Dried Blood Samples Research Articles

Tandemmassenspektrometrie Beitrag zum Neugeborenenscreening auf angeborene Störungen des Stoffwechsels

Hintergrund. Die Tandemmassenspektrometrie wurde als neue Analysetechnik fur die Fruherkennung angeborener Stoffwechselstorungen im Neugeborenenscreening erprobt. Ergebnisse. Die Untersuchung von 128.225 Blutproben von Neugeborenen deckte 11-mal eine Phenylketonurie (PKU) und 3-mal eine Hyperphenylalaninamie sowie 4 Falle von Citrullinamie und 1 Tyrosinamie auf. Eine Fehlfunktion der Dehydrogenase der mittellangkettigen Fettsauren (MCAD-Defekt) erwies sich mit 17 Fallen als haufig. Bei 2 Kindern wurde eine Abbaustorung der langkettigen hydroxylierten Fettsauren (LCHAD-Defekt) gefunden, bei 1 weiteren ein Defekt des trifunktionalen Proteins. Unter den Organazidamien zeigten der 3-Methylcrotonyl-CoA-Karboxylasemangel und die Isovalerianazidamie mit je 4 nachgewiesenen Fallen eine unerwartet hohe Inzidenz. Die Quote der notwendigen Zweitanforderungen von Blut zur Kontrolle betrug 0,38%. Sie war damit niedriger als die bisher fur das PKU-Screening allein angegebene. Schlussfolgerung. Die Tandemmassenspektrometrie kann nach diesen Erfahrungen den praventiven Nutzen des Neugeborenenscreenings weiter erhohen.

Infection rates of Amblyomma americanum (Acari: Ixodidae) by Ehrlichia chaffeensis (Rickettsiales: Ehrlichieae) and prevalence of E. chaffeensis-reactive antibodies in white-tailed deer in southern Indiana, 1997.

To monitor the percentage and stability of Ehrlichia chaffeensis-infected ticks in southern Indiana over time, pools of Amblyomma americanum (L.) ticks were screened for infection in southern Indiana for a 2nd time. Nested polymerase chain reaction (with 6% DMSO included only in the 2nd reaction) was performed on 920 ticks in pools of 5 individuals from 9 sites (5 sites previously examined and 4 new ones) in 6 counties. The average minimum infection rate for all sites for 1997 was 1.6%, lower than that of 4.9% previously observed for 1995. However, when only the 5 sites that were positive for infected ticks in 1995 were reexamined, the average minimum infection rate was even more disparate (1.4% in 1997 and 5.1% in 1995). To correlate the presence of infected ticks with the presence of exposed deer, which serve as a reservoir, dried blood samples collected from hunter-killed deer at 2 locations in southern Indiana were tested for E. chaffeensis-reactive antibodies using an indirect immunofluorescent assay. Antibodies were detected in 45 and 47% of 98 samples examined from the 2 stations. These data provide support to our previous report of a population of E. chaffeensis-infected A. americanum in southern Indiana and the high proportion of deer previously exposed to E. chaffeensis suggests a stable maintenance of E. chaffeensis in this tick-vertebrate zoonotic system.

Home Monitoring of 17 Hydroxyprogesterone Levels in Congenital Adrenal Hyperplasia with Filter Paper Blood Samples

Abstract Objective: The purpose of this study was to evaluate the usefulness of 17 hydroxyprogesterone (17OHP) determination in dried filter paper blood samples from patients with congenital adrenal hyperplasia caused by 21-hydroxylase deficiency. It was hypothesized that these home samples would enhance patient treatment. Study design: Results of 17OHP determination in simultaneously collected venous and dried filter paper blood samples were compared to establish assay reliability. Thereafter, parents mailed dried filter paper blood samples collected before each hydrocortisone dose. Results: The 17OHP levels in wet and dried blood samples correlated well (r = 0.98). Results did not change when stored for 2 weeks under various conditions. Blood sampling at different times of the day provided insights into the patterns of 17OHP secretion and identified times of inadequate adrenal suppression. Dose adjustments were then made considering the time of day when adrenal suppression was inadequate. Conclusion: Home monitoring of 17OHP is a reliable and practical approach for assessing adrenal steroid activity in patients with congenital adrenal hyperplasia. Considering the time of day of 17OHP elevations also facilitates hydrocortisone dosing adjustment. (J Pediatr 1999;134:185-9)

