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Background Non-cellulose dried matrix spotting (DMS) cards are an alternative to filter paper (FP) for bloodspots. We compared the interpatient distributions of bloodspot areas between DMS and FP for a fixed volume of application of whole blood, and examined correlations of areas with hematocrit. Methods EDTA-whole blood adult patient samples (n = 49; 25 males, 24 females) were utilized after routine measurement of hemoglobin and hematocrit. Replicate (4 ×) bloodspots were produced by bolus drop application of 50 μL whole blood via a fixed-volume pipettor to either FP or DMS. Dried bloodspot areas were determined by image analysis. Results Hematocrits (HCT) were normally distributed (HCT = 30.9 ± 5.3%). For both FP and DMS, bloodspot areas (a, cm2) across patients were normally distributed: for FP, a = 1.11 ± 0.056 cm2 (± 5.0%); for DMS, a = 0.378 ± 0.037 cm2 (± 9.9%). Relative bloodspot area differences across the population range were > 20% for both DMS and FP. Correlation of bloodspot areas to hematocrit was negative for FP (r = − 0.80) but positive for DMS (r = + 0.78). Conclusions Interpatient variation in blood volume per area is a preanalytical variable for both DMS and FP bloodspots. Hematocrit is but one interpatient variable, as correlations of fixed-volume bloodspot areas with hematocrit across patients were substantially inexact (r2

Creatine synthesis and transport disorders, Triple H syndrome and ornithine transcarbamylase deficiency are treatable inborn errors of metabolism. Early screening of patients was found to be beneficial. Mass spectrometry analysis of specific urinary biomarkers might lead to early detection and treatment in the neonatal period. We developed a high-throughput mass spectrometry methodology applicable to newborn screening using dried urine on filter paper for these aforementioned diseases.A high-throughput methodology was devised for the simultaneous analysis of creatine, guanidineacetic acid, orotic acid, uracil, creatinine and respective internal standards, using both positive and negative electrospray ionization modes, depending on the compound.The precision and accuracy varied by15%. Stability during storage at different temperatures was confirmed for three weeks. The limits of detection and quantification for each biomarker varied from 0.3 to 6.3 μmol/l and from 1.0 to 20.9 μmol/l, respectively. Analyses of urine specimens from affected patients revealed abnormal results. Targeted biomarkers in urine were detected in the first weeks of life.This rapid, simple and robust liquid chromatography/tandem mass spectrometry methodology is an efficient tool applicable to urine screening for inherited disorders by biochemical laboratories.

Background Mucopolysaccharidoses (MPSs) are a group of lysosomal storage diseases caused by the deficiency/absence of enzymes which catalyze the degradation of glycosaminoglycans (GAGs). The use of biological samples dried on filter paper has been increasing because it makes it easy to ship them to reference laboratories. Urinary GAGs are the main biomarkers of MPS and, thus, we studied the correlations of determinations to GAGs and creatinine, as well as compared the GAGs' profile on electrophoresis, between urine and dried urine in filter paper (DUFP) samples. We also assessed the GAG stability over time under different storage temperatures. Methods We quantified the GAG concentration in both sample types and compared the results by Pearson correlation. Results The results were very similar, with r = 0.97 for creatinine and with r = 0.94 and r = 0.98 for GAGs for controls and patients, respectively, with similar electrophoretic profiles. The GAG stability in DUFP was up to 30 days at − 20, 4, and 25 °C and up to 21 days at 37 °C. Conclusion Our proposal assessed urinary GAGs in DUFP and concluded that these samples can be used in the investigation of MPS, replacing urine samples in neonatal screening and monitoring of therapies, due to ease of transportation and storage.

