Long-term control of neuropathic pain in a non-viral gene therapy paradigm.

Traditional approaches to treating chronic neuropathic pain largely focus on manipulations directly altering neuronal activity or neuron-to-neuron communication. Recently, however, it has become clear that glial cells (including microglia and astroglia) play a significant role in pain expression in a variety of neuropathic pain models. Multiple aspects of the inflammatory response of glial cells, commonly observed in neuropathic pain conditions, have been implicated in pain expression. Thus, glial cell inflammation has emerged as a potential therapeutic target in neuropathic pain. Our laboratory has been exploring the use of an anti-inflammatory cytokine, interleukin-10 (IL-10), to control glial inflammatory activation thereby controlling neuropathic pain. IL-10 protein delivery is limited by a short half-life and an inability to cross into the central nervous system from the periphery, making a centrally delivered gene therapy approach attractive. We have recently characterized a non-viral gene therapy approach using two injections of naked DNA to achieve long-term (>3 months) control of neuropathic pain in a peripheral nerve injury model. Timing and dose requirements leading to long-term pain control are discussed in this review, as is recent work using microparticle-encapsulated DNA to achieve long-term therapeutic efficacy with a single injection.

Development of porous PEG hydrogels that enable efficient, uniform cell-seeding and permit early neural process extension.

Three-dimensional polymer scaffolds are useful culture systems for neural cell growth and can provide permissive substrates that support neural processes as they extend across lesions in the brain and spinal cord. Degradable poly(ethylene) glycol (PEG) gels have been identified as a particularly promising scaffold material for this purpose; however, process extension within PEG gels is limited to late stages of hydrogel degradation. Here we demonstrate that earlier process extension can be achieved from primary neural cells encapsulated within PEG gels by creating a network of interconnected pores throughout the gel. Our method of incorporating these pores involves co-encapsulating a cell solution and a fibrin network within a PEG gel. The fibrin is subsequently enzymatically degraded under cytocompatible conditions, leaving behind a network of interconnected pores within the PEG gel. The primary neural cell population encapsulated in the gel is of mixed composition, containing differentiated neurons, and multipotent neuronal and glial precursor cells. We demonstrate that the initial presence of fibrin does not influence the cell-fate decisions of the encapsulated precursor cells. We also demonstrate that this fabrication approach enables simple, efficient and uniform seeding of viable cells throughout the entire porous scaffold.

Biocompatibility of PEG-based hydrogels in primate brain.

Degradable polymers have been used successfully in a wide variety of peripheral applications from tissue regeneration to drug delivery. These polymers induce little inflammatory response and appear to be well accepted by the host environment. Their use in the brain, for neural tissue reconstruction or drug delivery, also could be advantageous in treating neurodegenerative disorders. Because the brain has a unique immune response, a polymer that is compatible in the body may not be so in the brain. In the present study, polyethylene glycol (PEG)-based hydrogels were implanted into the striatum and cerebral cortex of nonhuman primates. Four months after implantation, brains were processed to evaluate the extent of astrogliosis and scaring, the presence of microglia/macrophages, and the extent of T-cell infiltration. Hydrogels with 20% w/v PEG implanted into the brain stimulated a slight increase in astrocytic and microglial/macrophage presence, as indicated by a small increase in glial fibrillary acidic protein (GFAP) and CD68 staining intensity. This increase was not substantially different from that found in the sham-implanted hemispheres of the brain. Staining for CD3+ T cells indicated no presence of peripheral T-cell infiltration. No gliotic scarring was seen in any implanted hemisphere. The combination of low density of GFAP-positive cells and CD68-positive cells, the absence of T cells, and the lack of gliotic scarring suggest that this level of immune response is not indicative of immunorejection and that the PEG-based hydrogel has potential to be used in the primate brain for local drug delivery or neural tissue regeneration.

A new subtype of brachydactyly type B caused by point mutations in the bone morphogenetic protein antagonist NOGGIN.

