Newborn screening by DNA analysis of dried blood spots

Summary Amplification of DNA recovered from a dried blood spot was used to genotype individuals with sickle cell disease, sickle cell carriers, and controls. A single 200-l blood spot applied to a filter paper provides sufficient material for more than 20 genetic analyses. In addition, the stability of the DNA is such that adequate material for amplification can be isolated from dried blood spots up to a year following collection. The DNA analysis methods described in this study could be applied to large-scale screening of newborns for genetic disorders.     

Determination of vitamin A in dried human blood spots by high-performance capillary electrophoresis with laser-excited fluorescence detection

We have developed a high-performance capillary electrophoresis (HPCE) method to analyze the retinol (vitamin A) concentration as retinol-retinol binding protein (holo-RBP) from microvolumes of serum (5–10 μl) or one to two drops (∼20 μl) of blood collected and air-dried on blood collection filter paper. A 0.64-cm diameter disk was cut from the dried whole blood specimens and the samples were dissolved in a pretreatment buffer and filtered. Filtrate was injected onto the HPCE column for analysis. The separation was carried out in a 60 cm × 50 μm I.D. fused-silica capillary and the running voltage was 20 kV. A HeCd laser with a wavelength of 325 nm was used for excitation, and the fluorescence of the holo-RBP complex was monitored at 465 nm by a photodiode. A virtual linear relationship was obtained for the retinol concentrations between HPCE and HPLC for 28 serum samples, 19 dried venous blood samples and 9 capillary dried blood spot samples, indicating that valid measures of serum retinol can be obtained from one to two drops of capillary blood collected on filter paper. The absolute detection limit for retinol by HPCE is below 3 μg/l. The method is very useful for vitamin A level screening, especially for children and premature new-born babies.     

Automated high-performance liquid chromatographic method for the determination of guanidinoacetic acid in dried blood spots: a tool for early diagnosis of guanidinoacetate methyltransferase deficiency

A new automated method for the assay of guanidinoacetic acid (GAA) in dried blood spot (DBS) on filter paper is reported. The method, based on reversed-phase (RP)-HPLC, precolumn derivatisation with benzoin and fluorescence detection, has shown good precision and sensitivity and requires only minimal sample handling. The validity of the method was demonstrated by analysing the neonatal blood spot of a patient affected by guanidinoacetate methyltransferase (GAMT) deficiency. GAA concentration was found to be nearly 12-fold higher than the mean control value. We propose this method as an inexpensive and widely applicable tool for the diagnosis of GAMT deficiency.     

Increased sensitivity of malaria detection by nested polymerase chain reaction using simple sampling and DNA extraction

Malaria remains a disease of underdeveloped and remote regions of the world. The application of polymerase chain reaction (PCR) technology to malaria epidemiology has the potential for increasing our knowledge and understanding of this disease. In order to study malaria in all geographical locations it is important that specimen collection and DNA extraction for PCR be kept simple. Here we report a method for extracting DNA from dried blood spots on filter paper which is capable of detecting one Plasmodium falciparum and two Plasmodium vivax parasites/μl of whole blood by nested PCR without compromising the simplicity of specimen collection or DNA extraction.     

The development and validation of a fast and robust dried blood spot based lipid profiling method to study infant metabolism

Early life exposures and metabolic programming are associated with later disease risk. In particular lipid metabolism is thought to play a key role in the development of the metabolic syndrome and insulin resistance in later life. Investigative studies of metabolic programming are limited by the ethics and practicalities of sample collection in small infants. Dried blood spots on filter paper, derived from heel pricks are considered as the most suitable option for this age group. We validated a novel lipid profiling method, based on high resolution mass spectrometry to successfully determine the lipid composition of infants using dried blood spots. The spotting and air drying of blood on paper has noticeable effects on many of the lipids, leading to lipid oxidation and hydrolysis, which demand careful interpretation of the obtained data. We compared the lipid profiles from plasma or whole blood samples and the results from dried blood spots to determine if these revealed the same inter-subject differences. The results from dried blood spots were no less reproducible than other lipid profiling methods which required comparatively larger sample volumes. Therefore, lipid profiles obtained from dried blood spots can be successfully used to monitor infancy lipid metabolism and we show significant differences in the lipid metabolism of infants at age 3 versus 12 months.     

