all the trials. The reported and predicted percentages of screen positive individuals nearly always agreed. The homozygote prevalence was estimated to be 34 men and 40 women per 10,000 (prevalence predicted from family studies is 53 per 10,000). Clinical manifestations were present in 50% of male and 44% of female homozygotes. CONCLUSIONS: False positive rates, homozygote prevalences, and frequency of clinical manifestations were in general agreement with predictions from family studies. However, incomplete understanding about a number of issues requires that further pilot trials be carried out before screening can be considered part of routine medical practice.

Assigning risk for Smith-Lemli-Opitz syndrome as part of 2nd trimester screening for Down's syndrome.

OBJECTIVES: To design a reliable model in the context of prenatal screening for assigning the risk in an individual pregnancy of Smith-Lemli-Opitz syndrome (SLOS) and assess its performance. SETTING: A 2nd trimester screening programme for Down's syndrome that measures unconjugated estriol (uE3) along with other serum markers. METHODS: Development of individual risk estimates with a trivariate model incorporating measurements of maternal serum uE3, alpha-fetoprotein (AFP), and human chorionic gonadotropin (hCG) in both SLOS and unaffected pregnancies. RESULTS: Population parameters were computed for the three analytes, as were pairwise correlation coefficients and truncation limits, based on an unbiased collection of 29 affected pregnancies. Published parameters were used for unaffected pregnancies. With a cut off level of risk of 1:50, 62% of SLOS pregnancies can be detected by initially identifying 0.34% of unaffected pregnancies as screen positive. About 1 in 90 screen positive pregnancies will be affected. CONCLUSIONS: It is possible to screen for SLOS as an add on to existing 2nd trimester maternal serum screening, if uE3 is already being measured. A large, prospective trial is necessary to determine whether diagnostic testing can be performed in maternal urine or serum rather than amniotic fluid.