Neonatal Dried Blood Spot Samples Research Articles

P071 Neonatal levels of inflammatory markers in individuals who later develop early onset Inflammatory Bowel Disease

Abstract Background The early life period is suggested to be important in development of Inflammatory Bowel Disease (IBD). Studies in babies from mothers with IBD observed increased faecal calprotectin and changes in the microbial compartments compared to babies from healthy mothers1,2, which suggests that changes in early life might be associated with later disease development. Prospective studies of IBD risk following early life changes at the molecular level are sparse. We aimed to investigate if individuals with early onset (EO) IBD had changes in systemic inflammatory markers shortly after birth. Methods We measured the concentration of 9 cytokines in a cohort of 464 neonatal dried blood spot samples stored at the Danish National Biobank from individuals who later developed EO IBD (median age of diagnosis 11 years), as well as matched controls. Individuals who developed IBD were identified through the use of the Danish National Patient Registry. Uni- and multivariate logistic regression with adjustment for birth year, sex, and gestational age was conducted to assess the association between 6 of the inflammatory markers with later disease development (INF-γ, TNF-α, IL-1β, IL-4, IL-6 and IL-17a). Further exploratory analyses were performed investigating risk of very early onset (VEO) IBD (onset from 0-5 years) in 88 cases and their matched controls. Results No significant association with EO IBD was observed for any of the cytokines in uni- and multivariate models when adjusting for multiple testing. In the exploratory analysis of VEO IBD, increased IL-4 was associated with significantly reduced risk of IBD (OR=0.992, p-value=0.023) and increased IL-17a was borderline-significantly associated with increased risk of IBD (OR=1.002, p-value=0.063) in a multivariate logistic model including all 6 cytokines. This association did not remain significant when adjusting for multiple testing, which may reflect limited power. Conclusion In our unique study of 464 dried blood spots from later IBD patients and controls, we could not find an association between measured cytokines and EO IBD. In contrast, patients with VEO IBD had decreased levels of IL-4 and increased levels of IL-17a. This sub-cohort was small, and significance disappeared after adjusting for multiple testing. Still, we find it important to report these findings, as they are novel and may inspire future studies into the aetiology of VEO IBD. 1. Kim, E. S. et al. Longitudinal Changes in Fecal Calprotectin Levels Among Pregnant Women With and Without Inflammatory Bowel Disease and Their Babies. Gastroenterology (2021) 2. Torres, J. et al. Infants born to mothers with IBD present with altered gut microbiome that transfers abnormalities of the adaptive immune system to germ-free mice. Gut (2020)

Metabolic signature of the pathogenic 22q11.2 deletion identifies carriers and provides insight into systemic dysregulation

Large deletions at chromosome 22q11.2 are known to cause severe clinical conditions collectively known as 22q11.2 deletion syndrome. Notwithstanding the pathogenicity of these deletions, affected individuals are typically diagnosed in late childhood or early adolescence, and little is known of the molecular signaling cascades and biological consequences immediately downstream of the deleted genes. Here, we used targeted metabolomics to compare neonatal dried blood spot samples from 203 individuals clinically identified as carriers of a deletion at chromosome 22q11.2 with 203 unaffected individuals. A total of 173 metabolites were successfully identified and used to inform on systemic dysregulation caused by the genomic lesion and to discriminate carriers from non-carriers. We found 84 metabolites to be differentially abundant between carriers and non-carriers of the 22q11.2 deletion. A predictive model based on all 173 metabolites achieved high Accuracy (89%), Area Under the Curve (93%), F1 (88%), Positive Predictive Value (94%), and Negative Predictive Value (84%) with tyrosine and proline having the highest individual contributions to the model as well as the highest interaction strength. Targeted metabolomics provides insight into the molecular consequences possibly contributing to the pathology underlying the clinical manifestations of the 22q11 deletion and is an easily applicable approach to first-pass screening for carrier status of the 22q11 to prompt subsequent verification of the genomic diagnosis.