Pre-cut Filter Paper for Detecting Anti-Japanese Encephalitis Virus IgM from Dried Cerebrospinal Fluid Spots

BackgroundThe use of filter paper as a simple, inexpensive tool for storage and transportation of blood, ‘Dried Blood Spots’ or Guthrie cards, for diagnostic assays is well-established. In contrast, there are a paucity of diagnostic evaluations of dried cerebrospinal fluid (CSF) spots. These have potential applications in low-resource settings, such as Laos, where laboratory facilities for central nervous system (CNS) diagnostics are only available in Vientiane. In Laos, a major cause of CNS infection is Japanese encephalitis virus (JEV). We aimed to develop a dried CSF spot protocol and to evaluate its diagnostic performance using the World Health Organisation recommended anti-JEV IgM antibody capture enzyme-linked immunosorbent assay (JEV MAC-ELISA).Conclusions/SignificanceThe novel design of pre-cut filter paper saturated with CSF could provide a useful tool for JEV diagnostics in settings with limited laboratory access. It has the potential to improve national JEV surveillance and inform vaccination policies. The saturation of filter paper has potential use in the wider context of pathogen detection, including dried spots for detecting other analytes in CSF, and other body fluids.     

Effect of storage temperature and length on fatty acid composition of fingertip blood collected on filter paper

We have compared the fatty acids of the capillary and venous whole blood samples collected on the commercially developed blood collection paper and standard grade filter paper extracted by either the direct methylation or conventional method (including various blood lipids fractions). Also, reproducibility of fatty acids extracted from dried blood on the filter paper after storing at room temperature up to 2 months and at 4°C up to 6 months was assessed. In conclusion, the direct methylation of fingertip blood collected on both brand of papers produced fatty acids that reflected venous blood fatty acids extracted by the conventional method. Of the eight fatty acids evaluated, capillary DHA showed the strongest correlation with DHA of the venous whole lipids as well as various lipid fractions of the plasma and red cells. However, a prolonged storage of blood samples at 4°C had deleterious effect on the qualitative value of fatty acids, especially DHA.     

Discovery stage pharmacokinetics using dried blood spots

Early in the discovery stage, the measurement of drug candidates in biological fluids as a function time provides important information used in decision making for lead optimization. The detection methodology primarily used is liquid chromatography coupled to triple quadrupole mass spectrometry (LC-MS). Sample preparation is an important aspect of these experiments and robotic-based automation is commonly used. The often overlooked aspect of these experiments is the sample collection itself. Typically, several hundred microliters of whole blood is collected and the plasma fraction separated for each time-point. The plasma is then transferred to an appropriate vessel for subsequent aliquoting and processing. We describe a method for performing discovery stage pharmacokinetic analysis using whole blood dried onto filter paper. The use of dried blood spots is a well established technique for neo-natal screening, and its application to early screening of drug candidates proves to be robust, reliable and reproducible.     

用干血纸片扩增人雄激素受体基因

雄激素不敏感综合征(AIS)为一类主要与雄激素受体(AR)基因缺陷密切相关的X-连锁隐性遗传病.为进一步阐明AIS的发病机制,建立了用干血纸片直接PCR扩增或将干血纸片中的血细胞洗脱裂解后进行PCR扩增雄激素受体(AR)基因的方法,结合已建立的SSCP分析及DNA直接测序等方法,可对AR基因进行突变分析.干血纸片取样及保存容易,便于邮寄,适用于外地,特别是边远地区患者的取样.该法不仅为AIS患者的AR基因突变分析和家系调查提供简便易行的方法,也适用于PCR基础上的其他各种基因的突变分析.     

Antibodies to bluetongue and epizootic hemorrhagic disease viruses from white-tailed deer blood samples dried on paper strips.

