Filter Paper Cards Research Articles

Long-term Storage and Recovery of Buccal Cell DNA from Treated Cards

Economical methods for collecting and storing high-quality DNA are needed for large population-based molecular epidemiology studies. Buccal cell DNA collected via saliva and stored on treated filter paper cards could be an attractive method, but modest DNA yields and the potential for reduced recovery of DNA over time were unresolved impediments. Consequently, buccal cell DNA collection via oral mouthwash rinsing became the method of choice in epidemiologic studies. However, the amount of genomic DNA (gDNA) required for genotyping continues to decrease, and reliable whole genome amplification (WGA) methods further reduced the mass of gDNA needed for WGA to 10 ng, diminishing the obstacle of low DNA yields from cards. However, concerns about yield and DNA quality over time remained. We located and analyzed 42 buccal cell saliva samples collected and stored on treated cards for 7 years at room temperature, -20 degrees C, and -80 degrees C. We recovered DNA from the treated cards, estimated the concentration by a human-specific quantitative real-time PCR assay, and evaluated the quality by PCR amplification of 268-, 536-, and 989-bp fragments of the beta-globin gene and by AmpFlSTR Identifiler assay analysis. Most DNA yields per 3-mm punch were <10 ng, and most PCR amplicons failed to amplify, where size of the amplicon was negatively associated with successful amplification. Using these methods, treated cards did not consistently provide sufficient quantities of buccal cell gDNA after 7 years of storage for genotyping or WGA.

Triagem neonatal para hiperplasia adrenal congênita: experiência do estado do Rio de Janeiro

To determine the 17OH progesterone (17OHP) levels in the neonatal screening for Congenital Adrenal Hyperplasia due to 21 hydroxylase deficiency (CAH-21OHD). The evaluation was performed using 76,360 paper filter samples, obtained from newborn screening in Rio de Janeiro from June 1992 to December 2000. The 17OHP were assayed by fluoroimmunoassay method using the blood collected onto filter paper cards. The cut-off level was 10 ng/mL. The infants with 17OHP levels above this cut-off were recalled to undergo a new dosage. Thirty-eight patients presented 17OHP levels above the cut-off limit. In 11 (4 males, 6 females, 1 undefined gender) the diagnoses of CAH-21OHD were confirmed. Their 17OHP levels ranged from 25 to 254.5 ng/mL (mean: 133.84 ng/mL) on the first analysis and from 46.86 to 360 ng/mL (mean: 218.84 ng/mL) on the second analysis. The patients with the salt-wasting form showed higher 17OHP levels when compared to simple virilizers, both on the first analysis (mean: 169.21 ng/mL vs 27.46 ng/mL) and on the second one (mean: 227.16 ng/mL vs 110.95 ng/mL). Among the 27 infants without confirmed disease (false-positives), 17OHP levels ranged from 10.27 to 27.5 ng/mL (mean: 14.8 ng/mL) on the first sample and from 2.39 to 32.39 ng/mL (mean: 10.07 ng/mL) on the second. In this group, 8 children maintained borderline 17OHP levels during a variable period, but in 7 of them it was normalized before the first year of life. In the remaining case, who was asymptomatic after 8 years of follow-up, a cortrosin stimulation test was compatible with the non-classic form of the disease. These data confirm that 17OHP analysis was a reliable test to CAH-21OHD neonatal screening and was able to differentiate between normal infants and those with the classical form of CAH-21OHD.

Lack of correlation between fatty acid oxidation disorders and haemolysis, elevated liver enzymes, low platelets (HELLP) syndrome?

Fatty acid beta-oxidation defects comprise a heterogeneous group of disorders that may precipitate acute life threatening metabolic crises particularly during catabolic episodes. Several studies have demonstrated a possible association between fatty acid beta-oxidation defects, including long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency and severe pregnancy complications. However, the precise percentage of women with haemolysis, elevated liver enzymes, low platelets (HELLP) syndrome associated with foetal fatty acid beta-oxidation defects is not known. We carried out a multicentre retrospective study on 88 infants, born to women with HELLP syndrome. Acylcarnitine profiles from blood dried on filter paper cards were analysed by tandem mass spectrometry for the diagnosis of fatty acid beta-oxidation defects. In addition, we screened for the common long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency mutation using a standard restriction fragment length polymorphism polymerase chain reaction method. None of the infants studied carried the common long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency mutation. There was no evidence of fatty acid beta-oxidation defects, including long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency, as expected by unremarkable acylcarnitine profiles, while three infants with fatty acid beta-oxidation defects were diagnosed in the control group. Neither foetal long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency, including heterozygosity for the common long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency mutation, nor fatty acid beta-oxidation defects in general are a major risk factor for HELLP syndrome in Austria.