Determinants of Neonatal Vitamin D Levels as Measured on Neonatal Dried Blood Spot Samples

Background: Vitamin D deficiency is linked to adverse childhood health outcomes, yet data on the distribution and quantifiable determinants of neonatal 25-hydroxyvitamin D₃ (25OHD) concentration, a vitamin D biomarker, are limited. Objective: Our aim was to identify determinants of neonatal 25OHD concentration, measured using neonatal dried blood spots (DBS). Methods: A total of 259 ethnically diverse children aged 0-16 years born in Victoria, Australia, were recruited. Data included maternal sun exposure, skin type, 25OHD concentration on stored neonatal DBS, and genotypes at the target genes. Associations were investigated using multiple linear regression models. Results: The median 25OHD concentration was 29.2 nmol/l (IQR 18.0-47.4). Measured 25OHD was <50 nmol/l in almost half of the neonatal sample. Ambient ultraviolet radiation (UVR) 6 weeks before birth was the strongest predictor of neonatal 25OHD, accounting for 23% of its variation. A further 10% was explained by infant genetic variants at GC (rs2282679), the gene encoding the vitamin D binding protein, and DHCR7 (rs12785878), a gene required for synthesis of 7-dehydrocholesterol, a precursor to 25OHD. DBS age explained 7%, and patterns of maternal sun exposure and clothing choices accounted for 4%. A child's skin colour was strongly associated with GC gene variants and not independent of these variants in predicting 25OHD. The final model explained 43% of the total variance in neonatal 25OHD concentration. Conclusion: Maternal lifestyle factors and infant genetic variants predict neonatal 25OHD levels; the importance of maternal UVR exposure in late pregnancy is highlighted.

Quantification of 2-methylcitric acid in dried blood spots improves newborn screening for propionic and methylmalonic acidemias.

Background Newborn screening for propionic acidemia and methylmalonic acidurias using the marker propionylcarnitine (C3) is neither sensitive nor specific. Using C3 to acetylcarnitine (C3/C2) ratio, together with conservative C3 cut-offs, can improve screening sensitivity, but the false positive rate remains high. Incorporating the marker 2-methylcitric acid has been suggested, to improve the positive predictive value for these disorders without compromising the sensitivity. Methods Between July 2011 and December 2012 at the Newborn Screening Ontario laboratory, all neonatal dried blood spot samples that were reported as screen positive for propionic acidemia or methylmalonic acidurias based on elevated C3 and C3/C2 ratio were analyzed for 2-methylcitric acid, using liquid chromatography tandem mass spectrometry. Results Of 222,420 samples screened, 103 were positive for methylmalonic acidurias or propionic acidemia using C3 and C3/C2 ratio as markers. There were nine true positives: propionic acidemia (n = 3), Cobalamin (Cbl) A (n=1), and Cbl C (n = 5). Among false positives there were 72 neonates not affected, 20 with maternal B12 deficiency, and two incidental finding (transcobalamin II and unclassified Cbl defect). 2-Methylcitric acid was analyzed in all 103 samples and ranged between 0.1 and 89.4 µmol/l (reference range 0.04-0.36). Only 14 samples exceeded the set 2-methylcitric acid cut-off of 1.0 µmol/l, including the samples from all nine true positives. Conclusion By including 2-methylcitric acid in the screening algorithm, the positive predictive value of our primary and secondary screening targets improved from 8.7 to 64.3%. This would have eliminated 89 unnecessary referrals while maintaining 100% sensitivity.

High-Quality Exome Sequencing of Whole-Genome Amplified Neonatal Dried Blood Spot DNA.

Stored neonatal dried blood spot (DBS) samples from neonatal screening programmes are a valuable diagnostic and research resource. Combined with information from national health registries they can be used in population-based studies of genetic diseases. DNA extracted from neonatal DBSs can be amplified to obtain micrograms of an otherwise limited resource, referred to as whole-genome amplified DNA (wgaDNA). Here we investigate the robustness of exome sequencing of wgaDNA of neonatal DBS samples. We conducted three pilot studies of seven, eight and seven subjects, respectively. For each subject we analysed a neonatal DBS sample and corresponding adult whole-blood (WB) reference sample. Different DNA sample types were prepared for each of the subjects. Pilot 1: wgaDNA of 2x3.2mm neonatal DBSs (DBS_2x3.2) and raw DNA extract of the WB reference sample (WB_ref). Pilot 2: DBS_2x3.2, WB_ref and a WB_ref replica sharing DNA extract with the WB_ref sample. Pilot 3: DBS_2x3.2, WB_ref, wgaDNA of 2x1.6 mm neonatal DBSs and wgaDNA of the WB reference sample. Following sequencing and data analysis, we compared pairwise variant calls to obtain a measure of similarity—the concordance rate. Concordance rates were slightly lower when comparing DBS vs WB sample types than for any two WB sample types of the same subject before filtering of the variant calls. The overall concordance rates were dependent on the variant type, with SNPs performing best. Post-filtering, the comparisons of DBS vs WB and WB vs WB sample types yielded similar concordance rates, with values close to 100%. WgaDNA of neonatal DBS samples performs with great accuracy and efficiency in exome sequencing. The wgaDNA performed similarly to matched high-quality reference—whole-blood DNA—based on concordance rates calculated from variant calls. No differences were observed substituting 2x3.2 with 2x1.6 mm discs, allowing for additional reduction of sample material in future projects.