Background Lysosomal storage diseases (LSD) are a group of genetic conditions which could present a vast spectrum of abnormalities that may include skeletal abnormalities, organ dysfunction, neuronal involvement, and tissue accumulation of complex molecules, among other manifestations. Definitive diagnosis of LSD is generally obtained by specific enzyme assays performed in leukocytes, fibroblasts, or more recently, dried-blood filter paper (DBFP) samples. Methods We recently introduced dried-leukocytes filter paper (DLFP) as an alternative source of enzyme to assay heparan sulfamidase and galactocerebrosidase activities, which could not be measured in DBFP samples using fluorometric methods. We present a new fluorometric methods on DLFP samples, for evaluation of α-glucosidase (GAA), β-glucosidase (GBA), and N-acetylgalactosamine-6-sulfatase (GALNS) activities, key enzyme assays for the identification of patients with Pompe disease (PD), Gaucher disease (GD), and Morquio A disease (MD), respectively. Results We show a clear discrimination between confirmed PD, GD, and MD patients and healthy controls. Conclusions We conclude that the assays of GAA, GBA, and GALNS on DLFP are reliable and useful methods for the identification of PD, GD, and MD diseases, respectively. As sample preparation is feasible in standard biochemical laboratories and transportation is very simple, it could enable patients living in remote areas to be investigated, diagnosed and eventually treated with the specific therapies available for these diseases.

Mucopolysaccharidoses are complex lysosomal storage disorders caused by any of eleven different enzyme deficiencies resulting in the accumulation of substrates, mainly glycosaminoglycans (GAGs), in various tissues and biological fluids.We developed and validated a urine filter paper methodology for the analysis of GAGs using liquid chromatography-tandem mass spectrometry (LC-MS/MS) for mucopolysaccharidoses type I, type II and type VI patients. We focused on 2 objectives: first, its applicability to high-risk screening, and secondly, to facilitate the collection and shipping of samples to reference centers as part of diagnostic investigation, as well as from treated patients needing to be monitored for assessment of the efficacy of treatment. GAGs in urine dried onto filter paper were extracted and subjected to methanolysis to obtain the repeating disaccharides of the molecules. We devised a multiple reaction monitoring method in positive electrospray ionization mode.The use of deuterated internal standards for dermatan sulfate (DS) and heparan sulfate (HS) reduced a troubling matrix effect. The resulting CVs were14%. Linearity assessment showed Pearson correlation coefficients of 0.999 and 0.997, for DS and HS, respectively. The stability on filter paper was good for DS and HS for up to 6 weeks at various temperatures.We devised a robust and efficient LC-MS/MS methodology for GAGS quantification in urine dried on filter paper and subjected to environmental conditions likely to be encountered during collection, storage and shipping of specimens from referring physicians to medical centers.

Background Fluorometric and tandem mass spectrometry assays can be used to measure lysosomal enzyme activities in dried blood spots (DBS). The effect of DBS preparation, storage and shipping was evaluated on the activities of acid α-glucosidase, acid α-galactosidase, acid β-glucocerebrosidase, acid sphingomyelinase, and galactocerebrosidase. Methods Whole blood from normal donors was used to prepare DBS following Clinical and Laboratory Standards Institute guidelines and by several deviations. Some DBS were subjected to various treatments, storage and shipping conditions. The activity of 5 lysosomal enzymes (GAA, GLA, GBA, ASM, and GALC) was measured using tandem mass spectrometric and fluorometric (GAA only) assays with 2 distinct and commonly used synthetic substrates. Results Enzyme activities were strongly affected by the way DBS were prepared and stored. Exposure of DBS to elevated heat and humidity can destroy enzyme functions rapidly. DBS prepared from poorly mixed blood caused significant variation on enzyme activities. EDTA, but not heparin, as an anti-coagulant gave more precise results. Conclusions The study confirmed the importance of proper and consistent DBS preparation and storage when screening for deficiencies of lysosomal enzymes.

Background Fabry disease is an X-linked lysosomal storage disorder due to deficiency of alpha galactosidase A (AGAL, EC 3.2.1.22). Despite increasing utilization of dried blood spot (DBS) as samples for AGAL enzyme assays, the effects of blood sample collection techniques on enzyme activity have not been studied. Methods DBS samples were prepared by spotting blood collected into an ethylenediaminetetraacetic acid (EDTA) tube and by direct application of blood from a finger prick or a venipuncture syringe. AGAL activity was measured quantitatively by detecting the fluorescence of 4-methylumbelliferone (4-MU) generated using the substrate 4-methylumbelliferyl-α- d -glucopyranoside (4-MUGal) in an acidic pH for 20 h. N-acetyl- d -galactosamine (GalNAc) was used to inhibit α-galactosidase B (EC 3.2.1.49). Results We studied 88 previously diagnosed Fabry disease patients and 690 healthy controls. Average AGAL activity in DBS samples prepared using EDTA tubes was higher compared to those spotted directly irrespective of disease status. Conclusions The study confirms the need for collection method-specific reference ranges using DBS samples.

