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In 2019, investigations have found at least 40 blood bags, 5 cross-country skiers, 5 cyclists, and 1 physician involved in a highly sophisticated blood doping scandal (1). The findings highlight the need for improved detection of blood doping practices. Athletes use blood doping methods to increase hemoglobin mass and, thus, oxygen delivery to muscle cells to obtain an unfair advantage. Hemoglobin mass can be increased by a variety of methods, including blood transfusions (autologous or allogeneic), recombinant human erythropoietin preparations (EPO)2, or by a growing number of erythropoiesis-stimulating agents (2). Often the methods are combined or used in very small doses, called microdoses, to evade detection. Because of the wide variety of blood doping methods, an indirect method capable of detecting all forms of blood doping was developed. The method measures 2 blood parameters—hemoglobin concentration [Hb] and reticulocyte percentage (Ret%)—and the OFF score, which is defined as [Hb] −60 × √Ret% (3). Any method that stimulates erythropoiesis will cause a significant increase in immature red blood cells, called reticulocytes, as well as an increase in [Hb]. Blood withdrawal, before autologous transfusion, also stimulates erythropoiesis and will increase Ret%. Additionally, negative feedback mechanisms cause a prolonged suppression of Ret% after blood transfusion or upon cessation of EPO injections, which can last for up to 3 weeks (4–6). In 2009, the World Anti-Doping Agency (WADA) implemented longitudinal measurement of the 2 blood parameters as part of the hematological module of the athlete biological passport (ABP) program. In the ABP program, an athlete's blood parameters are measured by any antidoping laboratory and entered into a database that contains the previous blood data for that athlete. The ABP program uses individual baseline variation with a Bayesian statistical model to predict normal limits for each parameter with a 99.9% probability threshold …

Background Accurate discovery assay workflows are critical for identifying authentic circulating protein biomarkers in diverse blood matrices. Maximizing the commonalities in the proteomic workflows between different biofluids simplifies the approach and increases the likelihood for reproducibility. We developed a workflow that can accommodate 3 blood-based proteomes: naive plasma, depleted plasma and dried blood. Methods Optimal conditions for sample preparation and data independent acquisition-mass spectrometry analysis were established in plasma then automated for depleted plasma and dried blood. The mass spectrometry workflow was modified to facilitate sensitive high-throughput analysis or deeper profiling with mid-throughput analysis. Analytical performance was evaluated by the linear response of peptides and proteins to a 6- or 7-point dilution curve and the reproducibility of the relative peptide and protein intensity for 5 digestion replicates per day on 3 different days for each biofluid. Results Using the high-throughput workflow, 74% (plasma), 93% (depleted), and 87% (dried blood) displayed an inter-day CV Conclusion The standardized workflows established here allows for reproducible and quantifiable detection of proteins covering a broad dynamic range. We envisage that implementation of this standard workflow should simplify discovery approaches and facilitate the translation of candidate markers into clinical use.

Background Phlebotomy plays a key role in clinical laboratory medicine but poses certain challenges for the patient and the laboratory. Dried blood spots simplify collection and stabilize specimens effectively, but clinical reference intervals are based primarily on serum or plasma. We evaluated use of dried separated blood plasma specimens to simplify plasma sample collection via finger stick; however, this sampling technique posed substantial analytical challenges. We discuss herein our efforts to overcome these challenges and provide accurate and precise clinical measurements. Methods Microsamples of whole blood were collected via finger stick using a collection device employing laminar-flow separation of cellular blood and plasma fractions with subsequent desiccation. Samples were analyzed on modern autoanalyzers with FDA-approved reagent and calibration systems, as well as commercially available reagents with laboratory-developed assay parameters. Measured analyte concentrations from extracted dried plasma samples were normalized to a coextracted endogenous analyte, chloride. Results Chloride normalization reduced variability incurred through extraction and undefined plasma volume. Excellent correlation of normalized measurements from dried finger-stick samples (whole blood and plasma) versus matched venous samples facilitated developing mathematical transformations to provide concordance between specimen types. Independent end-to-end performance verification yielded mean biases Conclusion Challenges inherent with this microsampling technique and alternate sample matrix were obviated through capabilities of modern autoanalyzers and implementation of chloride normalization. These results demonstrate that self-collected microsamples from a finger stick can give results concordant with those of venous samples.

BACKGROUND Advancements in the quality and availability of highly sensitive analytical instrumentation and methodologies have led to increased interest in the use of microsamples. Among microsamples, dried blood spots (DBS) are the most well-known. Although there have been a variety of review papers published on DBS, there has been no attempt at describing the full range of analytes measurable in DBS, or any systematic approach published for characterizing the strengths and weaknesses associated with adoption of DBS analyses. CONTENT A scoping review of reviews methodology was used for characterizing the state of the science in DBS. We identified 2018 analytes measured in DBS and found every common analytic method applied to traditional liquid samples had been applied to DBS samples. Analytes covered a broad range of biomarkers that included genes, transcripts, proteins, and metabolites. Strengths of DBS enable its application in most clinical and laboratory settings, and the removal of phlebotomy and the need for refrigeration have expanded biosampling to hard-to-reach and vulnerable populations. Weaknesses may limit adoption in the near term because DBS is a nontraditional sample often requiring conversion of measurements to plasma or serum values. Opportunities presented by novel methodologies may obviate many of the current limitations, but threats around the ethical use of residual samples must be considered by potential adopters. SUMMARY DBS provide a wide range of potential applications that extend beyond the reach of traditional samples. Current limitations are serious but not intractable. Technological advancements will likely continue to minimize constraints around DBS adoption.