Sensitivity of electrospray-tandem mass spectrometry using the phenylalanine/tyrosine-ratio for differential diagnosis of hyperphenylalaninemia in neonates

The sensitivity of amino acid analysis by electrospray-tandem mass spectrometry (ESI-TMS) in differential diagnosis of the different forms of hyperphenylalaniemias (HPA) was studied in a total number of 78 dried blood samples from neonates with increased phenylalanine (Phe) concentrations (>150 μmol/l). Simultaneous quantitative analysis of Phe and tyrosine (Tyr) were performed by ESI-TMS. By means of the Phe/Tyr-ratio it was possible to differentiate between phenylketonuria (PKU; n=23) and nonPKU-HPA patients ( n=35; P<0.001) with a sensitivity of 100%. Neonates with transient HPA ( n=9) showed a lower Phe/Tyr-ratio compared to the nonPKU-HPA patients ( P<0.001). Amino acid analysis by ESI-TMS represents a powerful diagnostic tool in differential diagnosis of HPA and is an appropriate technique in the setting of neonatal screening based on dried blood spots.

Electrospray Ionization Mass Spectrometric Analysis of Blood for Differentiation of Species

The National Fish and Wildlife Forensic Laboratory is responsible for the determination of species of birds, reptiles, and mammals from the United States, as well as international species falling under the protection of CITES treaties. We have recently found electrospray ionization mass spectrometry to be an effective means of rapidly analyzing blood samples for species identification. Nearly 1000 individuals were analyzed which comprised 62 species represented by birds, mammals, and reptiles. Whole blood and dried blood samples were analyzed without purification to provide simultaneous molecular weights from the α- and β-proteins present in each sample's hemoglobin. The combination of the two molecular weights for the hemoglobin proteins (i.e., α/β-pairs) was used as species determining markers. In all, 133 distinctive α/β-pairs were observed from the individuals analyzed. Despite the variability in the hemoglobins evaluated, 86% of these α/β-pairs were found to be diagnostic for a particular species to the exclusion of all other species studied. No other single protein system studied by a single analytical technique can so effectively resolve species from a wide range of taxa as can the hemoglobin system when analyzed by electrospray ionization mass spectrometry.

Rapid method for screening dried blood samples on filter paper for human immunodeficiency virus type 1 DNA.

PCR is a highly sensitive method for the detection of human immunodeficiency virus type 1 (HIV-1) nucleic acids in blood mononuclear cells and plasma. However, blood separation techniques require extensive laboratory support systems and are difficult when a limited volume of blood is available, which is often the case for infants. The use of blood samples stored on filter paper has many advantages for the detection of perinatal HIV-1 infection, but current methods require extraction and purification of target DNA prior to PCR amplification. We report a highly sensitive and rapid method for the extraction and detection of HIV-1 DNA in infant blood samples stored on filter papers. Because this rapid protocol does not involve steps for the removal of potential inhibitors of the PCR, the highest sensitivity is achieved by testing the filter paper lysate in quadruplicate. Assays for HIV-1 DNA were done by using nested PCR techniques that amplify HIV-1 gag DNA from blood spot samples on filter paper and from corresponding viably frozen mononuclear cells separated from venous blood samples obtained from 111 infants born to HIV-1-seropositive mothers. PCR results with blood from filter papers showed 100% specificity (95% confidence internal [CI] 93.1 to 100%) and 96% (95% CI, 88.65 to 98.9%) and 88% (95% CI, 79.2 to 94.5%) sensitivity (for quadruplicate and duplicate tests, respectively) compared to PCR results with blood mononuclear cells. Moreover, this method could detect HIV-1 sequences of multiple subtypes.