Background Sample collection instructions for the bloodspot lead screening program conducted by the Nebraska Medical Center recommend continuous application of a single finger-stick blood drop per printed filter paper circle (a volume of approximately 50 μl). In this study, we assessed whether apparent blood volumes and geometries of finger-stick bloodspot samples submitted for lead testing were consistent with collection recommendations. Methods Samples were 422 extra bloodspots from 138 patients that were submitted for lead analysis. Using image analysis, apparent blood volumes were computed by comparison of bloodspot areas to bloodspot areas for standards of known volume. Circularity of samples was also assessed by image analysis. Results Mean blood volume (25 ± 13 μl) was approximately 50% of that needed to fill a printed circle. The distribution of volumes had three local maxima, consistent with bloodspot formation by multiple discrete applications of blood drops of small volumes (17 ± 6 μl) rather than by continuous application of blood. Multi-drop samples were also apparent from non-circular geometries. Conclusions Bloodspots submitted for lead analysis showed an apparently inherent drop volume of less than 20 μl per drop and the application of multiple drops. Non-ideality of such specimens indicates the need for continuing education of bloodspot collectors.

Background Lack of a second sample often precludes the ability to perform repeat lead measurements on filter paper bloodspot samples. We investigated whether annular specimens remaining from once-punched filter paper bloodspot specimens could provide accurate Pb measurements when measurements were scaled for the remainder area relative to the original punch area. Methods 50 μl bloodspot specimens were prepared using Pb-spiked EDTA whole blood. After removal of 6 mm punches, bitmap images of the remainder specimens were obtained using a scanner. Image analysis was used to determine the bloodspot area of the remainder sample relative to the area of the original punch. Measurement of Pb for punches and for remainder specimens was performed by ICP-MS. Results Area-corrected Pb measurements for remainder samples were significantly higher than for the punches, by an average factor of 1.52 ± 0.12 ( p n = 28). The difference was due to a discontinuity (an increase) in the per-area Pb at the bloodspot perimeter. Area-corrected results for annular specimens that excluded the perimeter were identical to those of the punch. Conclusion Area-corrected Pb measurement using annular once-punched bloodspot remainder specimens can accurately reproduce lead measurements obtained from the original punch when the bloodspot perimeter area is excluded.

Background Diagnoses of inherited lysosomal storage diseases are based on specific enzymatic assays performed on plasma, leukocytes, fibroblasts, and lately, dried-blood filter paper samples. We evaluated feasibility of detecting of patients with several inherited lysosomal storage diseases using dried-blood filter paper samples for appropriate enzyme assays. Methods Fluorometric methods were used to evaluate the activities of arylsulfatase B, α- N -acetylglucosaminidase, chitotriosidase, α and β-galactosidases, β-glucosidase, β-glucuronidase, total hexosaminidases, hexosaminidase A, α-iduronidase, and iduronate-2-sulfatase. A radiometric method was used for sphyngomyelinase determination. Single 3.0-mm diameter disks containing dried-blood samples were incubated at 37 °C with appropriate dilution buffers and artificial substrates, and the fluorescence or radioactivity was measured. Results Our results showed a statistically significant difference of the enzyme activity between affected individuals and controls, in all the assays performed. In contrast, we have not obtained a complete differentiation between heterozygotes and controls with these assays. Conclusions Enzyme assay on dried-blood filter paper is a suitable method to screen for several lysosomal storage diseases. Despite the low individual incidence of these pathologies, the incorporation of individual enzyme assays in neonatal screening programs could be justified to screen for diseases with relatively high local frequency and therapeutic measures available.