The feasibility of using dried blood samples for serologic testing of white-tailed deer (Odocoileus virginianus) for antibodies to bluetongue virus (BTV) and epizootic hemorrhagic disease virus (EHDV) was tested with matched samples of serum and eluted dried whole blood. Results from matched serum virus neutralization (SN) tests indicated that a 1-ml elution from a 1- x 2-cm section of filter paper strip containing dried blood approximated a 1:10 serum dilution. Neutralizing antibody titers detected from 34 matched titrations of serum and dried blood samples were equivalent in 25 (74%) titrations and were within a single dilution in the remaining nine (26%) titrations. Eluted blood samples from SN-positive deer, however, did not produce detectable precipitin lines on agar gel immunodiffusion tests for antibodies to either BTV or EHDV. In a trial using serum and dried blood samples from 108 hunter-killed deer from five locations in Georgia (USA), antibody prevalence and serotype distribution results were similar. Use of dried blood samples for serologic testing for antibodies to BTV and EHDV provides a reliable alternative to serum but should be considered only when serum collection is not feasible.     

A LAMP-SNP Assay Detecting C580Y Mutation in Pfkelch13 Gene from Clinically Dried Blood Spot Samples

Artemisinin resistance (ART) has been confirmed in Greater Mekong Sub-region countries. Currently, C580Y mutation on Pfkelch13 gene is known as the molecular marker for the detection of ART. Rapid and accurate detection of ART in field study is essential to guide malaria containment and elimination interventions. A simple method for collection of malaria-infected blood is to spot the blood on filter paper and is fast and easy for transportation and storage in the field study. This study aims to evaluate LAMP-SNP assay for C580Y mutation detection by introducing an extra mismatched nucleotide at the 3’ end of the FIP primer. The LAMP-SNP assay was performed in a water bath held at a temperature of 56°C for 45 min. LAMP-SNP products were interpreted by both gel-electrophoresis and HNB-visualized changes in color. The method was then tested with 120 P. falciparum DNA from dried blood spot samples. In comparing the LAMP-SNP assay results with those from DNA sequencing of the clinical samples, the 2 results fully agreed to detect C580Y. The sensitivity and specificity of the LAMP-SNP assay showed 100%. There were no cross-reactions with other Plasmodium species and other Pfkelch13 mutations. The LAMP-SNP assay performed in this study was rapid, reliable, and useful in detecting artemisinin resistance in the field study.     

Molecular genetic diagnosis of sickle cell disease using dried blood specimens on blotters used for newborn screening

Summary The protein-based technologies used to screen newborns for sickle cell disease require confirmation with a liquid blood specimen. We have developed a strategy for rapid and specific genotypic diagnosis using DNA extracted from a dried blood spot on the filter paper blotter used to screen newborns. DNA could be microextracted from a specimen as small as a 1/8 inch diameter punched disc representing the dried equivalent of approximately 3 μl of whole blood. We utilized the DNA from a 1/4 inch diameter specimen (12 μl equivalent) for polymerase chain reaction amplification of the β-globin region spanning the sickle cell mutation with detection by allele-specific oligonucleotide probes. Molecular confirmation of genotype from the original blotter would reduce the personnel costs associated with obtaining follow-up liquid blood specimens and would provide information to the family in a more timely and less equivocal manner.     