BACKGROUND Different work flows have been proposed to use miRNAs as blood-borne biomarkers. In particular, the method used for collecting blood from patients can considerably influence the diagnostic results. METHODS We explored whether dried blood spots (DBSs) facilitate stable miRNA measurements and compared its technical stability with biological variability. First, we tested the stability of DBS samples by generating from 1 person 18 whole-genome-wide miRNA profiles of DBS samples that were exposed to different temperature and humidity conditions. Second, we investigated technical reproducibility by performing 7 replicates of DBS again from 1 person. Third, we investigated DBS samples from 53 patients with lung cancer undergoing different therapies. Across these 3 stages, 108 genome-wide miRNA profiles from DBS were generated and evaluated biostatistically. RESULTS In the stability analysis, we observed that temperature and humidity had an overall limited influence on the miRNomes (average correlation between the different conditions of 0.993). Usage of a silica gel slightly diminished DBS' technical reproducibility. The 7 technical replicates had an average correlation of 0.996. The correlation with whole-blood PAXGene miRNomes of the same individual was remarkable (correlation of 0.88). Finally, evaluation of the samples from the 53 patients with lung cancer exposed to different therapies showed that the biological variations exceeded the technical variability significantly (P < 0.0001), yielding 51 dysregulated miRNAs. CONCLUSIONS We present a stable work flow for profiling of whole miRNomes on the basis of samples collected from DBS. Biological variations exceeded technical variations significantly. DBS-based miRNA profiles will potentially further the translational character of miRNA biomarker studies.

BACKGROUND We expanded the use of tandem mass spectrometry combined with liquid chromatography (LC-MS/MS) for multiplex newborn screening of seven lysosomal enzymes in dried blood spots (DBS). The new assays are for enzymes responsible for the mucopolysaccharidoses (MPS-I, -II, -IIIB, -IVA, -VI, and -VII) and type 2 neuronal ceroid lipofuscinosis (LINCL). METHODS New substrates were prepared and characterized for tripeptidyl peptidase 1 (TPP1), α-N-acetylglucosaminidase (NAGLU), and lysosomal β-glucuronidase (GUSB). These assays were combined with previously developed assays to provide a multiplex LC-MS/MS assay of 7 lysosomal storage diseases. Multiple reaction monitoring of ion dissociations for enzyme products and deuterium-labeled internal standards was used to quantify the enzyme activities. RESULTS Deidentified DBS samples from 62 nonaffected newborns were analyzed to simultaneously determine (run time 2 min per DBS) the activities of TPP1, NAGLU, and GUSB, along with those for α-iduronidase (IDUA), iduronate-2-sulfatase (I2S), N-acetylgalactosamine-6-sulfatase (GALNS), and N-acetylgalactosamine-4-sulfatase (ARSB). The activities measured in the 7-plex format showed assay response-to-blank-activity ratios (analytical ranges) of 102–909 that clearly separated healthy infants from affected children. CONCLUSIONS The new multiplex assay provides a robust comprehensive newborn screening assay for the mucopolysaccharidoses. The method has been expanded to include additional lysosomal storage diseases.

BACKGROUNDMetachromatic leukodystrophy (MLD) is an autosomal recessive disorder caused by deficiency in arylsulfatase A activity, leading to accumulation of sulfatide substrates. Diagnostic and monitoring procedures include demonstration of reduced arylsulfatase A activity in peripheral blood leukocytes or detection of sulfatides in urine. However, the development of a screening test is challenging because of instability of the enzyme in dried blood spots (DBS), the widespread occurrence of pseudodeficiency alleles, and the lack of available urine samples from newborn screening programs.METHODSWe measured individual sulfatide profiles in DBS and dried urine spots (DUS) from MLD patients with LC-MS/MS to identify markers with the discriminatory power to differentiate affected individuals from controls. We also developed a method for converting all sulfatide molecular species into a single species, allowing quantification in positive-ion mode upon derivatization.RESULTSIn DBS from MLD patients, we found up to 23.2-fold and 5.1-fold differences in total sulfatide concentrations for early- and late-onset MLD, respectively, compared with controls and pseudodeficiencies. Corresponding DUS revealed up to 164-fold and 78-fold differences for early- and late-onset MLD patient samples compared with controls. The use of sulfatides converted to a single species simplified the analysis and increased detection sensitivity in positive-ion mode, providing a second option for sulfatide analysis.CONCLUSIONSThis study of sulfatides in DBS and DUS suggests the feasibility of the mass spectrometry method for newborn screening of MLD and sets the stage for a larger-scale newborn screening pilot study.

BACKGROUND There is worldwide interest in newborn screening for lysosomal storage diseases because of the development of treatment options that give better results when carried out early in life. Screens with high differentiation between affected and nonaffected individuals are critical because of the large number of potential false positives. CONTENT This review summarizes 3 screening methods: (a) direct assay of enzymatic activities using tandem mass spectrometry or fluorometry, (b) immunocapture-based measurement of lysosomal enzyme abundance, and (c) measurement of biomarkers. Assay performance is compared on the basis of small-scale studies as well as on large-scale pilot studies of mass spectrometric and fluorometric screens. SUMMARY Tandem mass spectrometry and fluorometry techniques for direct assay of lysosomal enzymatic activity in dried blood spots have emerged as the most studied approaches. Comparative mass spectrometry vs fluorometry studies show that the former better differentiates between nonaffected vs affected individuals. This in turn leads to a manageable number of screen positives that can be further evaluated with second-tier methods.

BACKGROUNDDried blood spots (DBSs) have potential use in remote health applications for individual and population diagnosis, and can enable epidemiological surveillance for known and unknown diseases. The preparation and transportation of DBSs from remote settings often exposes these cards to extreme environmental stress that may impact the quality of the diagnostic data. Given these risks, it is essential to investigate the individual stabilities of biomarkers in DBSs. This paper details the stability of routinely-analyzed amino acids (AAs) on DBSs under environmental conditions that simulate a global health workflow.METHODSThe extractions of 21 AAs from three sets of DBSs prepared on cellulose and cotton filter paper were optimized for quantitation by dansylation-UPLC/MRM-MS. The effects of sunlight exposure, temperature, humidity, and storage time were studied.RESULTSThe AAs were stable in DBSs after 4-hour sunlight exposure, and after storage at -20 and 4 °C for 30 days. At 25 and 40 °C, only 7 AAs showed significant concentration decreases over time, while 2 showed concentration increases. The changes were accelerated by high humidity. Histidine was the least stable AA under the conditions tested.CONCLUSIONSThis study provides quantitative data on the short-term stabilities of 21 AAs in DBSs on cellulose and cotton-based filter paper, under environmental conditions that simulate a global-health workflow. These results highlight the importance of assessing the stability of clinically-relevant biomarkers in DBSs. Based on the measured stabilities, we recommend that higher-temperature and high-humidity storage of DBS samples be avoided for AA analysis in remote health applications.