CCND2, CTNNB1, DDX3X, GLI2, SMARCA4, MYC, MYCN, PTCH1, TP53, and MLL2 gene variants and risk of childhood medulloblastoma.

Recent studies have described a number of genes that are frequently altered in medulloblastoma tumors and that have putative key roles in the development of the disease. We hypothesized that common germline genetic variations in these genes may be associated with medulloblastoma development. Based on recent publications, we selected 10 genes that were frequently altered in medulloblastoma: CCND2, CTNNB1, DDX3X, GLI2, SMARCA4, MYC, MYCN, PTCH1, TP53, and MLL2 (now renamed as KMT2D). Common genetic variants (single nucleotide polymorphisms) annotating these genes (n = 221) were genotyped in germline DNA (neonatal dried blood spot samples) from 243 childhood medulloblastoma cases and 247 control subjects from Sweden and Denmark. Eight genetic variants annotating three genes in the sonic hedgehog signaling pathway; CCND2, PTCH1, and GLI2, were found to be associated with the risk of medulloblastoma (Pcombined < 0.05). The findings were however not statistically significant following correction for multiple testing by the very stringent Bonferroni method. The results do not support our hypothesis that common germline genetic variants in the ten studied genes are associated with the risk of developing medulloblastoma.Electronic supplementary materialThe online version of this article (doi:10.1007/s11060-015-1891-1) contains supplementary material, which is available to authorized users.

Hepatitis C virus seroprevalence in pregnant women delivering live-born infants in North Thames, England in 2012.

To estimate HCV seroprevalence in subpopulations of women delivering live-born infants in the North Thames region in England in 2012, an unlinked anonymous (UA) cross-sectional survey of neonatal dried blood spot samples was conducted. Data were available from 31467 samples from live-born infants received by the North Thames screening laboratory. Thirty neonatal samples had HCV antibodies, corresponding to a maternal seroprevalence of 0·095% (95% confidence interval 0·067-0·136). Estimated HCV seroprevalences in women born in Eastern Europe, Southern Asia and the UK were 0·366%, 0·162% and 0·019%, respectively. For women born in Eastern Europe seroprevalence was highest in those aged around 27 years, while in women born in the UK and Asia-Pacific region, seroprevalence increased significantly with age. HCV seroprevalence in UK-born women whose infant's father was also UK-born was 0·016%. One of the 30 HCV-seropositive women was HIV-1 seropositive. Estimated HCV seroprevalence for women delivering live-born infants in North Thames in 2012 (0·095%) was significantly lower than that reported in an earlier UA survey in 1997-1998 (0·191%). Data indicate that the cohort of UK-born HCV-seropositive women is ageing and that, in this area of England, most perinatally HCV-exposed infants were born to women themselves born in Southern Asia or Eastern Europe.

Gene expression profiling of archived dried blood spot samples from the Danish Neonatal Screening Biobank.

