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.

A point mutation in transthyretin increases affinity for thyroxine and produces euthyroid hyperthyroxinemia.

In a family expressing euthyroid hyperthyroxinemia, an increased association of plasma thyroxine (T4) with transthyretin (TTR) is transmitted by autosomal dominant inheritance and is secondary to a mutant TTR molecule with increased affinity for T4. Eight individuals spanning three generations exhibited the abnormality. Although five of eight individuals had elevated total T4 concentrations, all affected individuals were clinically euthyroid and all had normal free T4 levels. Purified TTR from the propositus had an affinity for 125I-T4 three times that of control TTR. Exons 2, 3, and 4 (representing greater than 97% of the coding sequence) of the TTR gene of DNA prepared from the propositus' peripheral blood leukocytes were amplified using the polymerase chain reaction (PCR) and were sequenced after subcloning. Exons 2 and 3 were indistinguishable from normal. In 50% of clones amplified from exon 4, a substitution of adenine (ACC) for guanine (GCC) in codon 109 resulted in the replacement of threonine-for-alanine, a mutation confirmed by amino acid sequencing of tryptic peptides derived from purified plasma TTR. The adenine-for-guanine substitution abolishes one of two Fnu 4H I restriction sites in exon 4. PCR amplification of exon 4 of TTR and restriction digestion with Fnu 4H I confirmed that five affected family members with increased binding of 125I-T4 to TTR are heterozygous for the threonine 109 substitution that increases the affinity of this abnormal TTR for T4.

Muscle weakness caused by an iodine-deficient diet: investigation of a nutritional myopathy.

Newborn rats raised on a commercially available iodine-deficient diet developed severe muscle weakness, affecting predominantly their proximal hind limbs, that electrophysiologically and morphologically was determined to be myopathic in type. Follow-up dietary studies, utilizing different combinations of vitamins, minerals, casein and elemental iodine, demonstrated that the myopathy was the result of a deficiency of multiple dietary constituents, particularly casein, and was not due to a deficiency of iodine alone. These findings were compared with those observed in earlier investigations of a variety of nutritional myopathies. In the laboratory study of animals raised on experimental diets, it becomes important to consider the possible contributions of multiple dietary deficiencies in the evaluation of any abnormalities found.

Prevalence of abnormal thyroid function test results in patients with acute medical illnesses.

We measured serum total and free thyroxine (T4) and triiodothyronine (T3) concentrations, free T4 and T3 indexes, thyroid-stimulating hormone (TSH), thyroxine-binding globulin (TBG) and thyroxine-binding prealbumin (TBPA) concentrations in 98 patients hospitalized for acute medical illnesses. The free thyroxine index (FT4I) or TSH level was abnormal in 16 percent, but only 3 percent had thyroid disease. Serum fre T4 measurements by equilibrium dialysis were abnormal in 25 percent, but no additional patients who initially had abnormal concentrations of serum free T4 were subsequently proved to have thyroid disease. Patients with supranormal serum free T4 concentrations (21 percent) ahd higher serum T4, lower serum T3, and higher serum reverse T3 (rT3) concentrations than other patients, but the measured changes in serum T4, TBG and TBPA levels could only partly account for the magnitude of the free T4 elevation. In these acutely ill patients, an accurate diagnosis of thyroid disease could be achieved by determination of FT4I and TSH level and a history of medication usage. We conclude that other tests are rarely necessary for this purpose in a patient population such as this.

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.

Serum triglyceride: a possible risk factor for ruptured abdominal aortic aneurysm.

BACKGROUND: We aimed to determine the relationship between ruptured abdominal aortic aneurysm (AAA) and serum concentrations of lipids and apolipoproteins. METHODS: A cohort of 21 520 men, aged 35-64 years, was recruited from men attending the British United Provident Association (BUPA) clinic in London for a routine medical examination in 1975-1982. Smoking habits, weight, height and blood pressure were recorded at entry. Lipids and apolipoproteins were measured in stored serum samples from the 30 men who subsequently died of ruptured AAA and 150 matched controls. RESULTS: Triglyceride was strongly related to risk of ruptured AAA. In univariate analyses the risk in men on the 90th centile of the distribution relative to the risk in men on the 10th (RO10-90) was 12 (95% confidence interval [CI] : 3.8-37) for triglyceride, 5.5 (95% CI: 1.8-17) for apolipoprotein B (apoB) (the protein component of low density lipoprotein [LDL]), 0.15 (95% CI : 0.04-0.56) for apo A1 (the protein component of high density lipoprotein [HDL]), 3.7 (95% CI: 1.4-9.4) for body mass index and 3.0 (95% CI: 1.1-8.5) for systolic blood pressure. Lipoprotein (a) (Lp(a)) was not a significant risk factor (RO10-90 = 1.6, 95% CI: 0.6-3.0). In multivariate analysis triglyceride retained its strong association. CONCLUSION: Triglyceride appears to be a strong risk factor for ruptured AAA, although further studies are required to clarify this. If this and other associations are cause and effect, then changing the distribution of risk factors in the population (by many people stopping smoking and adopting a lower saturated fat diet and by lowering blood pressure) could achieve an important reduction in mortality from ruptured AAA.

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.

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.

Perinatal management of ventral wall defects.