BACKGROUND Friedreich ataxia (FRDA) is caused by reduced frataxin (FXN) concentrations. A clinical diagnosis is typically confirmed by DNA-based assays for GAA-repeat expansions or mutations in the FXN (frataxin) gene; however, these assays are not applicable to therapeutic monitoring and population screening. To facilitate the diagnosis and monitoring of FRDA patients, we developed an immunoassay for measuring FXN. METHODS Antibody pairs were used to capture FXN and an internal control protein, ceruloplasmin (CP), in 15 μL of whole blood (WB) or one 3-mm punch of a dried blood spot (DBS). Samples were assayed on a Luminex LX200 analyzer and validated according to standard criteria. RESULTS The mean recovery of FXN from WB and DBS samples was 99%. Intraassay and interassay imprecision (CV) values were 4.9%–13% and 9.8%–16%, respectively. The FXN limit of detection was 0.07 ng/mL, and the reportable range of concentrations was 2–200 ng/mL. Reference adult and pediatric FXN concentrations ranged from 15 to 82 ng/mL (median, 33 ng/mL) for DBS and WB. The FXN concentration range was 12–22 ng/mL (median, 15 ng/mL) for FRDA carriers and 1–26 ng/mL (median 5 ng/mL) for FRDA patients. Measurement of the FXN/CP ratio increased the ability to distinguish between patients, carriers, and the reference population. CONCLUSIONS This assay is applicable to the diagnosis and therapeutic monitoring of FRDA. This assay can measure FXN and the control protein CP in both WB and DBS specimens with minimal sample requirements, creating the potential for high-throughput population screening of FRDA.

An important recent development in the field of HIV/AIDS care and treatment is the discovery that taking an antiretroviral medication 2–24 h before high risk sexual activity followed by daily doses for 2 days significantly lowers risk for HIV transmission (1). This strategy, called preexposure prophylaxis or PrEP,3 is naturally changing behavior amongst high-risk groups who are HIV negative. The particular approach of taking pills just before and after a high-risk encounter is known as “on demand” or “intermittent” use and has important practical implications. The conventional dictate for antiretroviral therapy has been that maximal viral suppression occurs when patients take at least 90% of their doses. For PrEP, although the recommendation is still that largest protection is upon daily dosing, high-risk individuals may take the medication around only high-risk encounters. To understand how we can know when PrEP users have adequate protection to guide clinical, or in this case, preclinical care, we spoke with Dr. Peter Anderson, professor of pharmaceutical sciences at the University of Colorado, who has had a long interest in nucleoside analogs and has developed an assay to measure adherence to PrEP. To determine if an HIV positive patient is taking …

Background: Succinylacetone (SUAC) is the primary metabolite accumulated in tyrosinemia type I—an inborn error of metabolism that, if untreated, can cause death from liver failure during the first months of life. Newborn screening laboratories measure SUAC in dried blood spot (DBS) samples to detect asymptomatic tyrosinemia type I. We used panels of SUAC-enriched DBSs to compare and evaluate the performance of these screening tests. Methods: We prepared sets of DBS materials enriched with predetermined SUAC concentrations and distributed samples of these materials, along with a screening practices questionnaire, to laboratories that perform SUAC tests. We compared their reported SUAC concentrations and questionnaire responses to identify screening practices that affect SUAC test outcomes. Results: Data from 2 pilot surveys showed large differences among laboratories in SUAC recoveries, reproducible within-laboratory recoveries, and stable performance of the DBS materials. Results from 257 proficiency test analyses contained a total of 6 false-negative misclassifications. Reported recoveries of added SUAC ranged from 0 to >200%. Low-biased SUAC recoveries were associated with 1 method used by 5 laboratories. All laboratories that reported SUAC recoveries ≥100% used DBS matrix calibrators. Conclusions: The wide ranges of SUAC concentrations reported for pilot and proficiency testing specimens demonstrate a need to harmonize quantitative results among laboratories. Although DBS matrix calibrators are important for optimizing SUAC recoveries, the preparation of these calibrators is not standardized among laboratories. Certified DBS-based SUAC calibrators are needed for accuracy and harmonization.

Background: Tyrosinemia type I (TYR 1) is a disorder causing early death if left untreated. Newborn screening (NBS) for this condition is problematic because determination of the diagnostic marker, succinylacetone (SUAC), requires a separate first-tier or only partially effective second-tier analysis based on tyrosine concentration. To overcome these problems, we developed a new assay that simultaneously determines acylcarnitines (AC), amino acids (AA), and SUAC in dried blood spots (DBS) by flow injection tandem mass spectrometry (MS/MS). Methods: We extracted 3/16-inch DBS punches with 300 μL methanol containing AA and AC stable isotope-labeled internal standards. This extract was derivatized with butanol-HCl. In parallel, we extracted SUAC from the residual filter paper with 100 μL of a 15 mmol/L hydrazine solution containing the internal standard 13C5-SUAC. We combined the derivatized aliquots in acetonitrile for MS/MS analysis of AC and AA with additional SRM experiments for SUAC (m/z 155–137) and 13C5-SUAC (m/z 160–142). Analysis time was 1.2 min. Results: SUAC was increased in retrospectively analyzed NBS samples of 11 TYR 1 patients (length of storage, 52 months to 1 week; SUAC range, 13–81 μmol/L), with Tyr concentrations ranging from 65 to 293 μmol/L in the original NBS analysis. The mean concentration of SUAC in 13 521 control DBS was 1.25 μmol/L. Conclusion: The inclusion of SUAC analysis into routine analysis of AC and AA allows for rapid and cost-effective screening for TYR 1 with no tangible risk of false-negative results.

BACKGROUND The noncalcemic actions of vitamin D in multiple organs are now widely recognized. Vitamin D status has been linked with a wide variety of conditions, which has led to an increasing demand for vitamin D screening. In particular, there is intense interest in the impact of vitamin D on a variety of developmental conditions. The most readily accessible pediatric samples are dried blood spots, and health organizations are increasingly archiving such samples for later assessment of the antecedents of disease. METHODS In 2009, we developed a method to quantify the major circulatory form of vitamin D, 25-hydroxyvitamin D, in archived dried blood spots. Over the last 6 years, we have made substantial alterations to the published method to enhance throughput, sensitivity, and assay robustness. RESULTS With the alterations, the assay was 3 times faster than the previously published assay and had a >10-fold increase in signal strength. Intraassay imprecision decreased from 13.4% to 6.9%, and there was a 5-fold reduction in interfering phospholipids. In actual use over 2 years, the assay showed an interassay imprecision of 11.6%. CONCLUSIONS This assay has performed reliably over the past 6 years. The practical changes we have made should allow clinical chemists to successfully adapt this method.