Home monitoring of 17 hydroxyprogesterone levels in congenital adrenal hyperplasia with filter paper blood samples

Objective: The purpose of this study was to evaluate the usefulness of 17 hydroxyprogesterone (17OHP) determination in dried filter paper blood samples from patients with congenital adrenal hyperplasia caused by 21-hydroxylase deficiency. It was hypothesized that these home samples would enhance patient treatment. Study design: Results of 17OHP determination in simultaneously collected venous and dried filter paper blood samples were compared to establish assay reliability. Thereafter, parents mailed dried filter paper blood samples collected before each hydrocortisone dose. Results: The 17OHP levels in wet and dried blood samples correlated well (r = 0.98). Results did not change when stored for 2 weeks under various conditions. Blood sampling at different times of the day provided insights into the patterns of 17OHP secretion and identified times of inadequate adrenal suppression. Dose adjustments were then made considering the time of day when adrenal suppression was inadequate. Conclusion: Home monitoring of 17OHP is a reliable and practical approach for assessing adrenal steroid activity in patients with congenital adrenal hyperplasia. Considering the time of day of 17OHP elevations also facilitates hydrocortisone dosing adjustment. (J Pediatr 1999;134:185-9)

PCR primer evaluation for the detection of trypanosome DNA in naturally infected goats

The buffy coat of 76 roaming goats from the Bansang and Missira regions in Gambia, was examined for the presence of trypanosomes. From these animals, extractions from dry blood samples on filter paper were subjected to PCR using three different primer sets, ORPHON5J, GOL and TVW, specific for Trypanosoma brucei/ Trypanosoma evansi, Trypanosoma congolense and Trypanosoma vivax, respectively. PCR results for T. congolense were 100% concordant with buffy coat examination. Besides the three T. vivax buffy coat-positive samples, another 15 yielded positive with the TVW primers. The ORPHON5J primers yielded no positive results. Analyses with the GOL primers of putatively negative samples, yielded aberrant band patterns whose diagnostic significance still remains to be determined.

Transient neonatal tyrosinemia: a frequent abnormality

OBJECTIVE: To evaluate the frequency of transient neonatal tyrosinemia, with or without secondary hyperphenylalaninemia,observed through neonatal screening for metabolic disorders, andthe need of monitoration and intervention with drugs and/or specialdiet in selected cases. METHODS: 457.870 dried blood samples obtained by heel stickfrom 3 to 20 days old babies were qualitatively evaluated by aminoacid thin-layer chromatography. Positive cases were quantitativelyconfirmed in serum samples by fluorimetric measurement of tyrosineand phenylalanine. RESULTS: 1.231 dried blood samples displayed positive resultsfor tyrosine in the cromatographic evaluation. The fluorimetricserum analysis disclosed normal levels of tyrosine and phenylalaninein 822 patients. The remained 409 patients showed hightyrosine levels and were placed in three groups according to thetyrosine concentration. In 118 of these cases serum phenylalaninewas also increased. CONCLUCIONS: Transient neonatal tyrosinemia is a very frequentdisorder in neonates (1/372); in some cases very high levels werefound, not only of tyrosine but also phenylalanine. As this findingis not yet accepted as absolutely harmless, the monitoration of thissituation and the use of measures to lower the tyrosine and phenylalaninelevels should be considered by the pediatrician.

