Residual Dried Blood Spot Samples Research Articles

Detection of glucosylsphingosine in dried blood spots for diagnosis of Gaucher disease by LC-MS/MS

IntroductionGaucher disease (GD) is caused by a deficiency of β-glucosidase (GCase), leading to accumulation of glucosylceramide (GlcC) and glucosylsphingosine (Lyso-Gb1). Lyso-Gb1 is a reliable biomarker for GD. ObjectivesThis study aims to develop a simple, effective and accurate method for the screening and diagnosis of GD using dried blood spot (DBS) samples. MethodsLyso-Gb1 in DBS was extracted by 50% acetonitrile aqueous solution containing isotope-labeled internal standard and analyzed using liquid chromatography tandem mass spectrometry (LC-MS/MS). A reference interval was established by analyzing samples from 277 healthy controls. Lyso-Gb1 was detected in the residual DBS samples from 142 high-risk patients with splenomegaly and/or thrombocytopenia. Based on GCase activity in DBS, samples were classified into four groups: confirmed GD patients (n = 52), GD carriers (n = 5), false positive (n = 36) and negative (n = 49). ResultsThe optimized Lyso-Gb1 assay showed intra- and inter-assay variations ranged between 2.0%–8.2% and 3.8%–10.2%, respectively. Accuracies ranged from 93.5% to 112.6%. The lowest limit of quantification was 1 ng/mL. The normal reference interval of Lyso-Gb1 in DBS ranged from 2.1 to 9.9 ng/mL. Among the 142 subjects, except for one GD patient (Lyso-Gb1 > 2500 ng/mL), the Lyso-Gb1 concentrations in 51 GD patients ranged from 190.5 to 2380.6 ng/mL (the median 614.8 ng/mL). Also, one negative patient was found to have an elevated Lyso-Gb1 level (684.5 ng/mL), while the other patients were normal. The negative case was then confirmed to be an atypical GD patient with a c.1091A > G (p.Y364C) homozygous variant in PSAP gene by next generation sequencing. ConclusionsThe optimized method to determine Lyso-Gb1 in DBS was demonstrated as a useful tool for the screening and diagnosis of GD.

α-Mannosidosis - An underdiagnosed lysosomal storage disease in individuals with an ‘MPS-like’ phenotype

Individuals affected by alpha-Mannosidosis suffer from similar clinical symptoms such as respiratory infections, skeletal changes as patients with mucopolysaccharidoses (MPS). α-Mannosidosis is considered as an ultra-rare disorders and also diagnostic testing is often limited. With the availability of novel therapies and easy-to-access diagnostic tests (e.g. Tandem mass spectrometry) using dried blood spots for both enzymatic and genetic testing, the chance for the development of a better understanding of disease and awareness may be triggered. In a pilot study, we have investigated 1010 residual dried blood spot samples from individuals suspicious to MPS. In these study cohort, 158/1010 individuals were genetically confirmed for MPS. Additional biochemical and genetic confirmatory testing for α-mannosidases revealed four individuals with a final diagnosis of α-mannosidosis. This unexpected high number of individuals with α-mannosidosis demonstrated the urgent need of taking this rare disorder in clinical and diagnostic consideration particularly in patients suspicious to MPS.

Velmanase alfa enzyme replacement therapy for alpha-mannosidosis improves patient outcomes over standard of care both in terms of clinically relevant improvement and disease stabilization

Individuals affected by alpha-Mannosidosis suffer from similar clinical symptoms such as respiratory infections, skeletal changes as patients with mucopolysaccharidoses (MPS). α-Mannosidosis is considered as an ultra-rare disorders and also diagnostic testing is often limited. With the availability of novel therapies and easy-to-access diagnostic tests (e.g. Tandem mass spectrometry) using dried blood spots for both enzymatic and genetic testing, the chance for the development of a better understanding of disease and awareness may be triggered. In a pilot study, we have investigated 1010 residual dried blood spot samples from individuals suspicious to MPS. In these study cohort, 158/1010 individuals were genetically confirmed for MPS. Additional biochemical and genetic confirmatory testing for α-mannosidases revealed four individuals with a final diagnosis of α-mannosidosis. This unexpected high number of individuals with α-mannosidosis demonstrated the urgent need of taking this rare disorder in clinical and diagnostic consideration particularly in patients suspicious to MPS.