Background: False-positive and false-negative results occur in current newborn-screening programs for hepatorenal tyrosinemia, which measure tyrosine concentrations in blood spots, sometimes in combination with other metabolites, including succinylacetone. We present our experience with a newly described method for succinylacetone quantification in routine newborn screening.Methods: Succinylacetone was extracted from blood spots that had already been extracted with absolute methanol for acylcarnitine and amino acid analysis. The solvent was acetonitrile–water (80:20 by volume) containing formic acid, hydrazine hydrate, and 100 nmol/L 5,7-dioxooctanoic acid as internal standard. Analysis was performed by tandem mass spectrometry in a separate run.Results: Of 61 344 samples, 99.6% had succinylacetone concentrations ≤5 μmol/L. With a cutoff of 10 μmol/L, no false-positive results were obtained. In 2 patients, the succinylacetone concentrations in the dried blood spots from the 36th and 56th hours of life were 152 and 271 μmol/L, respectively, and the tyrosine concentrations were 54 and 129 μmol/L. Hepatorenal tyrosinemia was subsequently confirmed in both patients. Retrospective analysis of the neonatal screening samples of 2 additional known patients revealed increased succinylacetone concentrations of 46 and 169 μmol/L, respectively.Conclusions: Tandem mass spectrometric quantification directly from residual blood spots is a useful method for the early detection of hepatorenal tyrosinemia in newborn-screening programs.

Background: Over the past decade laboratories that test for metabolic disorders have introduced tandem mass spectrometry (MS/MS), which is more sensitive, specific, reliable, and comprehensive than traditional assays, into their newborn-screening programs. MS/MS is rapidly replacing these one-analysis, one-metabolite, one-disease classic screening techniques with a one-analysis, many-metabolites, many-diseases approach that also facilitates the ability to add new disorders to existing newborn-screening panels.Methods: During the past few years experts have authored many valuable articles describing various approaches to newborn metabolic screening by MS/MS. We attempted to document key developments in the introduction and validation of MS/MS screening for metabolic disorders. Our approach used the perspective of the metabolite and which diseases may be present from its detection rather than a more traditional approach of describing a disease and noting which metabolites are increased when it is present.Content: This review cites important historical developments in the introduction and validation of MS/MS screening for metabolic disorders. It also offers a basic technical understanding of MS/MS as it is applied to multianalyte metabolic screening and explains why MS/MS is well suited for analysis of amino acids and acylcarnitines in dried filter-paper blood specimens. It also describes amino acids and acylcarnitines as they are detected and measured by MS/MS and their significance to the identification of specific amino acid, fatty acid, and organic acid disorders.Conclusions: Multianalyte technologies such as MS/MS are suitable for newborn screening and other mass screening programs because they improve the detection of many diseases in the current screening panel while enabling expansion to disorders that are now recognized as important and need to be identified in pediatric medicine.

The goal of newborn screening (NBS)3 for inherited disorders of metabolism is the early detection and confirmation of disease, thus enabling early medical intervention, treatment, and improved outcomes (1). Important characteristics of a screening method include analytical specificity and sensitivity, coupled with rapid, high throughput and timely reporting of abnormal results. Routine primary screening methods are designed to identify as many abnormal infants as possible, with diagnostic sensitivity favored over specificity for disorder detection. This approach not only increases the numbers of false-positive test results, thus adding to the cost of operating NBS programs, but also places unnecessarily increased stress, anxiety, and possibly parent–child dysfunction on families (2). As the number of disorders in the NBS test panels grows, however, so does the overall number of false-positive results, which has increased severalfold per true case (3). One solution to this problem is to use improved methods or to couple primary screening methods with second-tier tests that improve selectivity. The use of tandem mass spectrometry (MS/MS) for detecting phenylketonuria is an example of an NBS method that improves detection as a primary screen while also being more selective than older, classic NBS methods such as fluorometry. In one study, MS/MS analysis of newborn spot samples of dried blood collected ≤24 h after birth was compared with fluorometric analysis of the same samples. Because of this early time of collection, the decision level for an increased phenylalanine concentration was lowered by the public health laboratory using fluorometry to ensure that no infants with phenylketonuria were missed. MS/MS analysis of the identical samples demonstrated that disease detection could be sustained while improving selectivity (4). The ability to measure multiple analytes in the same analysis enabled the calculation of the phenylalanine/tyrosine molar ratio, which reduced false-positive rates a 100-fold. This screen for phenylketonuria …

Screening for α1-antitrypsin deficiency (AATD) can be performed with dried blood spots (DBS) on filter paper, which allows quantification of AAT by immunonephelometry (1), identification of the AAT phenotype by isoelectric focusing(2)(3), and/or AAT genotyping (4). In most diagnostic flow-charts, important decisions often depend on the AAT concentration determined from DBS; however, current methods are imprecise, and results from DBS are not identical to those obtained using serum samples(4). We wished to develop a method for quantifying AAT in DBS. We collected 75 specimens from healthy blood donors and 74 from patients with suspected AATD. Samples were centrifuged for 8 min at 1620 g , and the serum or plasma, without manifest hemolysis, was separated and stored at −80 °C. Blood was spotted on filter paper with dashed-line 13-mm printed circles (Schleicher & Schuell Grade 903; lot. W-011) and air-dried. Cards stored in plastic bags at room temperature can be kept for at least 1 year. We measured AAT in serum or plasma by nephelometry (Array 360 System) with a polyclonal anti-human AAT goat antibody and calibrator (CAL2, assigned AAT …