An Assessment of Dried Blood-Spot Technology for Identifying Iron Deficiency

The present study was undertaken to assess the feasibility of using ferritin and transferrin receptor measurements on dried capillary blood spots to identify iron deficiency (ID) in public health surveys. Measurements on serum and blood spots prepared from venous blood were performed in 71 healthy subjects, 41 of whom were iron-replete and 30 who had ID, either without (n = 20) or with (n = 10) anemia. Parallel measurements were performed on hemolyzed whole blood and washed hemolyzed red blood cells to assess the erythrocyte contribution of ferritin and transferrin receptor to dried blood samples. The concentration of ferritin in dried blood samples was threefold higher than serum assays due to the release of ferritin from hemolyzed erythrocytes, which diminished the usefulness of ferritin measurements for detecting ID. On the other hand, there was negligible erythrocyte contribution to the measurement of transferrin receptor in dried blood spots. The most sensitive parameter in dried blood spots was the ratio of receptor/ferritin, which was suitable for identifying iron-deficiency anemia (IDA), but less reliable than serum assays for detecting milder ID without anemia. We conclude that tandem measurements of serum ferritin and transferrin receptor in dried blood spots can be used to facilitate the identification of IDA in epidemiologic studies.© 1998 by The American Society of Hematology.

An Assessment of Dried Blood-Spot Technology for Identifying Iron Deficiency

The present study was undertaken to assess the feasibility of using ferritin and transferrin receptor measurements on dried capillary blood spots to identify iron deficiency (ID) in public health surveys. Measurements on serum and blood spots prepared from venous blood were performed in 71 healthy subjects, 41 of whom were iron-replete and 30 who had ID, either without (n = 20) or with (n = 10) anemia. Parallel measurements were performed on hemolyzed whole blood and washed hemolyzed red blood cells to assess the erythrocyte contribution of ferritin and transferrin receptor to dried blood samples. The concentration of ferritin in dried blood samples was threefold higher than serum assays due to the release of ferritin from hemolyzed erythrocytes, which diminished the usefulness of ferritin measurements for detecting ID. On the other hand, there was negligible erythrocyte contribution to the measurement of transferrin receptor in dried blood spots. The most sensitive parameter in dried blood spots was the ratio of receptor/ferritin, which was suitable for identifying iron-deficiency anemia (IDA), but less reliable than serum assays for detecting milder ID without anemia. We conclude that tandem measurements of serum ferritin and transferrin receptor in dried blood spots can be used to facilitate the identification of IDA in epidemiologic studies.© 1998 by The American Society of Hematology.

Diagnostic evaluation of PCR on dried blood samples from goats experimentally infected with Trypanosoma brucei brucei

In seven goats experimentally infected with a pleomorphic clone of Trypanosoma brucei brucei, parasitaemia was monitored weekly for 6 weeks by wet blood film and microhaematocrit buffy coat examination. Dried blood samples on filter paper were concomitantly collected and tested by PCR using three different primer sets, putatively specific for Trypanozoon, T. vivax and T. congolense. With the originally designed ORPHON5J Trypanozoon primers, PCR tests became positive after 1 week (six animals) or 2 weeks (one animal) of infection and remained consistently positive until the end of the experiments, thus yielding an overall positivity rate of 97%, as compared with 74% for all parasitological tests together. The T. vivax and T. congolense primers yielded no positive PCR results.

Evaluation of Highly Sensitive Thyrotropin Assay for Detecting Thyroid Diseases in Neonatal Screening: Preliminary Studies.

TSH concentrations in dried blood samples on filter paper were determined by a conventional enzyme-linked immunosorbent assay (ELISA), used for routine neonatal screening for primary hypothyroidism, and a highly sensitive bioluminescence ELISA (BL-ELISA) using firefly luciferase to examine whether central hypothyroidism and hyperthyroidism can be efficiently detected as cases of primary hypothyroidism. Samples were obtained from 3 patients with congenital central hypothyroidism, 5 patients with congenital primary hypothyroidism, 6 patients with hyperthyroidism, 31 neonatal babies with low birth weight (premature babies) and 242 newborn babies with normal birth weight from the general population (normal babies). The TSH values were low in central hypothyroidism and hyperthyroidism. Their deviations from the mean TSH value for normal babies by the BL-ELISA method (-3.12 SD and -4.79 SD in central hypothyroidism and hyperthyroidism respectively) were greater than those by the ELISA method (-2.00 SD and -2.97 SD respectively). The TSH values were high in primary hypothyroidism and normal in premature babies while deviations were the same when BL-ELISA and ELISA were used. These findings indicate that the highly sensitive TSH assay (BL-ELISA) can be used for detecting both primary and central hypothyroidism as well as hyperthyroidism in neonatal screening.