Background The mucopolysaccharidoses (MPS) are inherited metabolic disorders with bone, joint, and visceral abnormalities, leading to multi-organ dysfunction and, sometimes, neurological manifestations. These diseases are caused by storage of glycosaminoglycans (GAGs) and other complex molecules in tissues, among other pathogenic mechanisms. Definitive diagnosis of the affected individual is mainly based on the identification of the specific enzyme deficiency. New therapies are available or are in development for these pathologies, and early diagnosis seems to be important for the therapy outcomes. Almost all MPS patients have increased levels of GAGs in urine being their evaluation usually the first step in the screening of these conditions. Test on urine may be challenging as transportation of liquid urine samples in appropriate conditions for long distances, especially across international borders, could be difficult. Methods With the aim of overcoming the difficulties related to the use of liquid samples, we extended and validated previous studies about colorimetric determination of GAGs in dried-urine filter paper (DUFP) samples. Results In the conditions we described, there are no differences in the concentration of GAGs between urine and DUFP samples. Untreated patients with MPS and normal controls were well discriminated using any of the samples. Conclusions Dried-urine filter paper is a suitable sample for the colorimetric quantitation of GAGs, and that its incorporation as an additional tool for screening of MPS should be considered by reference laboratories.

Residual dried blood spots (DBSs) on filter paper from neonatal screening have been proposed as samples for population survey of lead contamination. We have investigated the EDTA effect on lead release in the eluting solution.Furnace atomic absorption spectrophotometry has been used for lead measurements. Standard, blank and sample solutions contained 2% m/v NH(4)H(2)PO(4), 0.5% v/v Triton X-100 and 0.2% v/v HNO(3) as matrix modifier solution (MMS) with or without EDTA. A calibration curve was established from aqueous standard solutions. Paper discs from DBS and blank, punched near the DBS, were eluted in MM solution and, where required, EDTA at different concentrations. Specimens were leftover DBSs with different storage times, matched samples from 20 adult patients consisting of liquid whole blood (LWB) containing 5 mmol/L EDTA, DBSs eluted in MM solution with 5 mmol/L EDTA or without EDTA.Optimal lead recovery from DBS required 5 mM EDTA in the eluting solution. Mean lead levels of LWB and DBSs eluted with EDTA were similar and higher than DBSs without EDTA (P0.001). Without EDTA, the median value of lead optical density was lower for 6-month-old DBSs than for blanks (P0.001).Residual DBSs can be used for population survey, but 5 mmol/L EDTA in the extracting solution is required to fully recover lead.

Background: Glycogen storage disease II is characterized by a deficiency of the lysosomal enzyme acid α-glucosidase. Currently, glycogen storage disease II is diagnosed by demonstrating the virtual absence or a marked reduction of acid α-glucosidase activity in muscle biopsies, cultured fibroblasts, or purified lymphocytes. Early diagnosis and treatment of glycogen storage disease II are considered to be critical for maximum efficacy of the enzyme replacement therapies that are in development. However, these existing diagnostic methods are not suited for newborn screening. We developed an assay useful for newborn screening for glycogen storage disease II. Methods: A series of three enzyme assays to measure the α-glucosidase activities in dried blood spots on filter paper was developed. The measurement of acid α-glucosidase activity with minimal interference by other α-glucosidases was accomplished using maltose as an inhibitor. The method was used on samples from glycogen storage disease II patients, obligate heterozygotes, and healthy controls. Results: Glycogen storage disease II patients were distinguished from carriers and healthy controls using the series of enzyme assays. Conclusions: We developed a simple and noninvasive screening method for glycogen storage disease II. The method could be incorporated into newborn screening.

Isothermal and isocratic HPLC chromatography for rapid quantitation of isoleucine and leucine (merged peak), tyrosine and phenylalanine from a single dried blood filter paper specimen ( 1 8“ dot blot) is described. The net chromatography time of the relevant peaks is less than 3 min. The results can be used to screen for PKU, MSUD, and tyrosinemia in one single assay. The advantage for early determination of PKU by simultaneous quantitation of both phenylalanine and tyrosine with the determination of their ratio is discussed. Using a dual column system this procedure might be suitable for large-scale newborn screening.

A method is described which enables the quantitative determination of both free and total carnitine levels in dried blood spots. This method is suitable for neonatal screening for either primary or secondary carnitine deficiency. The 95% confidence interval for free carnitine was 26–76 μmol/l (median = 44) and for total carnitine was 35–102 μmol/l (median = 60).