Newborn screening (NBS)3 programs are an anomaly in medicine—a universally applied, state-mandated evaluation of healthy individuals. The program had its origin in the presymptomatic diagnosis of phenylketonuria (1), but as technologies for the use of dried blood spots (DBS) have improved, there has been a rapid expansion of the number of disorders evaluated, as well as an increase in the governmental mandates for testing (2). DNA-based diagnosis is already a feature of NBS. The first integrated use of genetic testing was for the confirmation of hemoglobinopathies (3). Genetic testing is now used as a primary or confirmatory technique in nearly every NBS system in the US. In addition to the analysis of sequence, DNA-based testing can also be used to detect the normal maturation of cell-mediated immunity through the detection of T-cell receptor excision circles (TRECs) (4). Clearly, additional tests will be added over time. The expanded use of DNA-based testing in NBS requires a reliable extraction method that delivers genomic DNA of sufficient quality and quantity for downstream applications. In this issue of Clinical Chemistry , Saavedra-Matiz and colleagues report details of the DNA-extraction protocol used in the New York State Newborn Screening Program (5). The source of the DNA sample is DBS on 903 filter papers, which are commonly used in NBS. Therefore, changes in the collection technique (i.e., phlebotomy), the volume of blood collected, or the type of card used for collection would not be required for most screening programs. The key advantages of this protocol are its extremely low cost and simplified use of automation. The minimization of hands-on work reduces labor costs and the chances for the introduction of error. The authors' system is easily scalable to the number of samples that might be reasonably anticipated in a state …

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 …

Dried blood on filter paper has been found to be suitable in large-scale population screening programs (1)(2)(3), but to the best of our knowledge, dried blood or serum on filter paper has not been used for lipid measurements. In the present study, we evaluated the stability of cholesterol and triglycerides in serum dried on filter paper. Fifty-four patients coming to the centralized laboratory, CNC, All India Institute of Medical Sciences for lipid investigations were selected at random, and blood was collected by venipuncture into tubes without anticoagulant. Serum was separated within 2 h of collection. All procedures were in accordance with the ethics standards of our institution. An aliquot of each serum sample was analyzed immediately. Exact 10-μL replicates of the samples were also spotted onto 3M Whatman filter paper kept on a nonabsorbent surface (thermacol) and left at room temperature for 1 h for drying. The room temperature was 16–37 °C for the duration of the study. After drying, one aliquot was eluted and analyzed on the day of collection. The remaining filter discs were kept in a sealed plastic bag to protect them from dust and moisture and stored at room …

Diabetes affects >5% of US adults (15% of those older than 60 years) and is on the rise in adolescents. Glycosylated hemoglobin (Hb A1c), a cumulative marker of blood glucose concentrations over the previous 2 months, has become a powerful clinical tool for diabetes management (1)(2) and is predictive of future complications of diabetes (3). Large-scale community-based studies often preclude the option of phlebotomy and would benefit from simplified sample handling. Epidemiologic studies have begun to use Hb A1c in dried blood spots as a biomarker (4). We studied Hb A1c from dried blood spots by validating the storage conditions appropriate for large-scale epidemiologic/outreach studies to test the hypothesis that measurements of Hb A1c in dried blood spots are valid under low-intensity storage conditions. All participants were studied at the Brigham and Women’s Hospital, Boston, Massachusetts; all procedures were conducted in accordance with the Declaration of Helsinki. Blood samples were drawn via standard phlebotomy procedures into EDTA-containing tubes for duplicate HPLC analyses of Hb A1c (Tosoh G7 automated HPLC assay, Brigham and Women’s Hospital Hematology Laboratory). Intrarun imprecisions (CVs) were 0.5% and 0.9% at Hb A1c proportions of the total hemoglobin of 0.041 and 0.1335, respectively. Interrun CVs were 1.6% for control samples with Hb A1c values of 0.064 and 0.7% for control samples with Hb A1c values of 0.11. Blood for spotting was drawn into an identical …

Background: Blood samples for neonatal screening for inborn errors of metabolism are collected and shipped on standardized filter paper cards. Occasionally these samples are contaminated with EDTA, which is often used for anticoagulation. EDTA may interfere with newborn screening tests based on lanthanide fluorescence and thus lead to false-negative or false-positive results. Methods: We used tandem mass spectrometry (MS/MS) to detect EDTA in dried blood spots by use of an extra experiment that was integrated into the standard MS/MS neonatal screening and did not require an additional sample spot, nor extra time or work. We analyzed the influence of different blood sampling procedures on lanthanide fluorescence tests for thyroid-stimulating hormone (TSH) and 17-hydroxyprogesterone (17-OHP). Results: EDTA was increased in 138 of 190 000 newborn screening samples, 27 of which caused false- positive results in the immunoassay for 17-OHP. No false-negative TSH results were found. False-positive results in the 17-OHP test occurred when EDTA concentrations were >2.0 g/L; the TSH test, however, produced false negatives only when EDTA concentrations were >3.0 g/L. Using EDTA-containing devices the procedure of blood collection significantly influenced the concentration of the anticoagulant. Conclusion: Addition of EDTA quantification into standard MS/MS tests is a simple and useful method to avoid false-positive or false-negative neonatal screening results in lanthanide fluorescence–based tests.

We describe a direct, solid-phase time-resolved fluoroimmunoassay (TRFIA) for measuring 17 alpha-hydroxyprogesterone (17OHP) in serum and blood spots on filter paper. We used 17OHP-3-carboxymethyloxime (17OHP3CMO) coupled to polylysine as the label, which enabled incorporation of up to 34 atoms of europium per molecule of 17OHP, for a very high specific activity. The assay is based on competition between labeled 17OHP3CMO and 17OHP in blood specimens for polyclonal rabbit anti-17OHP antibodies. The antibody-label complex is separated by binding to anti-rabbit antibodies coated onto microtiter strips. The assay buffer contains danazol to displace 17OHP from steroid-binding proteins in serum. For serum samples, the assay is accomplished in 1 h of incubation at room temperature. The blood spot assay with filter paper discs involves incubation overnight at 4 degrees C. Results for both types of specimens from the same subjects correlated well. The lowest measurable concentrations of 17OHP (nmol/L) were 0.10 (3 SD) and 0.75 (3 SD) for serum and dried blood on filter paper, respectively. Intra- and interassay CVs were about 5-15% for both types of samples.