Filter paper cards contaminated with EMLA cream produce artefacts on acylcarnitine analysis

Electrospray ionization tandem mass spectrometry is a widely applied method for the analysis of acylcarnitines in blood samples spotted on filter paper cards (Guthrie cards). When the filter paper cards are contaminated by EMLA cream, highly intense signals at m/z 221 and 235 are detected under ESI-MS/MS conditions, monitoring for precursors of m/z 85. These signals correspond to the active ingredients prilocaine and lidocaine in EMLA and overlap with the signals from the isotopically labelled internal standards (2H3)propionyl carnitine and (2H3)butyrylcarnitine. This interference prevents the proper quantification of the two short-chain acylcarnitines when samples are analysed without derivatization.

Newborn screening for biotinidase deficiency in Brazil: biochemical and molecular characterizations.

Biotinidase deficiency is an inherited metabolic disorder characterized by neurological and cutaneous symptoms. Fortunately, it can be treated and the symptoms prevented by oral administration of the vitamin biotin. Using dried blood-soaked filter paper cards, biotinidase activity was determined in the sera of 225,136 newborns in Brazil. Mutation analysis performed on DNA from 21 babies with low serum biotinidase activity confirmed that 3 had profound biotinidase deficiency (less than 10% of mean normal sera biotinidase activity), 10 had partial biotinidase deficiency (10 to 30% of mean normal serum activity), 1 was homozygous for partial biotinidase deficiency, 4 were heterozygous for either profound or partial deficiency, and 3 were normal. Variability in serum enzyme activities and discrepancies with mutation analyses were probably due to inappropriate handling and storage of samples sent to the laboratory. Obtaining an appropriate control serum at the same time as that of the suspected child will undoubtedly decrease the false-positive rate (0.09%). Mutation analysis can be used to confirm the genotype of these children. The estimated incidence of biotinidase deficiency in Brazil is about 1 in 9,000, higher than in most other countries. Screening and treatment of biotinidase deficiency are effective and warranted. These results strongly suggest that biotinidase deficiency should be included in the newborn mass screening program of Brazil.

Dried-Serum Spot Assay for Folate

Recent interest in the folate status of diverse populations as a serious clinical and public health issue has been associated with substantial demands for blood folate analyses. Folate analysis by microbiological assays fulfills two important requirements for such screening by providing an acceptable detection limit and specificity at a low reagent cost. Dried blood spots (DBS) on special filter paper have been notably cost-effective as a blood sample collection device in epidemiologic field studies, allowing fingerstick peripheral blood sampling and ease of specimen transport and storage. We recently combined the advantages of a microbiological assay and DBS in an assay for erythrocyte folate (1). We now describe a dried-serum spot (DSS) assay for serum folate (SF). Filter-paper cards, each with 15 preprinted circles, were used (Type 903; Schleicher & Schuell). Individual fresh serum or EDTA-plasma samples from clinic patients or healthy donor volunteers were used throughout. DSS were prepared by pipetting 50–150 μL of serum onto the paper and drying the papers on a rack for at least 5 h at room temperature before storage at −80 °C in resealable plastic bags with desiccant sachets (1). Some paper was preimpregnated with ascorbic acid in an effort to extend DSS folate stability. These 15 × 10-cm paper cards were immersed flat in 10 mL of 10 g/L ascorbic acid for 1 min, followed by air-drying on sheets of aluminum foil. Whole DSS folates were analyzed after the entire serum spot (50 μL) was cut out using scissors. The spots were cut in half and placed in the base of a borosilicate tube (12 × 75 mm), covered with 2 mL of 5 g/L sodium ascorbate containing 0.5 mL/L Tween 80, mixed on a vortex-mixer, and sonicated for 15 min (Deacon Ultrasonics Ltd). The DSS eluate was considered to represent a 1:40 …

Stability of 17α-Hydroxyprogesterone in Dried Blood Spots after Autoclaving and Prolonged Storage

In the last few decades, management of patients with inborn errors of metabolism or congenital endocrine disorders has been greatly improved by the introduction of neonatal mass screening programs in many parts of the world. For this purpose, blood from newborns, ideally 4–7 days of age, is collected on filter-paper cards by means of a heel prick. A standard set of up to 20 tests is usually carried out at a centralized laboratory within a few days. Until recently, either when the tests were being performed or during the final step, the entire filter-paper card was autoclaved and then stored (1). Thus, leftover blood spots on filter paper from the entire young population in many countries are available in native or autoclaved form, although autoclaving has now become outdated because of a change in analytical methods (1). The stored cards provide a potentially valuable source for population studies or retrospective examinations. Documentation of the long-term stability of the analytes to be examined is a prerequisite for such studies. In many countries, the latest addition to the neonatal screening program is the examination for congenital adrenal hyperplasia (CAH), by measurement of 17α-hydroxyprogesterone (17-OHP) in a dried blood spot (2)(3)(4). The technique, first described by Pang et al. in 1977 (5), is based on a direct immunoassay and is used as the standard procedure in programs screening for CAH (2). In several trials, 17-OHP in dried blood spots on filter-paper cards was resistant to freezing, high ambient temperatures, humidity, and short- or intermediate-term (up to 1 year) storage (6)(7)(8)(9)(10). In the present study, we examined the impact of autoclaving (i.e., extremely high temperature, high humidity and pressure) and prolonged (more than a decade) storage under typical room conditions on the …