Brachydactyly type B (BDB) is characterized by terminal deficiency of fingers and toes, which is caused by heterozygous truncating mutations in the receptor tyrosine kinase-like orphan receptor 2 (ROR2) in the majority of patients. In a subset of ROR2-negative patients with BDB, clinically defined by the additional occurrence of proximal symphalangism and carpal synostosis, we identified six different point mutations (P35A, P35S, A36P, E48K, R167G, and P187S) in the bone morphogenetic protein (BMP) antagonist NOGGIN (NOG). In contrast to previously described loss-of-function mutations in NOG, which are known to cause a range of conditions associated with abnormal joint formation but without BDB, the newly identified BDB mutations do not indicate a major loss of function, as suggested by calculation of free-binding energy of the modeled NOG-GDF5 complex and functional analysis of the micromass culture system. Rather, they presumably alter NOG's ability to bind to BMPs and growth-differentiation factors (GDFs) in a subtle way, thus disturbing the intricate balance of BMP signaling. The combined features observed in this phenotypic subtype of BDB argue for a functional connection between BMP and ROR2 signaling and support previous findings of a modulating effect of ROR2 on the BMP-receptor pathway through the formation of a heteromeric complex of the receptors at the cell surface.

Late first-trimester invasive prenatal diagnosis: results of an international randomized trial.

OBJECTIVE: To assess, in a randomized trial, the safety and accuracy of amniocentesis and transabdominal chorionic villus sampling (CVS) performed at 11-14 weeks of gestation, given that this time frame is increasingly relevant to early trisomy screening. METHODS: We compared amniocentesis with CVS from 77 to 104 days of gestation in a randomized trial in a predominantly advanced maternal age population. Before randomization, the feasibility of both procedures was confirmed by ultrasonography, and experienced operators performed sampling under ultrasound guidance; conventional cytogenetic analysis was employed. The primary outcome measure was a composite of fetal loss plus preterm delivery before 28 weeks of gestation in cytogenetically normal pregnancies. RESULTS: We randomized 3,775 women into 2 groups (1,914 to CVS; 1,861 to amniocentesis), which were comparable at baseline. More than 99.6% had the assigned procedure, and 99.9% were followed through delivery. In contrast to previous thinking, in the cytogenetically normal cohort (n = 3,698), no difference in primary study outcome was observed: 2.1% (95% confidence interval 1.5, 2.8) for CVS and 2.3% (95% confidence interval, 1.7, 3.1) for amniocentesis. However, spontaneous losses before 20 weeks and procedure-related, indicated terminations combined were increased in the amniocentesis group (P =.07, relative risk 1.74). We found a 4-fold increase in the rate of talipes equinovarus after amniocentesis (P =.02) overall and in week 13 (P =.03, relative risk = 4.65), but data were insufficient to determine this risk in week 14. CONCLUSION: Amniocentesis at 13 weeks carries a significantly increased risk of talipes equinovarus compared with CVS and also suggests an increase in early, unintended pregnancy loss. LEVEL OF EVIDENCE: I

Quantifying stratospheric ozone in the upper troposphere with in situ measurements of HCl.

We have developed a chemical ionization mass spectrometry technique for precise in situ measurements of hydrochloric acid (HCl) from a high-altitude aircraft. In measurements at subtropical latitudes, minimum HCl values found in the upper troposphere (UT) were often near or below the detection limit of the measurements (0.005 parts per billion by volume), indicating that background HCl values are much lower than a global mean estimate. However, significant abundances of HCl were observed in many UT air parcels, as a result of stratosphere-to-troposphere transport events. We developed a method for diagnosing the amount of stratospheric ozone in these UT parcels using the compact linear correlation of HCl with ozone found throughout the lower stratosphere (LS). Expanded use of this method will lead to improved quantification of cross-tropopause transport events and validation of global chemical transport models.

Transplantation of brain cells assembled around a programmable synthetic microenvironment.

Cell therapy is a promising method for treatment of hematopoietic disorders, neurodegenerative diseases, diabetes, and tissue loss due to trauma. Some of the major barriers to cell therapy have been partially addressed, including identification of cell populations, in vitro cell proliferation, and strategies for immunosuppression. An unsolved problem is recapitulation of the unique combinations of matrix, growth factor, and cell adhesion cues that distinguish each stem cell microenvironment, and that are critically important for control of progenitor cell differentiation and histogenesis. Here we describe an approach in which cells, synthetic matrix elements, and controlled-release technology are assembled and programmed, before transplantation, to mimic the chemical and physical microenvironment of developing tissue. We demonstrate this approach in animals using a transplantation system that allows control of fetal brain cell survival and differentiation by pre-assembly of neo-tissues containing cells and nerve growth factor (NGF)-releasing synthetic particles.

Ratio of nuchal thickness to humerus length for Down syndrome detection.