Background: Retinol-binding protein (RBP) is accepted as a surrogate biochemical marker for retinol to determine vitamin A (VA) status. A recently developed enzyme immunoassay for RBP uses serum or whole blood stored as dried blood spots (DBS). However, the stability of RBP in DBS has not been examined. Methods: RBP stability was studied in a laboratory and in field conditions in northern Kenya. For the laboratory study, 63 DBS collected by finger prick and stored sealed in a plastic bag with desiccant were exposed to 1 of 5 time/storage-temperature treatments: (a) baseline, (b) 30 °C/7 days, (c) 30 °C/14 days, (d) 30 °C/28 days, and (e) 4 °C/38 days. Baseline RBP concentrations were compared to those obtained after the storage treatments. For the field study, 50 paired DBS and serum specimens were prepared from venous blood obtained in northern Kenya. DBS were stored in a sealed plastic bag with desiccant at ambient temperature (12 °C–28 °C) for 13–42 days, and sera were stored at −20 °C to −70 °C. Recovered RBP concentrations were compared with serum retinol for stability, correlation, sensitivity, and specificity. Results: RBP in DBS stored in the laboratory at 30 °C remained stable for 2–4 weeks, but specimens stored at 4 °C for 38 days produced values below baseline (P = 0.001). DBS stored under field conditions remained stable for 2–6 weeks, as demonstrated by good correlation with serum retinol, a result that suggests that RBP in DBS will have good sensitivity and specificity for predicting VA deficiency. Conclusion: RBP in DBS can withstand storage at a relatively high ambient temperature and thus facilitate accurate VA assessments in populations in locations where serum collection and storage are unfeasible.

There is interest in newborn screening of lysosomal storage diseases (LSDs) because of the availability of treatments. Pilot studies have used tandem mass spectrometry with flow injection of samples to achieve multiplex detection of enzyme products. We report a multiplexing method of 9 enzymatic assays that uses HPLC-tandem mass spectrometry (MS/MS).The assay of 9 enzymes was carried out in 1 or 2 buffers with a cassette of substrates and internal standards and 1 or 2 punches of a dried blood spot (DBS) from a newborn screening card as the source of enzymes. The pre-HPLC-MS/MS sample preparation required only 4 liquid transfers before injection into a dual-column HPLC equipped with switching valves to direct the flow to separation and column equilibration. Product-specific and internal standard-specific ion fragmentations were used for MS/MS quantification in the selected reaction monitoring mode.Analysis of blood spots from 58 random newborns and lysosomal storage disease-affected patients showed that the assay readily distinguished affected from nonaffected individuals. The time per 9-plex analysis (1.8 min) was sufficiently short to be compatible with the workflow of newborn screening laboratories.HPLC-MS/MS provides a viable alternative to flow-injection MS/MS for the quantification of lysosomal enzyme activities. It is possible to assay 9 lysosomal enzymes using 1 or 2 reaction buffers, thus minimizing the number of separate incubations necessary.

Blood dried on filter paper is widely used for screening of inherited metabolic disorders (1). In Sweden, such filters from all newborns have been permanently stored since 1975. It has been shown that proteins and DNA may be recovered from these cards after extended periods of storage (2)(3)(4). RNA, however, has been considered too vulnerable to degradation by ribonucleases to be recovered from these filters. Despite this, Zhang and McCabe (5) and Matsubara et al.(6) reported that mRNA could be isolated from such filters after up to 4 years of storage. The stability of viral RNA on filters has also been reported (7)(8), although this was not tested over extended periods of time. The purpose of the present study was to investigate whether RNA could be recovered from filters that had been stored since 1975 and be amplified by reverse transcription-PCR. After approval by the local ethics committee, we randomly selected filter papers (specimen collection paper 2992; Schleicher & Schuell) that had been stored for 1 month, 21 years, and 27 years; for each time point, we selected five filters. One-fourth of a spot (∼0.3 cm2) containing dried blood was cut out of each filter with a sterile razor …

Concentrations of phytanic acid and pristanic acid were measured in stored dried blood spots collected at neonatal screening from patients with peroxisomal disorders, and compared with concentrations in control blood spots. In blood spots from two patients with Zellweger syndrome both phytanic acid and pristanic acid concentrations were increased but their concentration ratio was normal. In the blood spot from a patient with rhizomelic chondrodysplasia punctata, the concentration of phytanic acid was increased, whereas pristanic acid was within the control range, resulting in a low pristanic acid/phytanic acid ratio. In the blood spot from a patient with X-linked adrenoleukodystrophy, the concentrations of the acids and their ratio were normal. These findings are consistent with results for these acids in plasma from such patients. Measurement of phytanic acid and pristanic acid and their ratios in stored dried blood collected at neonatal screening can therefore be used in the diagnosis of peroxisomal disorders, especially for those cases in which, owing to early death of the patient, no other material is available for biochemical investigations.

Lipoprotein(a) [Lp(a)] is considered an additional, independent, and largely genetically determined risk factor for the development of premature coronary heart disease. Analogous with increased Lp(a) concentrations that represent an additional risk factor in adults, above-normal concentrations of Lp(a) can be detected in five- to seven-day-old newborns. We describe a simple enzyme-linked immunosorbent assay for measuring Lp(a) in dried blood spots collected by heel-prick in five- to seven-day-old infants. Lp(a) could be quantitatively recovered from blood spots. We chose a cutoff value of 100 mg/L for identifying the newborns at risk, based on the Lp(a) distribution in 180 such infants.

Fujimoto et al. (1) reported that the stability of galactose 1-phosphate in dried blood spots for neonatal screening was adversely affected by humidity and temperature, especially when low-value samples are evaluated. We extend these findings to glucose-6-phosphate dehydrogenase (G-6-PD; EC 1.1.1.49) activity, deficiency of which is by far the most common genetic disease worldwide (2) and accounts for more than one-half of the cases of severe jaundice among male newborns in Greece, China, and in Sephardic Jewish groups (3). Tests for G-6-PD deficiency are thus included in newborn screening programs in some regions. We collected whole-blood specimens from …

The concept of screening newborns for inherited metabolic disorders was the brainchild of Robert Guthrie, an upstate New York microbiologist with a passion to prevent the devastating and irreversible neurologic damage sustained by victims of untreated phenylketonuria (PKU). The solution he developed was a simple and inexpensive bacterial inhibition assay for phenylalanine in blood (1). He also invented a unique method of specimen collection, in which peripheral blood was collected from a newborn’s pricked heel onto a special cotton fiber filter-paper known as a “PKU card” or “Guthrie card”. After the blood had dried, the specimen was mailed to a laboratory that would identify any child at risk for PKU from a concentration of phenylalanine above an age-matched control limit. Further diagnostic testing was then required to determine whether the disease was present. Treating affected children with a phenylalanine-depleted diet, started within the first month of life, was effective in preventing mental retardation. Unable to persuade the state of New York to conduct newborn screening with the test, Guthrie convinced Massachusetts that the cost of screening the entire population of newborns and treating affected children with the special diet was less than the cost to society of untreated PKU cases. Thus, in 1963 began state-mandated newborn screening for PKU (2). The test was gradually adopted by other states and eventually by countries all over the world. The success of PKU screening in dried blood spots (DBS), which identifies ∼200 new cases annually in the United States alone, prompted the addition of tests for other disorders that fit the PKU paradigm. By the …