Application of an enzyme chip to the microquantification of l-phenylalanine

We describe here a new microquantification method of l-phenylalanine concentration in an extract from a dried blood spot by using the diaphorase-resazurin system. To miniaturize the fluorometric enzymatic microplate assay for the diagnosis of phenylketonuria, an enzyme chip immobilized with His-tag fused phenylalanine dehydrogenase (PheDH) was developed. His-tag fused PheDH was immobilized on the surface of nickel-coated slide glass. A microarray sheet (8 x 30 well) was fabricated with poly(dimethylsiloxane) (PDMS) using the photolithographic technique. An enzyme reaction chamber in a double-layered structure was constructed with different types of microarray PDMS sheets on the surface of Ni-coated slide glass immobilized with His-tagged PheDH. To evaluate the affinity toward the Ni-chelating ligand, eight kinds of His-tagged PheDH variants were constructed and expressed. (His)(6)- and (His)(9)-PheDH variants at the N terminus showed high adsorption ratio to Ni-chelating ligand. The V(max) and k(cat) values of the (His)(6)-PheDH variant at the N terminus for l-phenylalanine were higher than those of the (His)(9)-PheDH variant, and the (His)(6)-PheDH variant was found to be most suitable for immobilization onto nickel-coated slide glass. Fluorescence formed by resazurin-coupled enzymatic reaction (in a 0.2-microl reaction mixture) on the enzyme chip exhibited good linearity and a correlation coefficient up to 12.8 mg/dl of the l-phenylalanine-containing sample extracted from a dried blood spot on filter paper.     

Using dried blood spots stored on filter paper to measure cholinesterase activity in wild avian species.

Birds of prey that are poisoned by cholinesterase inhibitors (e.g. organophosphate and carbamate insecticides) are often cared for at animal shelters, rehabilitation centres and wildlife diagnostic facilities. Plasma cholinesterase (ChE) activity is a recognized method of assessing exposure to these insecticides, but standard blood-handling protocols are difficult to follow in non-laboratory settings. The primary objective of this study was to expand upon a method for storing human blood on filter paper without the need for complicated equipment or refrigeration, and to test its utility for measurement of ChE activity in avian blood. ChE activity from whole blood, plasma, and dried blood spots was analysed from 169 wild birds and comparisons made among sample types. ChE activity measured in whole blood haemolysates and dried blood spots were significantly correlated (r = 0.74, p < 0.001), as was ChE activity measured in plasma and dried blood spots (r = 0.68, p < 0.001). This study demonstrated that monitoring pesticide exposure in birds could be conducted using elementary blood sampling, preserving and shipping techniques.     

Preanalytical standardization of amino acid and acylcarnitine metabolite profiling in human blood using tandem mass spectrometry

Quantitative metabolite profiling in biological samples has the potential to reflect physiological status and to identify disease associated disturbances in metabolic networks. However, this approach is hampered by a wide range of preanalytical variables. Hence, the aim of our study was to develop a standardized preanalytical protocol for metabolite profiling of amino acids and acylcarnitines in human blood. Amino acids and acylcarnitines were simultaneous analyzed after butylation of 3 μL dried blood or 10 μL whole blood, serum and anticoagulated plasma using electrospray tandem-mass spectrometry. The influence of exogenous and endogenous preanalytical variables was investigated in healthy volunteers. Different sampling materials and anticoagulants for blood taking were investigated. Concentrations of long-chain acylcarnitines were 5-fold higher in EDTA-whole blood or dried whole blood compared to serum and anticoagulated plasma. Significant differences in amino acid concentrations were found for capillary versus venous blood taking. Fasting for 8 h before specimen collection minimized the nutritional influence. Physical activity significantly alters amino acid and short chain acylcarnitine concentrations. As a result of our preanalytical investigation we developed a pre-treatment protocol based on EDTA whole blood dried on filter paper to reduce the preanalytical variability and facilitate reproducible quantitative metabolite profiling in clinical trials.     

Dried blood spot liquid chromatography assay for therapeutic drug monitoring of metformin

The use of blood spot collection cards is a simple way to obtain specimens for analysis of drugs for the purpose of therapeutic drug monitoring, assessing adherence to medications and preventing toxicity in routine clinical setting. We describe the development and validation of a microanalytical technique for the determination of metformin from dried blood spots. The method is based on reversed phase high-performance liquid chromatography with ultraviolet detection. Drug recovery in the developed method was found to be more than 84%. The limits of detection and quantification were calculated to be to be 90 and 150 ng/ml, respectively. The intraday and interday precision (measured by CV%) was always less than 9%. The accuracy (measured by relative error, %) was always less than 12%. Stability analysis showed that metformin is stable for at least 2 months when stored at -70 degrees C. The small volume of blood required (10 microL), combined with the simplicity of the analytical technique makes this a useful procedure for monitoring metformin concentrations in routine clinical settings. The method is currently being applied to the analysis of blood spots taken from diabetic patients to assess adherence to medications and relationship between metformin level and metabolic control of diabetes.     