Analysis of guanidinoacetate and creatine by isotope dilution electrospray tandem mass spectrometry

Guanidinoacetate methyltransferase (GAMT) deficiency is a disorder of creatine metabolism characterized by low plasma creatine concentrations in combination with elevated guanidinoacetate (GAA) concentrations. Although rare, GAMT deficiency has been identified in children with seizures, extrapyramidal movements, developmental delay, myopathies and behavioral abnormalities. Treatment with creatine monohydrate has been proven to be effective. We describe an isotope dilution electrospray tandem mass spectrometry (ES-MS/MS) assay for the simultaneous determination of plasma GAA and creatine using multiple reaction monitoring (MRM), d 3-creatine as the internal standard and derivatization of GAA and creatine as butyl-esters. We analysed plasma of 16 healthy adults and 20 healthy children as well as three affected children. Plasma GAA concentrations were 5.02±1.84 μmol/l (mean±S.D.) in adults, 3.91±0.76 μmol/l in children age 5–10 years and 11.57, 15.16, 14.36 μmol/l in children with GAMT deficiency. Plasma creatine concentrations were 34.7±15.25 μmol/l in adults, 58.96±22.30 μmol/l in children and 5.37, 8.15, 403.5 μmol/l in two untreated children and one treated child with GAMT deficiency, respectively. GAA can also be reliably measured from filter cards, which is sufficient to make the correct diagnosis while creatine is consistently falsely elevated probably secondary to liberation of red cell creatine. In nine healthy newborn infants, GAA concentrations from filter cards were 4.83±1.43 and 5.04±1.84 μmol/l in 16 healthy adults. We conclude that isotope dilution ES-MS/MS is ideal for rapid high-throughput diagnosis of GAMT deficiency both from plasma and filter paper cards. Using this technique neonatal screening is feasible for this treatable inborn error of creatine metabolism.

Neonatal screening for congenital toxoplasmosis in the Poznań region of Poland by analysis of Toxoplasma gondii-specific IgM antibodies eluted from filter paper blood spots

The aims of the study were to determine the prevalence of congenital toxoplasmosis at birth in the Poznań region of Poland, the value of the serologic examination of filter paper blood specimens collected from newborns for the diagnosis of congenital Toxoplasma infection and the duration of anti-Toxoplasma-specific IgM antibodies in infants' sera. All neonates born in the maternity wards of the University Hospital of Gynaecology and Obstetrics in Poznań and in 10 selected obstetrics wards in the district hospitals were included. Blood samples were collected on filter paper cards, between the first and sixth day of life, screened for anti-Toxoplasma-specific IgM antibodies by an immunocapture enzyme-linked immunosorbent assay and if positive further analyzed for specific IgG and IgA antibodies. Between June, 1996, and October, 1998, filter paper samples from 27,516 liveborn infants were tested, which constituted approximately 75% of all births and 83% of liveborn neonates from the Poznań region. Anti-T. gondii-specific IgM antibodies were found in 13 newborns, equivalent to a prevalence of Toxoplasma-specific IgM in newborns of 1 per 2,117 liveborn children (0.47 per 1,000) or 1 per 870 children (1.15 per 1,000) born to seronegative women at risk of primary T. gondii infection during pregnancy. We identified two congenitally infected infants who were IgM-negative at birth, had a classic triad of clinical symptoms during the first year of life and had high levels of specific IgG. The birth prevalence of congenital toxoplasmosis in the Poznań region was at least 1 per 1,834 live births (0.55 per 1,000) or 1 per 754 live neonates born to seronegative women (1.33 per 1,000). The sensitivity of the IgM assay on eluate from filter paper was not more than 86.7%, and the mean duration of IgM detectable by enzyme-linked immunosorbent assay in serum samples was the first 4.8 weeks of life. In Poland the screening for congenital toxoplasmosis detecting one case per each 2,000 live births could be considered for inclusion in existing national neonatal screening programs for phenylketonuria and congenital hypothyroidism.

Automated tandem mass spectrometry for mass newborn screening for disorders in fatty acid, organic acid, and amino acid metabolism.