Newborn screening prevents thousands of premature illnesses and deaths every year and is one of the most successful public health programs in the US. Lately, there has been considerable controversy regarding the retention and use of residual newborn blood samples after their initial use in screening without obtaining parental informed consent. The concerns have focused on the confidentiality, privacy, and autonomy rights of parents to decide whether to permit the secondary use of these samples for research. This ongoing controversy regarding the preservation and use of dried blood samples in research has led to the destruction of several million archived samples and potentially more in the near future if no further actions are taken. Left out of the discussion, however, have been the benefits of making the residual blood …

In their letter, Lukacs et al. present an interesting modification and improvement of the procedure described previously by Bellisario et al. (1), using the Luminex Xmap system to simultaneously assay for thyroxine (T4) and thyrotropin (TSH) in a Guthrie specimen. Their innovation builds on …

We present a new, simple, and inexpensive sandwich-type double-antibody enzyme immunoassay for alpha 1-antitrypsin in dried blood collected on the fifth day post-partum. The method is very sensitive, having a detection limit of 2.84 fmol/well. Intra- and interassay CVs are 6.1% and 10.3%, respectively, for assay of 5-mm-diameter blood spots eluted into 7 mL of phosphate buffer. Since February 1984, we have used this method to systematically screen 39 289 consecutive births: 336 of these newborns (0.085%) showed values for alpha 1-antitrypsin below the cutoff value of 800 mg/L (50th percentile, 1470 mg/L). Of these 336 we were able to obtain 0.5 mL of serum from 161 for further testing. Four presented with a ZZ phenotype and 15 with a SZ phenotype, which indicates a deficiency in alpha 1-antitrypsin. Our data suggest a prevalence of 1.4% and 3.6% of Z and S alleles, respectively, in the French-speaking community of Belgium.

Congenital hypothyroidism causes a decreased growth rate and skeletal development and can lead to severe mental retardation (1). In the nearly three decades since measurement of thyroxine in filter paper dried blood disks (Guthrie cards) was introduced as a method to screen newborns for congenital hypothyroidism (2), new procedures for thyroxine and thyrotropin (TSH), such as ELISA, fluoroimmunoassay, or chemiluminescence (3) have been developed or adapted from commercial kits, most of them requiring overnight incubation. Previously, we described a manual adaptation of a commercial immunochemiluminometric assay kit based on an acridinium label to measure TSH in dried blood spots (4). This method is used routinely in our laboratory, and we have detected 176 congenital hypothyroidism cases in the 480 000 newborn samples tested. Studies concerning its correlation with the DELFIATM neonatal TSH kit (Wallac Oy) were also described (5). Here, we present results obtained with a rapid and automated third-generation TSH chemiluminescent assay for serum samples adapted to measure TSH in Guthrie cards (ACS:180TM, Chiron Diagnostics). This fully automated chemiluminescence immunoassay system uses paramagnetic particles as the solid phase (6). We measured TSH in 2200 dried blood spot samples obtained in filter paper (Schleicher and Schuell, cat. no. 903) by heel stick from newborns routinely referred to our service. The samples were dried at room temperature, and the TSH concentrations were measured using our routine chemiluminescent method (4) and also by the technique described here. We used calibrators at 2.2, 22.0, 55.0, 110.0, 220.0, and 550.0 mIU TSH/L serum equivalent and controls at 33.0 and 132.0 mIU TSH/L serum equivalent provided with the DELFIA …

We describe a double-antibody enzyme immunoassay for determination of free thyroxin (FT4) in dried blood samples on filter paper, with use of a T4-beta-D-galactosidase complex. The measurable range of FT4 concentration in two 3-mm blood discs, each of which contained about 2.7 microL of blood, was 1.9 to 93 ng/L, as determined by comparison with concentrations of FT4 in known serum standards. FT4 in blood samples dried on filter paper was stable for at least four weeks when kept dry at -20 degrees C, room temperature, or 37 degrees C. The mean coefficients of variation were 7.6% (within assay) and 6.4% (between assays). Results for FT4 by this method correlated well with those for serum determined by radioimmunoassay (r = 0.98). The proposed method can be used to differentiate persons with hyper- and hypothyroidism from normal subjects and those with abnormal concentrations of thyroxin-binding globulin. The procedure seems suited for screening studies.

We evaluated the effect of sampling of capillary blood on filter paper on the later analysis for glucose. We found the method simple and reliable. Determination at the central laboratory of glucose in blood collected onto filter paper and comparison of the results with those obtained with test strips read in reflectometers at outpatient units is easy. Collecting duplicate samples on filter paper facilitates quality control by avoiding the complications that arise from using quality-control solutions that are not directly comparable with fresh blood, and it avoids the disturbances of test strip chemistry attributable to the glycolysis inhibitors added to blood samples intended for quality control.

We adapted a commercial RIA kit to measure alpha-fetoprotein (AFP) in 0.785-microliters portions of 60-microliters spots of dried blood from newborns. We evaluated sample elution, temperature and time stability, between- and within-assay variability, sensitivity, and use of liquid vs dried specimens of blood. Also, we present normal AFP concentrations for healthy neonates during the first postnatal week. Our procedure permits measurement of AFP concentrations both in maternal liquid plasma and in spots of dried blood from the infant with the same RIA kit reagents and standards. The sensitivity, precision, stability, and simplicity of this procedure makes more practical the routine measurement of AFP in dried blood specimens from newborns than measurement in liquid plasma or serum. Blood-sample collection by heel stick suffices for this simple, efficient, inexpensive determination of AFP concentration in the newborn.