A large part of the human genome is transcribed into various forms of RNA, and the global gene expression profile (GEP) has been studied for several years using technology such as RNA-microarrays. In this study, we evaluate whether neonatal dried blood spot (DBS) samples stored in the Danish Neonatal Screening Biobank (DNSB) can be used for GEP. This paper is divided into sub-studies examining the effects of: 1) different whole transcriptome amplification kits (WTA); 2) years of storage and storage in room temperature (RT) versus freezers (-20°C) on DNSB DBS samples; 3) effects of RT storage vs freezer storage on DBS samples from the USA and DNSB, and 4) using smaller disc sizes, thereby decreasing DBS use. We present evidence that reliable and reproducible GEPs can be obtained using neonatal DBS samples. The main source of variation is the storage condition. When samples are stored at -20°C, the dynamic range is increased, and Pearson correlations are higher. Differential analysis reveals no statistically significant differences between samples collected a decade apart and stored at -20°C. However, samples stored at RT show differential expression for a third of the gene-specific probes. Our data also suggests that using alternate WTA kits significantly changes the GEP. Finally, the amount of input material, i.e., the size and number of DBS discs used, can be reduced to preserve this valuable and limited material. We conclude that DNSB DBS samples provide a reproducible resource for GEP. Results are improved if the cards are stored at -20°C. Furthermore, it is important to use a single type of kit for analysis because using alternate kits introduces differential expression.

Compound heterozygotes and beta‐thalassemia: Top‐down mass spectrometry for detection of hemoglobinopathies

We have shown previously that liquid extraction surface analysis of dried blood spots coupled to high resolution top-down MS may be applied for the diagnosis of hemoglobin (Hb) variants FS, FAS, FC, FAC, FAD in newborn samples. The objective of the current work was to determine whether the structural variant HbE, compound heterozygote variants FSC and FSD, and β-thalassemia were amenable to diagnosis by this approach. Anonymized residual neonatal dried blood spot samples, taken as part of the routine newborn screening program, were analyzed by liquid extraction surface analysis coupled to high resolution MS/MS. The samples had been previously screened and were known to be FAE, FSC, FSD, or β-thalassemia. Manual analysis of the mass spectra revealed that, in all cases, the variants may be confirmed. Direct surface sampling MS should be considered as an alternative to current screening techniques for the diagnosis of Hb variants.

Archived neonatal dried blood spot samples can be used for accurate whole genome and exome-targeted next-generation sequencing

Dried blood spot samples (DBSS) have been collected and stored for decades as part of newborn screening programmes worldwide. Representing almost an entire population under a certain age and collected with virtually no bias, the Newborn Screening Biobanks are of immense value in medical studies, for example, to examine the genetics of various disorders. We have previously demonstrated that DNA extracted from a fraction (2×3.2mm discs) of an archived DBSS can be whole genome amplified (wgaDNA) and used for accurate array genotyping. However, until now, it has been uncertain whether wgaDNA from DBSS can be used for accurate whole genome sequencing (WGS) and exome sequencing (WES).This study examined two individuals represented by three different types of samples each: whole-blood (reference samples), 3-year-old DBSS spotted with reference material (refDBSS), and 27- to 29-year-old archived neonatal DBSS (neoDBSS) stored at −20°C in the Danish Newborn Screening Biobank. The reference samples were genotyped using an Illumina Omni2.5M array, and all samples were sequenced on a HighSeq2000 Paired-End flow cell. First, we compared the array single nucleotide polymorphism (SNP) genotype data to the single nucleotide variation (SNV) calls from the WGS and WES SNV calls. We also compared the WGS and WES reference sample SNV calls to the DBSS SNV calls.The overall performance of the archived DBSS was similar to the whole blood reference sample. Plotting the error rates relative to coverage revealed that the error rates of DBSS were similar to that of their reference samples. SNVs called with a coverage<×8 had error rates between 1.5 and 35%, whereas the error rates of SNVs called with a coverage≥8 were <1.5%. In conclusion, the wgaDNA amplified from both new and old neonatal DBSS perform as well as their whole-blood reference samples with regards to error rates, strongly indicating that neonatal DBSS collected shortly after birth and stored for decades comprise an excellent resource for NGS studies of disease.

DNA methylome profiling using neonatal dried blood spot samples: A proof-of-principle study