Diagnostic evaluation of PCR in goats experimentally infected with Trypanosoma vivax

Six goats were experimentally infected with a stock of Trypanosoma vivax. Parasitaemia was weekly monitored by buffy coat and wet blood film examination during a period of 15 weeks and another 3 weeks following drug-treatment. Dried blood samples were tested by the polymerase chain reaction (PCR), using an extraction method with Chelex® 100 (BioRad). PCR proved consistently more sensitive than the parasitological techniques.

Rapid self testing for HIV infection

Home access testing for HIV infection has been recently approved by the Food and Drug Administration (FDA), USA.1 This type of testing entails sending a dried blood sample to a laboratory and receiving the results by telephone from a trained counsellor. The issues raised in the licensing of the home-access test were complex and contentious,2 but these issues are dwarfed by those of a true self test for HIV infection. A self test would enable people to find out their HIV status independently of another party and without professional involvement.

Biotinidase activity in patients with phenylketonuria

In patients with biotinidase deficiency, an autosomal recessive condition (McKusick 253260) (Wolf et al 1985), skin rashes, alopecia, seizures, hypotonia, developmental regression and ataxia occur usually after the age of 3 months. Additionally, seborrhoeic or atopic dermatitis (eczema) are common symptoms in untreated phenylketonuria patients (PKU) (McKusick 261600), and Burton et al (1975) have found that there is an abnormality in their sebaceous gland function. We therefore measured biotinidase activity in plasma of PKU children under dietary treatment . . . . . . Fifty-three patients with classic phenylketonuria were classified into two groups according to their annual mean plasma phenylalanine concentration (Phe mean). Group A (n--27), mean age 4.8 +3.5 years, had good compliance with the special diet; their Phe mean was 192+ll .5/nnol/L. Group B (n=26), mean age 7.9+3.7 years, did not adhere very well to this diet; their Phe mean was 597+ 187/maol/L. Group C (n=21), mean age 6.7+2.3 years, was the control group with Phe mean 109_+24/zmol/L. All PKU patients had been put on the special Phe-restricted diet during their neonatal period. Nine (38%) of the patients in group B suffered from seborrhoeic dermatitis or eczema. Blood (5ml) was withdrawn from each individual for biotinidase assay and liver function tests. Phe was measured with a quantitative enzymatic assay on dried blood samples on filter paper (2992 Schleicher-Schull) with Porton Cambridge reagents. Biotinidase activity was assayed with a fluorimetric method (Ebrahim and Dekisnamur 1986). At the time of blood collection for biotinidase assay, the mean phenylalanine concentration was 133+24/nnol/L in group A, which was statistically significantly different from that of group B at 473+127/zmol/L. Biotinidase activity in group A (5 .58+l .77nmol /min per L) was not significantly different from the controls (5.98+2.08nmol/min per L). However, the mean biotinidase activity in group B (4.21 + 1.68nmol/min per L) was significantly decreased (p--0.006) compared to that of group A as well as to that of controls (p<0.001). Biotinidase activity in group A did not correlate with mean phenylalanine concentration (r=0.08) nor that at the time of investigation (r=0.23). In contrast, in group B biotinidase activity correlated negatively with Phe mean ( r=-0 .59) and Phe concentration at the time of investigation (r---0.60). Vegetables and fruits, which are the main part of the PKU diet (group A), contain watersoluble forms of biotin. The PKU children in group B, who do not adhere well to the special diet, consume more animal products, in which biotin is in firmly bound complexes insoluble in water. However, biotin deficiency does not alter biotinidase activity in vitro: the activities in the sera of several patients who became biotin-deficient were normal (Wolf