OBJECTIVE: Ultrasonographic biometry markers are now being used clinically to adjust Down syndrome risk. The limitations are that the definitions of "abnormal" measurements used are arbitrary, thus reducing screening performance, and also that patient-specific Down syndrome risks cannot be calculated. We report a new ultrasonographic algorithm that is sensitive for Down syndrome detection and that estimates individual risk. STUDY DESIGN: Overall in fetal populations with Down syndrome the humerus length is decreased, whereas the nuchal thickness is increased relative to that of a normal population. The nuchal thickness/humerus length ratio therefore shows an even greater increase and magnifies the separation between Down syndrome and healthy groups. Prospective data were collected in midtrimester amniocentesis cases. A regression equation for the median nuchal thickness/humerus length ratio based on biparietal partial diameter was generated. The Down syndrome likelihood ratio, or the odds on the basis of the nuchal thickness/humerus length ratio (multiples of the median), was multiplied by the age-related risk to give the posterior Down syndrome risk. Charts for rapid estimation of individual Down syndrome risk on the basis of maternal age and the nuchal thickness/humerus length ratio were constructed. RESULTS: There were 94 cases of Down syndrome and 4700 cases in which the karyotype was normal. The mean (+/-SD) gestational age of the study population was 16.1 +/- 1.6 weeks. Thirty-three fetuses with Down syndrome and 68 karyotypically normal fetuses had gross anomalies. The equation for the expected median nuchal thickness/humerus length ratio was as follows: 10e(1.7163 - 0.0292) x BPD + 0.0003 x BPD2, where BPD is the biparietal diameter. In the overall study population the nuchal thickness/humerus length ratio and maternal age had a 79.8% detection rate at a 22.1% false-positive rate, compared with maternal age plus humerus length (sensitivity, 55.1%) or maternal age plus nuchal thickness (sensitivity, 66.7%) at the same false-positive rate. For women > or =35 years old the values were 80% and 22.0%, respectively. CONCLUSIONS: We report an ultrasonographic biometry algorithm that, in combination with maternal age, detects 79.6% of Down syndrome cases in a high-risk group. Individual Down syndrome risk can be quickly calculated at the bedside and made available to women who desire this information before making a decision on amniocentesis. On the basis of published standards, ultrasonographic biometry as described would be a cost-effective alternative to amniocentesis in this high-risk group.

Molecular systematics of Plethodon and Aneides (Caudata: Plethodontidae: Plethodontini): phylogenetic analysis of an old and rapid radiation.

The closely related salamander genera Plethodon and Aneides (Plethodontidae) differ in morphology, behavior, and ecology. Although the systematics of these taxa has been the focus of much study, many details remain unresolved. To generate an hypothesis for the relationships among these taxa, I sequenced a segment of the mitochondrial protein-coding gene ND4 and portions of mitochondrial tRNAs. Taxa sampled were 5 species of Aneides, 7 species of western Plethodon, and 13 species of eastern Plethodon. Ensatina eschscholtzii was used as the outgroup. Phylogenetic analyses using maximum-parsimony, neighbor-joining, and maximum-likelihood consistently recovered some relationships. The eastern species of Plethodon are a robust, well-supported clade. Sister taxon relationships of P. elongatus and P. stormi, of P. dunni and P. vehiculum, and of A. hardii and the three west coast species of Aneides were also consistently resolved with good support. The monophyly of Aneides was only weakly supported in some analyses and there is no evidence for the monophyly of Plethodon or of the western species of Plethodon. Excluding the relatively distant outgroup, down-weighting saturated substitutions, and analyzing conserved data partitions did not yield additional resolution or support among the lineages of western Plethodon and Aneides. These results are consistent either with saturation of sequences, due to the age of the lineages, or with relatively rapid radiation. An old, rapid radiation is consistent with the results of previous studies. An analysis of current taxonomy within the phylogenetic framework presented here retains Aneides and recognizes Plethodon as a metataxon (indicated with an asterisk, Plethodon*).

Comparison of urinary hyperglycosylated human chorionic gonadotropin concentration with the serum triple screen for Down syndrome detection in high-risk pregnancies.