Identification of Hb D-Punjab gene: application of DNA amplification in the study of abnormal hemoglobins.

Hemoglobin D-Punjab (or D-Los Angeles) is a common variant worldwide. It is also the most frequent abnormal hemoglobin in Xinjiang Uygur Autonomous Region of China. A large survey of hemoglobinopathy, including 142,171 people and 21 national/ethnic groups, was carried out in Xinjiang and indicated Hb D-Punjab accounted for 55.6% of the total hemoglobin variants there. Here we describe a simple way--EcoRI mapping of the amplified beta-globin DNA sampling from dried blood spots on filter paper blotters--of identifying the Hb D-Punjab gene. The primers were designed and synthesized to emzymatically amplify a 144-bp fragment of beta-globin gene which included codons beta 121 (GAA) and 122 (TTC) representing an EcoRI recognition site. The Hb D-Punjab gene could be easily detected by EcoRI digestion of the amplified DNA sequence on agarose gel because of a single base change at codon 121. The analysis of amplified DNA sampling from dried blood provides a very useful method for population study of Hb D-Punjab and will be of significance for demonstration of the occurrence of the Hb D-Punjab gene and for understanding of the relations among various nationalities.     

Galactosaemia: estimated live birth incidence in New Zealand.

194 cord blood samples were studied in both a fluorimetric assay and an electrophoresis method for Gal-1-PUT. From this the expected live birth incidence was 1:37000. The New Zealand neonatal screening programme has detected 5 cases in 223326 live births--an apparent incidence of 1:44600. The Beutler testing of dried blood spots collected on filter paper cards is a satisfactory method of detecting galactosaemia in the neonate.     

Using dried blood spots stored on filter paper to measure cholinesterase activity in wild avian species

Birds of prey that are poisoned by cholinesterase inhibitors (e.g. organophosphate and carbamate insecticides) are often cared for at animal shelters, rehabilitation centres and wildlife diagnostic facilities. Plasma cholinesterase (ChE) activity is a recognized method of assessing exposure to these insecticides, but standard blood-handling protocols are difficult to follow in non-laboratory settings. The primary objective of this study was to expand upon a method for storing human blood on filter paper without the need for complicated equipment or refrigeration, and to test its utility for measurement of ChE activity in avian blood. ChE activity from whole blood, plasma, and dried blood spots was analysed from 169 wild birds and comparisons made among sample types. ChE activity measured in whole blood haemolysates and dried blood spots were significantly correlated (r=0.74, p<0.001), as was ChE activity measured in plasma and dried blood spots (r=0.68, p<0.001). This study demonstrated that monitoring pesticide exposure in birds could be conducted using elementary blood sampling, preserving and shipping techniques.     

Buccal DNA samples for DNA typing: new collection and processing methods

With the recent expansion of DNA database laws in many states, there is a critical need for the rapid and simple collection of DNA samples and streamlined processing for downstream applications. The Buccal DNA Collector was developed to address the need for a reliable, practical alternative to blood collection that is compatible with high-throughput operations. The collection area consists of filter paper that is placed against the inside of the cheek, and the sample is taken by swiping the cheek several times while pulling the device out of the mouth. Using this method, DNA profiles have been obtained from samples stored for 2 years at room temperature. Cells are collected on all regions of the filter paper with the maximum DNA recovery from the tip. The processing of DNA for DNA typing is accomplished with BodeElute, a new product that prepares DNA for amplification in a single 30-min heating step. Extracted DNA samples were successfully amplified with four commonly used multiplex short tandem repeat (STR) amplification kits. These products provide simplified approaches for collecting and processing buccal cell samples.