Development of acylcarnitine and amino acid profiling using tandem mass spectrometry, and its application for use with dried blood specimens collected on filter-paper cards, has introduced an innovative new technology for detecting inborn errors of fatty acid, organic acid, and amino acid metabolism. From November 1, 1992 through June 30, 1999 we screened more than 700,000 newborns in Pennsylvania, Ohio, North Carolina, and Louisiana. We have prospectively detected 163 inborn errors of metabolism. Eighty-six patients have amino acid metabolism errors. Among them are phenylketonuria, hyperphenylalaninemia, maple syrup urine disease, and several urea cycle disorders. Thirty-two have organic acid metabolism errors, including glutaric aciduria type 1; 3-methylcrotonyl coenzyme A (CoA) carboxylase deficiency, propionic acidemia, methylmalonic acidemia, and 3-hydroxy-3-methylglutaryl-CoA lyase deficiency; and 45 have fatty acid oxidation errors, including 36 with medium-chain acyl-CoA dehydrogenase deficiency. Details of the methodology are presented and the potential of this screening technology is discussed.

HFE gene mutation and transferrin saturation in very low birthweight infants

AIMTo determine if there is an association between high transferrin saturation and the C282Y HFE gene mutation in very low birthweight (VLBW) infants.METHODSOne hundred and forty three VLBW infants receiving...

Comparison of the effects of season and prematurity on the enzymatic newborn screening tests for galactosemia and biotinidase deficiency

In the Commonwealth of Virginia, 299,420 newborns were screened for biotinidase deficiency and galactosemia during the years 1989–1991 using enzymatic assays for galactose-l-phosphate uridyl transferase activity and biotinidase activity on dried blood filter paper cards. Retrospectively, false-positive initial samples for galactosemia or biotinidase deficiency were identified, and monthly frequencies of false-positive results were determined. The false-positive rates for galactosemia and biotinidase deficiency screening decreased over the 3-year period and were significantly higher in galactosemia screening for each year examined P < 0.001). For galactosemia screening, monthly false-positive rates were highest during May through September, seasonal differences in false-positive rates were significant for each year evaluated ( P < 0.001). and seasonal false-positive rates for the 3 years combined were highest during the summer and lowest during the winter. For biotinidase deficiency, differences in seasonal false-positive rates were significant in 1989 ( P<0.001) and 1991 ( P<0.01), and the highest seasonal false-positive rate for the 3 years combined occurred in the spring. Average monthly temperature and year of measurement were significant factors in galactosemia screening only ( P< 0.0001) and accounted for much of the variance in false-positive rates ( r 2 = 0.43). Approximately half of the infants with false-positive results for biotinidase deficiency were premature, significantly more than the incidence of prematurity in Virginia (7–8%) during the 3 years ( P<0.001). Prematurity in false-positive galactosemia screens was not significantly different from expected rates.

Galactose and galactose-1-phosphate spot test for galactosemia screening

A simple spot test to measure galactose and galactose-1-phosphate in bloodimpregnated filter paper was studied as a screening test for galactosemia in the newborn. A 3-mm disc punched from blood-impregnated filter paper card was fixed with acetone-methanol and incubated in a reaction mixture containing galactose dehydrogenase and alkaline phosphatase. This reaction mixture was then spotted on DEAE-cellulose paper, dried, and observed under a UV-lamp. The minimum amount of galactose detected by this procedure was 2 mg/dl. Estimation of galactose and galactose-1-phosphate with this procedure correlated well with estimation by bacterial assay.

Blood-spot thyrotropin radioimmunoassay in a screening program for congenital hypothyroidism.

We describe a highly sensitive and precise radioimmunoassay for thyrotropin in dried blood spots on filter paper cards. In a screening program for congenital hypothyroidism, blood-spot thyrotropin concentrations are measured in infants whose blood-spot thyroxine concentrations are in the lower 10%, and this strategy has reduced the recall rate from 1.7% (thyroxine assay alone) to 0.17%. Thyrotropin assay samples consist of discs 4.5-mm in diameter, containing about 6 microL of blood, punched from blood spots. By appropriate attention to assay conditions, a mean least-detectable thyrotropin concentration equivalent to 2.5 milliunits/L plasma has been achieved. Concomitant measurement of thyrotropin by plasma and blood-spot assays in 91 subjects yielded a Spearman rank correlation coefficient of 0.9732. An analysis of variance of the distribution volume of thyrotropin in blood spots and a covariance analysis of factors affecting blood-spot thyroxine results are presented.

A simple rapid gaschromatographic method for the determination of theophylline in dried whole blood on filter paper cards.

A simple rapid gaschromatographic method for the determination of theophylline in dried whole blood on filter paper cards.

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.