DNA methylation is the most common DNA modification and perhaps the best described epigenetic modification. It is believed to be important for genomic imprinting and gene regulation and has been associated with the development of diseases such as schizophrenia and some types of cancer.Neonatal dried blood spot samples, commonly known as Guthrie cards, are routinely collected worldwide to screen newborns for diseases. Some countries, including Denmark, have been storing the excess neonatal dried blood spot samples in biobanks for decades. Representing a high percentage of the population under a certain age, the neonatal dried blood spot samples are a potential alternative to collecting new samples to study diseases. As such, neonatal dried blood spot samples have previously been used for DNA genotyping studies with excellent results. However, the amount of material available for research is often limited, challenging researchers to generate the most data from a limited quantity of material.In this proof-of-principle study, we address whether two 3.2mm disks punched from a neonatal dried blood spot sample contain enough DNA for genome-wide methylome profiling, measuring 27,578 loci at the same time. We selected two subjects and carried out the following with each: 1) collected an adult whole-blood sample as reference, 2) spotted a fraction of the whole-blood sample onto a similar type of filter paper as used in the newborn screening and stored it for 3years to serve as a dried blood spot reference, and 3) identified the archived neonatal dried blood spot samples, stored for 26–28years, in the Danish Newborn Screening Biobank as a representative of the archived samples. For comparison, we used two different kits for DNA extraction.The DNA, extracted using the Extract-N-Amp Blood PCR kit, was analyzed, and no statistically significant differences were observed (P<0.001) when we compared the methylation profile of the reference whole-blood samples to the dried blood spot references. This indicates that two 3.2mm disks contain enough material for reliable methylome profiling and that storing the whole-blood sample on neonatal dried blood spot filter paper for 3years does not interfere with the outcome of the analysis.Furthermore, we compared the adult DNA methylation profile to the neonatal dried blood spot sample profile. Approximately 50 sites in the subjects were significantly (P<0.001) different in the newborn sample compared with the adult sample. Both being healthy adults and the high quality of the DNA methylation array led to the conclusion that the archived neonatal dried blood spot samples can be used for methylome profiling, despite decades of storage and DNA degradation.In conclusion, we show that reliable methylome data can be obtained from old neonatal dried blood spot samples, by using a reasonable amount of the limited resource. This further adds to the use of neonatal dried blood spot samples in genetic research and screening and paves the way for unique population-based studies of epigenetic modifications after birth.

Top-Down Proteomics and Direct Surface Sampling of Neonatal Dried Blood Spots: Diagnosis of Unknown Hemoglobin Variants

We have previously shown that liquid microjunction surface sampling of dried blood spots coupled with high resolution top-down mass spectrometry may be used for screening of common hemoglobin variants HbS, HbC, and HbD. In order to test the robustness of the approach, we have applied the approach to unknown hemoglobin variants. Six neonatal dried blood spot samples that had been identified as variants, but which could not be diagnosed by current screening methods, were analyzed by direct surface sampling top-down mass spectrometry. Both collision-induced dissociation and electron transfer dissociation mass spectrometry were employed. Four of the samples were identified as β-chain variants: two were heterozygous Hb D-Iran, one was heterozygous Hb Headington, and one was heterozygous Hb J-Baltimore. The fifth sample was identified as the α-chain variant heterozygous Hb Phnom Penh. Analysis of the sixth sample suggested that it did not in fact contain a variant. Adoption of the approach in the clinic would require speed in both data collection and interpretation. To address that issue, we have compared manual data analysis with freely available data analysis software (ProsightPTM). The results demonstrate the power of top-down proteomics for hemoglobin variant analysis in newborn samples.

Detecting 22q11.2 Deletions by Use of Multiplex Ligation-Dependent Probe Amplification on DNA from Neonatal Dried Blood Spot Samples

The 22q11 deletion syndrome, which is caused by a 1.5- to 3.0-megabase hemizygous deletion in chromosome 22q11.2, has a prevalence of 1/2000 to 1/4000. However, the syndrome presents with highly variable phenotypes and thus may be underestimated among Danish newborns. To establish a true incidence of 22q11.2 deletions among certain manifestations, eg, congenital heart disease, on selected Danes, a multiplex ligation-dependant probe amplification (MLPA) analysis was designed. The analysis was planned to be performed on DNA extracted from dried blood spot samples (DBSS) obtained from Guthrie cards collected during neonatal screening programs. However, the DNA concentration necessary for a standard MLPA analysis (20 ng) could not be attained from DBSS, and a novel MLPA design was developed to permit for analysis on limited amounts of DNA (2 ng). A pilot study is reported here that validates the new MLPA design using nine patients diagnosed with the 22q11.2 deletion and 101 controls. All deletions were identified using DNA extracted from DBSS, and no copy number variations were detected in the controls, resulting in a specificity and sensitivity of 100%. It is thereby concluded that the novel MLPA probe design is successful and reliable using minimal amounts of DNA. This allows for use of DBSS samples in a retrospective study of 22q11.2 deletion among certain manifestations associated with DiGeorge Syndrome.