Glucosylsphingosine for the screening, diagnosis, monitoring and prediction in Gaucher disease

Gaucher disease (GD) is caused by a deficiency of β-glucosidase (GCase), leading to accumulation of glucosylceramide (GlcC) and glucosylsphingosine (Lyso-Gb1). Lyso-Gb1 is a reliable biomarker for GD.This study aims to develop a simple, effective and accurate method for the screening and diagnosis of GD using dried blood spot (DBS) samples.Lyso-Gb1 in DBS was extracted by 50% acetonitrile aqueous solution containing isotope-labeled internal standard and analyzed using liquid chromatography tandem mass spectrometry (LC-MS/MS). A reference interval was established by analyzing samples from 277 healthy controls. Lyso-Gb1 was detected in the residual DBS samples from 142 high-risk patients with splenomegaly and/or thrombocytopenia. Based on GCase activity in DBS, samples were classified into four groups: confirmed GD patients (n = 52), GD carriers (n = 5), false positive (n = 36) and negative (n = 49).The optimized Lyso-Gb1 assay showed intra- and inter-assay variations ranged between 2.0%–8.2% and 3.8%–10.2%, respectively. Accuracies ranged from 93.5% to 112.6%. The lowest limit of quantification was 1 ng/mL. The normal reference interval of Lyso-Gb1 in DBS ranged from 2.1 to 9.9 ng/mL. Among the 142 subjects, except for one GD patient (Lyso-Gb1 > 2500 ng/mL), the Lyso-Gb1 concentrations in 51 GD patients ranged from 190.5 to 2380.6 ng/mL (the median 614.8 ng/mL). Also, one negative patient was found to have an elevated Lyso-Gb1 level (684.5 ng/mL), while the other patients were normal. The negative case was then confirmed to be an atypical GD patient with a c.1091A > G (p.Y364C) homozygous variant in PSAP gene by next generation sequencing.The optimized method to determine Lyso-Gb1 in DBS was demonstrated as a useful tool for the screening and diagnosis of GD.

The prevalence of hepatitis B virus infection in HIV-positive and HIV-negative infants: KwaZulu-Natal, South Africa.

BackgroundThe prevalence of hepatitis B virus (HBV) amongst South African infants and children has been reported in the pre-HIV era. Despite the reported high prevalence of HIV in the general population of South Africa, the rate of HIV/HBV co-infection amongst infants and children remains poorly reported.ObjectivesWe describe the prevalence of HBV infection amongst HIV-positive and HIV-negative infants by molecular methods of diagnosis using dried blood spot samples.MethodsThis retrospective cross-sectional study was conducted between July 2011 and December 2011 in an academic referral laboratory offering viral diagnostic services to the entire KwaZulu-Natal province of South Africa. A total of 322 study samples were collected from discarded residual dried blood spot samples following routine infant diagnosis of HIV. Equal proportions of HIV-positive and HIV-negative infant specimens were studied. Statistical differences in the prevalence of HBV between the HIV-positive and HIV-negative samples were calculated using the Pearson chi-square test, and a p-value < 0.05 was considered statistically significant. Further testing for HBV DNA using a nested polymerase chain reaction method was performed.ResultsThe overall prevalence of HBV was 10%. In the HIV-positive group, 21 of 161 infants tested positive for HBV compared with 12 of 161 HIV-negative infants who tested positive for HBV. The proportion of infants infected with HBV was marginally higher amongst HIV-positive infants (13.0%; 95% CI 6.8–19.9) compared with HIV-negative infants (7.5%; 95% CI 2.5–13.7; P = 0.098), though not statistically significant.ConclusionThe finding of a 10% HBV prevalence in this infant cohort is clinically significant. The non-statistically significant difference in HBV prevalence between the HIV-positive and HIV-negative infants suggests that high prevalence of HBV infection in children may be a problem independent of HIV.