OBJECTIVE: Both modest screening performance and declining patient and physician acceptance have stimulated interest in alternative markers to the triple screen for the detection of Down syndrome. Our purpose was to compare the concentration of a single urinary analyte, hyperglycosylated human chorionic gonadotropin, with the serum triple screen (alpha-fetoprotein, human chorionic gonadotropin, and unconjugated estriol concentrations combined with age) for second-trimester Down syndrome detection. STUDY DESIGN: Urine and blood were obtained from pregnant women in the second trimester undergoing genetic amniocentesis. Urinary hyperglycosylated human chorionic gonadotropin concentration and serum triple-screen values were measured. Individuals undergoing amniocentesis because of abnormal triple-screen results were excluded. Individual Down syndrome risks on the basis of urinary hyperglycosylated human chorionic gonadotropin concentration plus maternal age and on the basis of the triple-screen results were calculated. For each algorithm the sensitivity and false-positive rate for Down syndrome detection at different risk thresholds were determined. From these values receiver operating characteristic curves were constructed, and the area under the curve was determined for each algorithm. Finally, the performance of a new combination in which urinary hyperglycosylated human chorionic gonadotropin concentration replaced serum human chorionic gonadotropin concentration in the triple screen was ascertained. RESULTS: We studied 24 pregnancies complicated by Down syndrome and 500 unaffected pregnancies between 14 and 22 weeks' gestation in a mostly white (93.5%) population undergoing amniocentesis primarily because of advanced maternal age. The sensitivity and false-positive rate for urinary hyperglycosylated human chorionic gonadotropin concentration were 75. 0% and 5.6%, respectively, whereas those for the triple screen were 75.0% and 33.2%, respectively. Urinary hyperglycosylated human chorionic gonadotropin concentration was superior to the triple screen (area under the curve, 0.9337 vs 0.7887; P =.02). The substitution of urinary hyperglycosylated human chorionic gonadotropin concentration for serum human chorionic gonadotropin concentration in the triple screen resulted in a 91.7% sensitivity at a 10.0% false-positive rate, versus a 54.2% sensitivity for the traditional triple screen at the same false-positive rate. CONCLUSION: The performance of urinary hyperglycosylated human chorionic gonadotropin concentration was statistically superior to that of the serum triple screen in a high-risk population. The use of urinary hyperglycosylated human chorionic gonadotropin concentration as an alternative test or substitution of this measurement for serum human chorionic gonadotropin concentration in the triple screen would improve diagnostic accuracy and address many current concerns related to the triple screen.

Combined ultrasound biometry, serum markers and age for Down syndrome risk estimation.

OBJECTIVE: To compare Down syndrome screening efficiency of the standard serum triple analyte screen to that of a four-component screen consisting of ultrasound biometry and serum markers in the second trimester. METHODS: The Down syndrome screening efficiency of the triple screen, i.e. alpha-fetoprotein (AFP), unconjugated estriol (E3), hCG and maternal age, was compared with the four-marker algorithm, i.e. humerus length, nuchal thickness, AFP and hCG plus maternal age. A quadrivariate Gaussian algorithm was used to calculate individual Down syndrome odds. Receiver operating characteristic (ROC) curves plotting sensitivity against false-positive rate were constructed for each algorithm and the areas under the curves were compared to determine which was superior. Sensitivity and false-positive rates at different Down syndrome risk thresholds were also compared. RESULTS: There were 46 cases of Down syndrome (1.9%) with 2391 normal singleton pregnancies in a referral population in which triple screen, fetal biometry and karyotype had been done. The gestational age range for the study was 14-24 completed weeks. The median maternal age for the study group was 35.0 years (14.0-46.0 years). The areas (SE) under the ROC curves were 0.75(0.04) and 0.93(0.02) for the standard triple and the four-marker screen, respectively (P < 0.001). At a 10% false-positive rate, detection was 45.7% for the triple and 80.4% for the four-marker screen. CONCLUSIONS: A new algorithm combining humerus length and nuchal thickness measurement with serum AFP, hCG and maternal age substantially improved Down syndrome screening efficiency compared with the traditional triple screen. The model appears promising and should be evaluated in an independent data set.

Cytogenetical diagnosis in paraffin-embedded fetoplacental tissue using comparative genomic hybridization.

Comparative genomic hybridization (CGH) is a FISH-related technique used to assess global chromosomal aberrations in a variety of human tumours. Recently CGH has been applied to cytogenetic analysis of fresh frozen fetoplacental tissues. Here we report the application of CGH to paraffin-embedded placental samples. Ten samples from paraffin-embedded blocks of 6 control placentas and fetoplacental tissue from 10 aneuploidies, and 2 unbalanced aberrations were evaluated. Balanced karyotype profiles were obtained from samples of healthy placentas and all samples from the same placenta appeared to have similar confidence intervals. CGH analysis of four cases of trisomy 21, three cases of trisomy 18, one case of trisomy 13, one case of trisomy 15 and one case of trisomy 7 all showed overrepresentation of the respective trisomic chromosome. The CGH profile was also in accordance with the karyotyping of a case with isochromosome 21. The CGH profile of a case with der (2)t(2;6)(q37.3;q22.2) revealed partial trisomy for chromosome 6 between q21 and q27. CGH may be a useful adjunct in prenatal genetic diagnosis when retrospective diagnosis is needed from archival samples.