Reported is the analysis of morbidity, mortality, and mode of delivery in 38 cases of ventral wall defects identified from among 128,500 consecutive live births in Maine (January 1975 to December 1982). Thirteen of the ventral wall defects were classified as gastroschisis, and only one had an additional defect not directly attributable to the ventral wall defect itself. By contrast, 16 of the 25 omphalocele cases had additional defects, including eight congenital heart lesions, four genitourinary malformations, two neural tube defects, and three trisomies. Ten cases of omphalocele and one of gastroschisis died, all as a result of independent defects or involvement of adjacent structures. Intrauterine growth retardation was prominently associated with gastroschisis. Vaginal delivery occurred in three of the six ventral wall defects diagnosed antenatally and in 28 of the 32 ventral wall defects not diagnosed until delivery. The only episode of birth trauma to ventral wall defect sac or abdominal viscera occurred during cesarean section in an undiagnosed case. The present data provide a basis for prognosis and management of antenatally diagnosed ventral wall defects and suggest that these defects are not, a priori, an indication for abdominal delivery.

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.

Data from an alpha-fetoprotein pilot screening program in Maine.

In Maine, maternal serum alpha-fetoprotein (AFP) values equaled 2 or more multiples of the median in 4.8% of screened women. Between 2.0 and 2.9 multiples of the median repeat maternal serum AFP testing and sonography were comparable as the next diagnostic step; at 3 multiples of the median or higher sonography was superior. Sonography moved dates back in only ten singleton viable pregnancies with maternal serum AFP elevations; three of these had open fetal defects. Among singletons, all five anencephaly cases, one of two open spina bifida lesions, and all three open ventral wall defects were identified. Three closed singleton neural tube defects and two open spina bifida defects in twins were not detected. Nineteen of 36 multiple gestations had maternal serum AFP 2 or higher multiples of the median. In singletons, maternal serum AFP of 3 or higher multiples of the median indicated a thirtyfold increased risk for fetal death and a sevenfold increased risk for birth weight under 2500 g.

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.

A cellulose sandwich fluoroimmunoassay for Factor VIII-related antigen.

We describe a cellulose sandwich fluoroimmunoassay for Factor VIII-related antigen (FVIIIR:Ag). Plasma is incubated with cellulose-antibody for 2 hours, fluorescent-labelled antiserum is added and the samples are allowed to react for an additional 2 hours. The fluorescence associated with the cellulose-antibody is measured after the tubes have been centrifuged, and the deposit washed and then digested with sodium hydroxide. Ninety-seven samples are assayed and the results are compared with radioimmunoassay (correlation coefficient 0.960) or electroimmunoassay (correlation coefficient 0.882). Between-run coefficients of variation are 8.0% to 9.9%. The cellulose-antibody reagent and the fluorescent-labelled antiserum were both stable over a period of 1 year, demonstrating a major advantage of fluoroimmunoassay over radioimmunoassay.

Disparity in antiserum avidity as measured by nephelometry, radial immunodiffusion, and column-binding strength.

When antiserum potency is being assessed, avidity is often inferred from affinity measurements or from measurements obtained from completely unrelated immunoassay techniques. The present experiments describe an instance where antibodies that have very high column-binding strength, obtained by elution from immobilized antigen with dissociating agents, have low avidity in nephelometry and radial immunodiffusion. This low avidity appears not to be due to molecular damage since the eluted antibodies do not contain molecular aggregates and retain their column-binding strength when reapplied to the immobilized antigen. We theorize that in nephelometry and radial immunodiffusion, lattice formation may require a heterogeneous population of antibodies and that the low avidity fractions may have too limited a specificity. We support this theory by combining two different populations of low avidity antibodies and finding that the combination produces a nephelometric response greater than that of either fraction alone.

Preventing manifestations of hereditary haemochromatosis through population based screening.

OBJECTIVE: To evaluate the efficacy of identifying presymptomatic hereditary haemochromatosis through population based screening. DESIGN: Review the hereditary pattern, prevalence, and clinical manifestations of haemochromatosis. Estimate the detection and false positive rates associated with available screening and diagnostic tests. Develop examples of screening protocols and other components that would be necessary for proper implementation. Identify potential barriers and objections. CONCLUSIONS: Hereditary haemochromatosis, an autosomal recessive disorder with a prevalence of three to five per thousand in the general population, is associated with a wide variety of clinical manifestations, usually beginning in mid to late adult years. Identifying and treating this disorder after symptoms appear can arrest its progress but usually cannot reverse existing damage to joints, liver, pancreas, pituitary gland, and other organs. Measuring transferrin saturation in serum is now known to be a reliable screening test for haemochromatosis when applied to a general population of healthy adults, detecting about 80% of cases, with a 0.3% false positive rate. Liver biopsy with iron staining and total iron concentration is the recommended diagnostic test for subjects with positive screening tests. Treatment with phlebotomy can then prevent manifestations. Effective systematic identification and management of presymptomatic haemochromatosis in the general population is best accomplished within the framework of an organised screening programme. Potential barriers include accessibility of young adult populations and attitudes in the health community that severe clinical manifestations are relatively uncommon. It is recommended that pilot programmes be undertaken to determine the feasibility of introducing screening for haemochromatosis as part of routine health care.

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.

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.