Michigan BioTrust for Health: Public Support for Using Residual Dried Blood Spot Samples for Health Research

Purpose: Focus groups were utilized as a mechanism to solicit input from the public in developing the Michigan BioTrust for Health, a program of the Michigan Department of Community Health to improve storage conditions and promote use of residual newborn screening dried blood spots in health research. Methods: In 2008–2009, 10 diverse communities representative of the general public that might have special concerns as stakeholders in the BioTrust were identified, and 4 discussion questions were developed for use with a standard agenda. Focus group discussions were audio-recorded and transcribed by department staff. Qualitative conclusions resulting from the group discussions were compared with written, quantitative pre- and post-survey responses completed by individual participants. Results: Overall, there was considerable concurrence of opinion across the focus groups. Participants were generally positive about the BioTrust; a sentiment that held true across different demographic populations with over 85% of participants stating they would support use of residual dried blood spots in health research. Conclusion: The focus group process and findings played an important role for public health policy makers in affirming the importance of engaging and informing the public and led to concrete steps to foster community support.

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.

Storage policies and use of the Danish Newborn Screening Biobank

After routine newborn screening, residual dried blood spot samples (DBSS) are stored at -20 degrees C in the Danish Newborn Screening Biobank (NBS-Biobank), which contains DBSS from virtually all newborns in Denmark since 1982--about 1.8 million samples. The purpose of the storage is: (1) diagnosis and treatment of congenital disorders including documentation, repeat testing, quality assurance, statistics and improvement of screening methods; (2) diagnostic use later in infancy after informed consent; (3) legal use after court order; (4) the possibility of research projects after approval by the Scientific Ethical Committee System in Denmark, The Danish Data Protection Agency and the NBS-Biobank Steering Committee. The operation and use of the NBS-Biobank has until recently been regulated by an executive order of 1993 from the Danish Ministry of Health. The Ethical Council, the Central Scientific Ethical Committee and the National Board of Health were also involved in the regulations. These regulations have now been replaced by detailed general operational guidelines for biobanks in Denmark according to Acts on Processing of Personal Data, Patient's Rights, Health 546/2005 and the Biomedical Research Ethics Committee System. No specific Act on biobanks per se has been made in Denmark, but the new regulations and guidelines make the operations of the Danish NBS-Biobank even more clear-cut and safe. The Danish NBS-Biobank has been used in several research projects for aetiological studies of a number of disorders, recently employing new sensitive multiplex technologies and genetic analyses utilizing whole-genome amplified DNA.

Guidelines for the retention, storage, and use of residual dried blood spot samples after newborn screening analysis: Statement of the council of regional networks for genetic services

Guidelines for the retention, storage, and use of residual dried blood spot samples after newborn screening analysis: Statement of the council of regional networks for genetic services

Guidelines for the Retention, Storage, and Use of Residual Dried Blood Spot Samples after Newborn Screening Analysis: Statement of the Council of Regional Networks for Genetic Services

These guidelines provide scientific information for policy development by state health departments considering appropriate use of newborn screening specimens after screening tests are finished. Information was collected, debated, and formulated into a policy statement by the Newborn Screening Committee of the Council of Regional Networks for Genetic Services (CORN), a federally funded national consortium of representatives from 10 regional genetics networks. Newborn screening programs vary widely in approaches and policies concerning residual dried blood spot samples (DBS) collected for newborn screening. Recognition of the epidemiological utility of DBS samples for HIV seroprevalence surveys and a growing interest in DBSs for DNA analysis has intensified consideration of issues regarding retention, storage, and use of residual DBS samples. Potentially these samples provide a genetic material “bank” for all newborns nationwide. Their value as a resource for other uses has already been recognized by scientists, administrators, and judicial officials. Programs should promulgate rules for retention and use of residual newborn screening DBS samples based on scientifically valid information. Banking of newborn samples as sources of genetic material should be considered in light of potential benefit or harm to society.