Hyperglycosylated human chorionic gonadotropin (invasive trophoblast antigen) immunoassay: A new basis for gestational Down syndrome screening.

BACKGROUND: Serum human chorionic gonadotropin (hCG) and hCG free beta-subunit tests are used in combination with unconjugated estriol and alpha-fetoprotein in the triple screen test, and with the addition of inhibin-A in the quadruple marker test for detecting Down syndrome in the second trimester of pregnancy. These tests have a limited detection rate for Down syndrome: approximately 40% for hCG or free beta-subunit alone, approximately 60% for the triple screen test, and approximately 70% for the quadruple marker test, all at 5%, or a relatively high, false-positive rate. New tests are needed with higher detection and lower false rates. Hyperglycosylated hCG (also known as invasive trophoblast antigen or ITA) is a new test. It specifically detects a unique oligosaccharide variant of hCG associated with Down syndrome pregnancies. We evaluated this new Down syndrome-directed test in prenatal diagnosis. METHODS: Hyperglycosylated hCG was measured in urine samples from women undergoing amniocentesis for advanced maternal age concerns at 14-22 weeks of gestation, 1448 with normal karyotype and 39 with Down syndrome fetuses. RESULTS: The median hyperglycosylated hCG value was 9.5-fold higher in Down syndrome cases (9.5 multiples of the normal karyotype median). The single test detected 80% of Down syndrome cases at a 5% false-positive rate. Urine hyperglycosylated hCG was combined with urine beta-core fragment (urine breakdown product of serum hCG free beta-subunit), serum alpha-fetoprotein, and maternal age-related risk. This urine-serum combination detected 96% of Down syndrome cases at a 5% false-positive rate, 94% of cases at a 3% false-positive rate, and 71% of cases at a 1% false-positive rate. These detection rates exceed those of any previously reported combination of biochemical markers. CONCLUSIONS: Hyperglycosylated hCG is a new base marker for Down syndrome screening in the second trimester of pregnancy. The measurement of hyperglycosylated hCG can fundamentally improve the performance of Down syndrome screening protocols.

Synthesis and biological activity of polyethylene glycol-mouse nerve growth factor conjugate.

Conjugation of poly(ethylene glycol) derivatives with therapeutic proteins is a promising approach for enhancing protein stability and, therefore, effectiveness. An N-hydroxysuccinimidyl ester of fluorescein-PEG 2000 was used for chemical modification of mouse nerve growth factor (mNGF), a dimeric protein with therapeutic potential for Alzheimer's disease. The mNGF-PEG2000-fluorescein conjugate was characterized by RP-HPLC, spectrofluorometry, and SDS-PAGE and was biologically active, as determined by two independent NGF-specific assays (enhancement of ChAT activity in fetal neurons and neurite outgrowth in PC12 cells). The conjugate was not detectable by a standard NGF ELISA, suggesting a fortuitous reduction in protein recognition by antibodies.

The meanings and correlates of spirituality: suggestions from an exploratory survey of experts.

There has been an increasing interest in spirituality among health care professionals over the last several decades. Specialists in the areas of trauma, grief, and death and dying have been among those who have shown particular interest in religious and spiritual issues. Recent efforts to distinguish religiosity from spirituality have stimulated inquiries into the changing meanings of these dimensions. Drawing on prior and parallel works, the authors created a questionnaire and asked for responses to it from convenience samples of experts in death studies (n = 22) and spiritual studies (n = 13). Our findings are suggestive of possible lines of convergence and divergence. Both groups considered themselves to be spiritual but not religious, and there was consensus that the meaning of the term spirituality is currently changing. There was also general agreement that spiritual experiences are meaningful learning opportunities and that spiritual individuals tend to be more hopeful and to experience more meaning or purpose in life than their nonspiritual peers. The themes most strongly associated with spirituality in both groups were charity, community or connectedness, compassion, forgiveness, hope, meaning, and morality. Future research should be directed toward clarifying what people mean by "spiritual" and how they experience and express this dimension of their lives.

Elevated maternal urine level of beta-core fragment of human chorionic gonadotropin versus serum triple test in the second-trimester detection of down syndrome.