A technique designed to measure quantitatively succinylacetone (4,6-dioxoheptanoic acid) is presented. It essentially involves the inhibition of delta-aminolevulinate dehydratase (EC 4.2.1.24) by succinylacetone. Prior to their use in the assay, the samples are heated at 100 degrees C for 30 min in order to transform all succinylacetoacetate (3,5-dioxooctanedioic acid) to succinylacetone. By this transformation of the first abnormal metabolite specific to hereditary tyrosinemia to the second and last one, which is a powerful inhibitor of delta-aminolevulinate dehydratase, we determine in one sensitive assay the total amount of both. Succinylacetone was measured in sera and urines from 19 patients with hereditary tyrosinemia. All sera and urines contained succinylacetone at concentrations ranging, respectively, from 2 to 100 mumol/l and from 190 to 6000 mumol/g creatinine. The technique was also adapted to dried blood spots on paper and was used as a test complementary to blood tyrosine determination in mass screening for hereditary tyrosinemia. A total of 2412 samples having concentrations of 60 mg/l or more of tyrosine were assayed, and ten showed the presence of succinylacetone. These were all from newborns with hereditary tyrosinemia. The test has proven to virtually eliminate false positives, and, thereby, much clerical work and parental anxiety.

An immunoradiometric two-site assay for thyrotrophin (TSH) in dried blood filter paper spots is described. The assay is automated by means of the Kemtek 3000 automated immunoassay system. The technique uses a 6.0 mm disc punched from the dried blood samples collected as part of the screening programme for phenylketonuria. The method is sensitive and precise, and results correlate well with those obtained in TSH assays of serum samples. The procedure is rapid, results being available within 24 h of receipt of samples. Of 25204 specimens so far screened by this assay, 99.9% have TSH levels less than 15 mU/1. One false positive result has been obtained and six confirmed cases of neonatal hypothyroidism detected, giving a prevalence of 1 in 4200.

A simple method for quantifying lysozyme in serum and urine has been established by modifying the procedures of sample application in the lysoplate method reported by Osserman and Lawlor [1]. A strip of filter paper is partially immersed in a liquid sample, dried at room temperature and cut into discs which are later placed on the surface of an agarose gel containing Micrococcus lysodeikticus. The following procedures for determination are carried out as described in the lysoplate method. There was no statistically significant loss of enzyme activity during the sample preparation. Lysozyme dried on filter paper is so stable that it can be stored at room temperature for at least 13 weeks and can be mailed. The sensitivity of the method is increased by pretreatment of the filter paper with Triton X-100 and consequently corresponds to that of the lysoplate method. The reproducibility of our method is practically good since the variation coefficient of the diameters of lytic zones within assays is around 1%. There is a very close correlation between lysozyme levels determined by this method and by the lysoplate method with serum samples obtained from patients with various hematological diseases (r = + 0.994). The method can be utilized for routine clinical determinations of lysozyme.

Blood spots on filter paper are used to screen for phenylketonuria. We have developed a high pressure liquid Chromatographic assay of phenylalanine and tyrosine after elution of these amino acids from filter paper. The analysis time is 20 min. The amino acids are separated using an ion-exchange resin with post-column ortho-phthalaldehyde derivatization and subsequent fluorescence detection. Other common amino acids do not interfere. The assay is linear from 20 pmol to at least 2000 pmol with a coefficient of variation of 3%. The assay is as accurate as the determination of phenylalanine by ion-exchange ninhydrin-reactive methods currently in use, but is 100 times more sensitive. The method has the advantages of avoiding venipuncture, is cost effective, has a high sensitivity, and a rapid turnaround.

An evaluation was made of 8 atomic absorption systems for the determination of lead in capillary blood in order to determine their suitability for the routine analysis of 100 specimens per day. The precision of results obtained with the Delves cup or tantalum ribbon was better than that with the graphite crucible. Using the Delves cup, results with the filter paper disc technique were higher and less precise than those with liquid blood samples. Other systematic differences in accuracy between methods were found which could not be related to either instrumental configuration or sample form.

We have described the protein, enzyme, and isoenzyme characteristics of three gross fractions of both normal and abnormal human sera, as obtained by application of high centrifugal force. In the absence of solution gradient, such fractions represent pure and undiluted elements of the serum; they are reproducible and sufficiently voluminous for extensive analytical study. Specific features of these fractions may be of interest in planning additional or peripheral investigations of serum. Fraction I appears to be a fair sample of native serum water; Fraction II appears to segregate the pre-β-globulin lipoprotein component observed in some sera; Fraction III is a concentrated solution of LDH, CPK, and SGOT enzymes, and γ-globulin-rich protein, which is hpoprotein-free. More than 95% of the LDH, CPK, and SGOT is located in Fractions II and III. Quantitative activity and qualitative nature of these enzymes appears to survive the stress of the ultracentrifuge technique relatively well.