In this sensitive, simple method for measuring "free thyroxin" (FT4) in eluates of dried blood spots on filter paper by use of a radioimmunoassay kit (Amerlex Free T4 RIA), the measurable range of FT4 is 1.8 to 57 ng/L (equivalent to the concentration in serum), or 7 to 237 fg/tube. The mean coefficients of variation for within assay-within spots, within assay-between spots, and between assays were 5.3%, 5.0%, and 6.2%, respectively. FT4 in blood spotted on filter paper is stable for at least a month when dried and kept at either -20 degrees C, 4 degrees C, room temperature (about 25 degrees C), or 37 degrees C. The results for FT4 in dried blood spots correlated closely with the free-T4 concentration in serum (r = 0.99). The method can be used to differentiate cases of primary and secondary hypothyroidism from normal subjects and those with subnormal thyroxin-binding globulin. This method may be useful in screening for congenital hypothyroidism, because sample-retesting is not necessary.

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.

We used dried blood spots successfully as alternative specimens for quantifying concentrations of theophylline in the circulation by a modified fluorescence polarization immunoassay (FPIA) with the Abbott TDx instrument. The method described is simple, rapid, and acceptably precise. More importantly, it can provide results comparable with those of the conventional serum assay. Results for 64 pairs of dried blood spots (y) and serum (x) specimens analyzed by the respective FPIA methods yielded the following regression parameters: y = 0.94x + 0.71, r = 0.988, and Sxy = 0.92. A major advantage of FPIA is that it requires only basic laboratory skills. When coupled with the use of dried blood spots, this system can be effective in remote theophylline monitoring, particularly suited for domiciliary care.

We evaluated the usefulness, in routine newborn screening for congenital hypothyroidism, of a time-resolved fluoroimmunoassay kit (DELFIA Neonatal TSH) for the determination of thyrotropin (TSH) in dried blood spots. A total of 11 531 dried blood samples from newborns were tested in parallel in each of two Swiss screening laboratories, by RIA and DELFIA. Six cases of confirmed congenital hypothyroidism were detected during the study period. The rate of false-positive results, after single TSH determination in the DELFIA assay, was 0.16%. Correlation of RIA and DELFIA results for TSH was very good in both laboratories (0.959 and 0.97, respectively). The new method fulfills the criteria for precision and sensitivity of a screening assay. Screening results are usually available the day after the sample arrives in the laboratory, thus favoring early diagnosis and allowing treatment to begin by the seventh or eighth postnatal day.

We prepared whole-blood pools to contain various concentrations of phenylalanine (Phe), thyroxin (T4), and thyrotropin (TSH) and applied them to six different lots of Schleicher & Schuell Grade 903 filter paper, two of which represented extremes for serum-absorbancy. Individual measured T4 values showed minimal overlap among all pools for each individual filter-paper lot and for all lots combined, but Phe values overlapped considerably among the high-concentration pools within and among lots. Individual TSH values also showed considerable overlap among the high-concentration pools for all lots combined, but little overlap within each lot. Maximum differences in mean observed values among lots ranged from 6% to 36% for all analytes. Assay results from hemolyzed blood specimens generally were lower than from intact-cell blood specimens for T4 and TSH, but slightly higher for Phe. Maximum among-lot differences in mean values ranged from 13% to 29% for all analytes when each tested lot was used for assay calibration. Lot-to-lot differences in measured values were not strongly related to serum absorbancy values. We conclude that routinely encountered within- and among-lot filter paper variability, as measured by serum-absorbancy, is not alone sufficient to cause gross quantification errors in neonatal screening programs.

A double-antibody enzyme immunoassay was developed for determination of thyroxin-binding globulin in dried blood samples on filter paper. The measurable concentration range of thyroxin-binding globulin in two 3-mm blood discs was 3.3 to 52 mg/L equivalent of serum (i.e., equivalent to the concentrations in known serum standards). Thyroxin-binding globulin in dried blood samples on filter paper was stable for at least four weeks when kept dry at -20 degrees C, 4 degrees C, or room temperature. The mean coefficients of variation were 6.6% (within assay) and 5.9% (between assays). The concentrations of thyroxin-binding globulin in dried blood samples determined by this method correlated well with those in serum determined by radioimmunoassay (r = 0.95) and by enzyme immunoassay (r = 0.96). This method is applicable for detecting cases of thyroxin-binding globulin deficiency and avoids the false-positive results for neonatal hypothyroidism obtained by measuring thyroxin.

Aliquots (0.1 mL) of whole-blood pools prepared to contain various concentrations of phenylalanine were applied to filter-paper collection cards, dried, and stored in sealed bags. We measured the phenylalanine content of the dried blood spots by bioassay, fluorometry, and "high-performance" liquid chromatography, and found that the concentrations remained constant for two years when samples were kept at -20 degrees C or lower. Intra- and interlaboratory studies showed that results for phenylalanine were greater for laboratories using bioassay procedures than for those using fluorometric procedures. Further, CVs (both among- and within-laboratory) obtained with fluorometric procedures were nearly half as great as the CVs obtained by laboratories using bioassay techniques.

We have developed a highly sensitive radioimmunoassay of thyroxine and thyrotropin for mass screening for neonatal hypothyroidism. This assay involves a single disc (3 mm diameter) of dried blood on filter paper. The minimum detectable concentrations are 15 pg/tube (10 microgram/L) for thyroxine and 15 nano-int. units/tube (10 milli-int. units/L) for thyrotropin; intra- and interassay CV’s are < 15% in both assays. The high sensitivity of this method is due to use of labeled thyroxine with high specific activity (3 kCi/g) and of an anti-thyrotropin serum with high affinity (Keq = 7.8 × 10(11) L/mol). With this method, 11337 newborns were screened; a follow-up study revealed that only newborns with both high thyrotropin and low thyroxine concentrations had permanent hypothyroidism. We conclude that this method is sensitive, simple, and reliable and that the recall rate with this method is much lower than that of tests for measuring thyroxine or thyrotropin alone.

We describe a fluorometric micromethod for measuring adenosine deaminase activity in dried blood spots on filter paper. Earlier methods require venipuncture and preparation of washed erythrocytes; in the present method, whole capillary blood, spotted on filter paper and mailed (dried) to a central laboratory, is used. The stability of the enzyme in dried blood on filter paper was assessed. The results were compared with those of a spectrophotometric method. The presence of serum appears not to affect the estimation of the activity and the method may be useful in early detection of severe combined immunodeficiency disease and hereditary hemolytic anemia.