Genome-wide scans using archived neonatal dried blood spot samples

BackgroundIdentification of disease susceptible genes requires access to DNA from numerous well-characterised subjects. Archived residual dried blood spot samples from national newborn screening programs may provide DNA from entire populations and medical registries the corresponding clinical information. The amount of DNA available in these samples is however rarely sufficient for reliable genome-wide scans, and whole-genome amplification may thus be necessary. This study assess the quality of DNA obtained from different amplification protocols by evaluating fidelity and robustness of the genotyping of 610,000 single nucleotide polymorphisms, using the Illumina Infinium HD Human610-Quad BeadChip. Whole-genome amplified DNA from 24 neonatal dried blood spot samples stored between 15 to 25 years was tested, and high-quality genomic DNA from 8 of the same individuals was used as reference.ResultsUsing 3.2 mm disks from dried blood spot samples the optimal DNA-extraction and amplification protocol resulted in call-rates between 99.15% – 99.73% (mean 99.56%, N = 16), and conflicts with reference DNA in only three per 10,000 genotype calls.ConclusionWhole-genome amplified DNA from archived neonatal dried blood spot samples can be used for reliable genome-wide scans and is a cost-efficient alternative to collecting new samples.

Genotyping whole‐genome‐amplified DNA from 3‐ to 25‐year‐old neonatal dried blood spot samples with reference to fresh genomic DNA

Stored surplus of dried blood spot (DBS) samples from neonatal screening programs constitute a vast potential for large genetic epidemiological studies. However, age of the samples and the small amounts of DNA available may limit their usage. In this study we validate genotyping accuracy and efficiency of whole-genome-amplified DNA (wgaDNA) obtained from stored DBS samples, with reference to fresh genomic DNA from the same individuals. DBS samples from 29 volunteers, stored for up to 25 years, in the Danish Neonatal Screening Biobank were included and three DNA extraction methods, each using one 3.2 mm disk, were evaluated. Four whole-genome amplification kits, and one re-amplification kit, were used. Thirty-one SNPs were genotyped using the Sequenom platform and the wgaDNA samples calls were compared with their references for accuracy and efficiency evaluation. The genotype calls done blinded by the user had in many setups a 100% call- and concordance rate. Our results showed that genotyping performance is dependent on the combination of extraction procedure and amplification method, whereas years of storage did not seem to influence in this study. Based on these results we conclude that DBS samples should be considered a reliable and potential resource for future genotyping studies.

Adiponectin levels measured in dried blood spot samples from neonates born small and appropriate for gestational age

Adiponectin levels measured in neonatal dried blood spot samples (DBSS) might be affected by both prematurity and being born small for gestational age (SGA). The aim of the study was to measure adiponectin levels in routinely collected neonatal DBSS taken on day 5 (range 3-12) postnatal from infants. A retrospective case-control study. One hundred and twenty-two infants: 62 very premature (34 SGA) and 60 mature infants (27 SGA). Adiponectin concentrations were determined in stored neonatal DBSS using a sandwich immunoassay based on flow metric Luminex xMap technology. Adiponectin was measurable in all samples, and repeated measurements correlated significantly (r = 0.94). Adiponectin concentrations were negatively associated with both SGA (B = -0.283, P = 0.04) and prematurity (B = -2.194, P < 0.001), independently of each other. In the premature but not the mature group, adiponectin levels increased with increasing postnatal age at blood sampling (B = 0.175, P < 0.001). Reliable quantification of adiponectin in stored DBSS is feasible and may be used to study large populations of routinely collected samples. Low levels of adiponectin in neonatal DBSS are associated with SGA as well as prematurity. Blood adiponectin levels increase with postnatal age in premature infants, suggesting a rapid yet unexplained metabolic adaptation to premature extrauterine life.

