Urinary screening tests for fetal Down syndrome: I. Fresh beta-core fragment

Variable results have been reported using urine beta-core fragment as a mar ker for fetal Down syndrome. Initial studies by Cuckle ct al. (1994) and Ca nick et al. (1995) indicated that beta-core fragment was an outstanding mar ker, detecting >80 percent of Down syndrome cases. Since these reports, wid ely varying results have been published, indicating between 20 per cent and 66 per cent detection of cases at 5 per cent false-positive rate. The wide variation in the reported data has led to a loss of enthusiasm for this ma rker as a useful test for Down syndrome screening. Here we report the results of a three-year prospective study in which urine samples were collected daily from women undergoing fetal karyotype analysi s for advanced maternal age. Samples were tested within one week of collect ion and then frozen. We also investigated the likely causes of the variabil ity observed in beta-core fragment data. We collected 1157 urine samples over 955 days, beta-core fragment levels we re measured. A regression line was calculated for the weekly medians of the 1134 control samples and multiples of the control median (MoM) were determ ined. The median MoM for the controls was 1.0 and the logarithmic standard deviation (log SD) was 0.41. The median MoM for the 23 Down syndrome cases was 5.44 and the log SD was 0.45. Over the study period, 65 per cent of Dow n syndrome cases exceeded the 95th centile of the control group. The median MoM of control samples and the proportion of Dawn syndrome cases detected by the test was relatively constant during the study period. The unaffected cases were divided into three equal divisions, corresponding to approximat ely the first, second and third year of sample collection. No trend was fou nd in the median control MoM values in three sample collection periods (r(2 )=0.04). A similar number of cases exceeded the 95th centile of control sam ples in the three sample collection periods, 63 per cent, 66 per cent and 6 6 percent. Consistent results were indicated during the three years of samp le testing. Levels of total oestriol were determined in urine samples and MoM statistic s derived. The median oestriol level in Down syndrome cases was 0.59 MoM. O nly 12 per cent of cases bad MoM levels below the fifth centile. Gaussian m odels were prepared combining biochemical data and maternal age distributio n. While beta-core fragment by itself detected 65 per cent of Down syndrome cases, beta-core fragment modelled with maternal age detected 66 per cent, and modelled with age and total oestriol levels detected 82 per cent of ca ses at 5 per cent false-positive rate. At the completion of the study, we thawed and reassayed 20 random urine sam ples (10 control and 10 Down syndrome) collected at different times during the study period. While the control samples (74-1700 ng/ml) had slightly in creased values when reassayed (mean value 137 per cent of original prospect ive value), the Down syndrome samples (360-20 500 ng/ml) all had decreased values when reassayed (mean=53 per cent, t-test,controls versus cases, p=0. 0003). The Down syndrome samples were decreased to between 93 per cent and 12 per cent of the original value. A relationship was identified between th e magnitude of the original beta-core fragment value and the change in immu noreactivity when reassayed (r(2)=0.998). The higher the initial beta-core fragment value the greater the loss of immunoreactivity. We considered the possibility that the beta-core fragment molecules aggregate upon storage in the freezer. We repeated the assay of the 20 samples after treatment with a high salt buffer. Down syndrome samples recovered half of the lost beta-c ore fragment immunoreactivity (mean increase in beta-core fragment levels 5 6 per cent, t-test, controls versus cases, p=0.004). We infer that aggregat ion of beta-core fragment upon storage interferes with beta-core fragment m easurements. This may be the cause of the poor beta-core fragment screening performance reported using stored/frozen urine samples. We conclude that urine beta-core fragment, or combinations of beta-core fra gment and total oestriol need to be considered as viable alternatives to cu rrent serum protocols for;screening for Down syndrome. We also conclude tha t urine beta-core fragment has no consistency or variability limitations, p rovided samples are tested within one week of collection, as occurs in clin ical practice. Copyright (C) 1999 John Wiley & Sons, Ltd.