PSA assay of dried blood samples from the ear lobe on a filter paper with special reference to prostatic mass screening

To eliminate complicated procedures for taking blood samples in the field work for prostatic mass screening, we developed a method for PSA testing using blood samples taken from the ear lobe and dried on a filter paper. The stability, the diagnostic efficacy, and the cost-benefit effect of the method were confirmed in hospitalized patients, as well as in subjects from our mass screening program.

A prenatal trisomy 21 screening program using α-fetoprotein, human chorionic gonadotropin, and free estriol assays on maternal dried blood

OBJECTIVE: The feasibility of large-scale Down syndrome maternal screening with dried blood samples and nonradioactive methods was examined. STUDY DESIGN: A prospective observation study was performed on a nonselected population of 11,241 pregnant women sampled between January 1991 and September 1992, between 14 and 24 weeks' gestation (ultrasonographic scanning available for 91.6%), through a multicenter collaborative network. Enzyme-linked immunosorbent assays for α-fetoprotein, human chorionic gonadotropin, and free estriol were performed on dried blood samples. Risk determination was made with an in-house software implementing the multivariate gaussian log likelihood method. RESULTS: A total of 10,450 samples were eligible for the study. Mean age at term was 27.9 years. A total of 6.84% of the patients were ≥35 years old with a prior risk of trisomy 21 > 1:350. The general positive rate of our sample ws 8.15%. After calculation 31.7% with prior risk >1:350 were still in the high-risk group; 6.36% of the low-risk group were found to be at high risk for Down syndrome. Fifteen trisomic pregnancies were observed, of which 11 had a calculated risk higher than the selected cutoff value (1:350). The overall detection rate was 73%, specificity was 92%, and positive predictive power was 1.2%. CONCLUSION: Our pilot study has shown performances within the range of conventional serum screening programs. Dried blood assays are a handy alternative to serum assays. Blot paper cards represent a simple method of sampling, well fitted for large population screening. Combined with nonradioactive methods, this method appears to be both low cost and effective. The current work apparently is the first large-scale Down screening program performed with dried blood.

Measurement of HbA1c Using dry Blood Sample Spotted on Filter Paper

Measurement of HbA<SUB>1c</SUB> Using dry Blood Sample Spotted on Filter Paper

12 HYPOTHYROIDISM NEONATAL SCREENING IN CORD BLOOD, EVALUATION OF 2 TSH ASSAYS

Cord blood neonatal screening for congenital hypothyroidism was carried out from October 1991 to December 1992 in Maternidad Ramón Sardá. It has been determined by TSH RIA. A total of 7365 dried blood samples from newborns were tested. Two hypothyroids (incidence 1:3682)and 2 transient hypothyroids(incidence 1: 3682) and 6 false positives (with TSH between 30 and 50 uU/ml) were detected. The recall rate was 0.14%versus 0.04%that is the rate we have in our screening after 48 hs of life. In order to decrease the recall rate in cord blood 76 samples from newborns were tested in parallel with RIA (DPC) and IRMA (ICN). The interassay coefficients of variation of RIA and IRMA were 20% and 5%, with a detection limit of 25 uU/ml and 2.5 uU/ml respectively. Distribution of TSH concentration (uU/ml) determined by RIA and IRMA was a follows:RIA results were lower than IRMA(p< 0.001)(Wilcoxon's test). No correlation was found between the 2 mothods (r:0.13 Spearman rank). We suggest that IRMA neonatal assay represents a true alternative to RIA and has better sensitivity and specificity for cord blood samples allowing to decrease false positive results.