OBJECTIVE: This study was undertaken to compare the Down syndrome screening efficiency of elevated maternal urine level of the beta-core fragment of human chorionic gonadotropin with that of the traditional serum triple test. STUDY DESIGN: Urinary beta-core fragment and serum analyte levels were measured prospectively in women with singleton pregnancies who were undergoing second-trimester genetic amniocentesis. Urinary analyte levels were measured within a week of specimen collection. In some cases only alpha-fetoprotein was measured initially and human chorionic gonadotropin and unconjugated estriol levels were subsequently determined from the stored serum specimens. The Down syndrome screening efficiency of urinary concentration of beta-core fragment plus maternal age was compared with that of the traditional triple test. Receiver operating characteristic curves were generated for each algorithm and the areas under the curves were compared to determine which algorithm was superior. RESULTS: There were a total of 926 study patients, of whom 21 (2.3%) carried fetuses with Down syndrome. The mean (+/-SD) gestations at amniocentesis were 16.6 +/- 1.5 weeks for the fetuses without Down syndrome and 17.7 +/- 2.3 for the fetuses with Down syndrome. A total of 539 women (4 of whom carried fetuses with Down syndrome) had serum alpha-fetoprotein alone measured initially. Urinary concentration of beta-core fragment had a 61.9% detection rate with a 4.9% false-positive rate for Down syndrome, whereas the values for the triple screen were 57. 1% and 11.2%, respectively. The areas under the receiver-operating characteristic curves were 0.8744 for elevated urinary beta-core fragment level and 0.7504 for the triple screen (P =.1116). When the false-positive rate was fixed at an ideal threshold value (

Cystic hygroma in the fetus and newborn.

Cystic hygromas are developmental abnormalities of the lymphoid system that occur at sites of lymphatic-venous connection, most commonly in the posterior neck. They are frequently associated with karyotypic abnormalities, various malformation syndromes, and several teratogenic agents. The disease course of an infant with cystic hygroma is unpredictable. When diagnosed prenatally, the overall prognosis is poor. Cystic hygroma diagnosed after birth is usually associated with a good prognosis. This article reviews the embryologic, genetic, and pathologic correlates of these lymphatic system abnormalities, as well as the clinical course and outcome of the fetus and newborn with a cystic hygroma. Management strategies are reviewed, including newer nonsurgical therapies for the neonate with a cystic hygroma.

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

Variable results have been reported using urine beta-core fragment as a marker for fetal Down syndrome. Initial studies by Cuckle et al. (1994) and Canick et al. (1995) indicated that beta-core fragment was an outstanding marker, detecting >80 per cent of Down syndrome cases. Since these reports, widely 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 marker 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 analysis for advanced maternal age. Samples were tested within one week of collection and then frozen. We also investigated the likely causes of the variability observed in beta-core fragment data. We collected 1157 urine samples over 955 days. Beta-core fragment levels were measured. A regression line was calculated for the weekly medians of the 1134 control samples and multiples of the control median (MoM) were determined. 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 Down syndrome cases exceeded the 95th centile of the control group. The median MoM of control samples and the proportion of Down syndrome cases detected by the test was relatively constant during the study period. The unaffected cases were divided into three equal divisions, corresponding to approximately the first, second and third year of sample collection. No trend was found in the median control MoM values in three sample collection periods (r2=0.04). A similar number of cases exceeded the 95th centile of control samples in the three sample collection periods, 63 per cent, 66 per cent and 66 per cent. Consistent results were indicated during the three years of sample testing. Levels of total oestriol were determined in urine samples and MoM statistics derived. The median oestriol level in Down syndrome cases was 0.59 MoM. Only 12 per cent of cases had MoM levels below the fifth centile. Gaussian models were prepared combining biochemical data and maternal age distribution. 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 cases at 5 per cent false-positive rate. At the completion of the study, we thawed and reassayed 20 random urine samples (10 control and 10 Down syndrome) collected at different times during the study period. While the control samples (74-1700 ng/ml) had slightly increased values when reassayed (mean value 137 per cent of original prospective 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 the magnitude of the original beta-core fragment value and the change in immunoreactivity when reassayed (r2=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-core fragment immunoreactivity (mean increase in beta-core fragment levels 56 per cent, t-test, controls versus cases, p=0.004). We infer that aggregation of beta-core fragment upon storage interferes with beta-core fragment measurements. This may be the cause of the poor beta-core fragment screening performance reported using sto