The electrophoresis and immunodiffusion steps for an open, atmospheric-un-equilibrated system in dilute agar gel on vegetable parchment paper has been described. Studies were carried out on resolution and precipitin identification. Figures showing the attempt to identify individual components of serum were presented.

A method is described for rapid analysis of phenylalanine and tyrosine using eluates of whole blood that has been impregnated into filter paper. This method employs semi-automated ion-exchange column chromatography (amino acid analyzer) and will yield reasonably accurate and consistent quantitative values for phenylalanine and tyrosine concentrations within two hours. The technique has significant value in rapidly confirming the presence of increased phenylalanine with or without a concomitant increase in tyrosine concentration in autoclaved or unautoclaved blood filter paper specimens. Thus phenylketonuria can be differentiated from hyperphenylalaninemia due to tyrosinemia within hours after the initial identification of an elevated phenylalanine concentration by a screening method such as the Guthrie test. In addition, further evaluations of phenylalanine and tyrosine concentrations can be facilitated in infants on whom only blood filter paper specimens are obtainable.

The effect of Millipore filtration on four methods of bilirubin determination in amniotic fluid was investigated. Millipore filtration is unacceptable as a method of amniotic fluid clarification when the methods of Ovenstone and Connon6 or Fleming and Woolf5 are used. Results obtained from the methods of Liley1 and Freda2 were not much affected by this type of filtration.

Serum α1-antitrypsin, α1-acid glycoprotein, baptoglobin, ceruloplasmin and C-reactive protein are increased, albumin, transferrin, α1-and β-lipoprotein are decreased as an early response to tissue trauma and inflammatory disease. In infection these changes are followed by a rise of γ-globulins.

A method is described for the preparation of linearising cam. It is based upon the mathematical treatment of the response obtained from an initial scan using the stepwedge prepared from human serum protein on rules, siliconised paper. The form of the equation connecting protein concentration on the paper strip to the reflected light complied with the requirements of a rectangular hyperbola.

Bisalbuminaemia in two members of a Kashmiri family is reported. This, the first Asian variant, is described in terms of its electrophoretic mobilities and dye-binding properties.

A two-dimensional electrophoretic technique has been described, based on the different mobilities of haemoglobin on paper at alkaline pH and on agar at acid pH. The method is very sensitive for the detection of small or minute amounts of haemoglobin A in the presence of large amounts of haemoglobin F, and for the detection and closer characterization of other minor components of human haemoglobin solutions.

A method for lipoprotein electrophoresis on cellulose acetate is described. Features which distinguish this method are: (1) It affords complete resolution of chylomicrons, β-lipoproteins, pre-β-lipoproteins, and α-lipoproteins into distinct bands and allows the relative magnitude of each to be estimated by transmission microden-sitometry. (2) Lipoproteins can be eluted quantitatively from separated bands for analyses. (3) Intact chylomicrons can be recovered by ultrasonic treatment. (4) The procedure is rapid, reproducible, and does not require addition of albumin to the buffer.

Electrophoresis of sugars is proposed for the identification of unknown melliturias in clinical laboratories. The sugars which we encountered most frequently in non-diabetic urines were lactose, galactose, glucose, arabinose and xylose. Glucosuria, galactosuria, lactosuria, fiuctosuria and pentosurias can be easily identified within a few hours. No purification nor concentration of urine is necessary; salts and organic constituents of urine do not inhibit the migration of sugars.

The separation of a mixture of quinine, strychnine and nicotine when added to blood is described. The procedure involves protein precipitation by hot tungstic acid and extraction of the alkaloids with ether. Ascending chromatography, which takes only 14 minutes, on cellulose citrate ion-exchange paper at pH 4.5 is used to separate the alkaloids from a chloroform solution of the dried ether extract. The alkaloids, which are clearly separated from each other and from basic compounds present in normal blood are detected by examination in ultraviolet light.