HIV-1 subtypes in pregnant women in the UK

We analyse the distribution of HIV-1 subtypes in HIV-1-seropositive samples from 333,270 residual neonatal dried blood spot samples tested for routine newborn screening tests in the UK between July 1999 and December 2002. Of the 813 antibody-positive samples shown to contain passively acquired, maternal HIV-1 for which subtyping was attempted, 333 (41%) could not be subtyped due to cross-reactivity or low values of the assay results, and 480 (59%) were classified as B (35, 7.3%) or non-B (445, 92.7%). The proportions of subtyped B samples differed significantly (P=0.004) between those from neonates whose mothers were born in the UK (21.4%) and those from neonates whose mothers were known to be born abroad (7%). Using a serological approach to establish viral serotype, we document the distribution of HIV-1 subtypes in infected pregnant women in the UK.

HIV prevalence in pregnant women in an ethnically diverse population in the UK: 1998-2002.

To describe trends in the prevalence of HIV in an ethnically diverse population of pregnant women in the United Kingdom. Data on parental country of birth from national birth registration records were linked to neonatal dried blood spot samples routinely collected for neonatal screening in the North Thames region between 1998 and 2002. Identifiers were subsequently irreversibly deleted prior to establishing maternal HIV status by testing the neonatal samples. A total of 491 213 dried blood spot samples were collected, and 490 879 (99.93%) were tested for HIV. Of these, 1029 were seropositive. There was an overall significant increasing trend (P-value = 0.001) between 1998 and 2002. Maternal region of birth was available for 89.8% of HIV-infected samples, and, among these, 80.5% of mothers were born in sub-Saharan Africa and 11.1% in the UK. The highest prevalences of HIV were in women born in sub-Saharan Africa (2.09%). If both parents were born in the UK, overall seroprevalence was 0.016%. HIV infection in pregnant women in the UK continues to occur predominantly in women born in sub-Saharan Africa with prevalence in this group increasing significantly. Although the absolute number of HIV-infected women rose in some other groups, there was no evidence for a statistically significant rise in HIV prevalence in women born outside sub-Saharan Africa. Over 93% of children at risk of vertical transmission of HIV had at least one parent born abroad. This paper underlines the value of data linkage in monitoring HIV prevalence in a diverse population.

Technical aspects of neonatal screening using tandem mass spectrometry. Report from the 4th meeting of the International Society for Neonatal Screening.

Quantitative analysis of amino acids (AA) and acylcarnitines using tandem mass spectrometry is an emerging technology used to screen neonatal dried blood spot samples for disorders in the metabolism of AA, organic acids and fatty acids. This paper provides a brief review of some of the technically oriented issues which emerged at the 4th meeting of the International Society for Neonatal Screening in Stockholm, 1999. The information covers sample preparation, instrumentation, data acquistion modes, internal standards, interpretation, confounding factors and practical screening experience.

Use of dried blood spots for the detection and confirmation of HTLV-I specific antibodies for epidemiological purposes.

AIMS--To modify and evaluate a gelatin particle agglutination test that could provide a sensitive, specific and inexpensive method for the detection of HTLV-I antibody in dried blood spot samples (DBS) collected on filter paper. METHODS--A set of 26 reference samples confirmed as HTLV-I antibody positive were assembled from patients with tropical spastic paraparesis or adult T cell leukaemia and blood donors. Serum samples and simulated antibody positive dried blood spot eluates were tested using the Serodia assay together with two confirmatory tests: HTLV BLOT 2.3, a western blot, and Select-HTLV, an enzyme immunoassay (EIA). Both confirmatory tests use synthetic peptides to differentiate between antibodies to HTLV-I and -II. The modified Serodia assay was then used to test anonymously 10,135 DBS collected from neonates from London. Samples reactive in the modified Serodia test producing a positive result were titrated to an end point and confirmed as before. RESULTS--All 26 eluates made from simulated DBS derived from positive reference samples were identified as positive by the modified Serodia HTLV-I test and were confirmed as anti-HTLV-I positive by EIA. Two eluates derived from relatively low titre reference samples gave indeterminate results on western blotting. Screening of the 10,135 neonatal DBS resulted in six repeat reactives, five of which were confirmed. The remaining reactive sample gave an indeterminate result on western blotting and there was insufficient eluate for testing by EIA. The overall seroprevalence of HTLV-I in this population was 0.05% (five of 10,135). CONCLUSION--The modified Serodia HTLV-I assay provides a sensitive, specific and inexpensive (10 pence/test) method for screening large numbers of DBS. The format of the assay makes it ideally suited for simultaneous screening of antibodies to HIV-1, HIV-2 and HTLV-I using semi-automated equipment.