Achieving Congruence among Reference Laboratories for Absolute Abundance Measurement of Analytes for Rare Diseases: Psychosine for Diagnosis and Prognosis of Krabbe Disease.

Measurement of the absolute concentration of the biomarker psychosine in dried blood spots (DBS) is useful for diagnosis and prognosis of Krabbe disease and to support newborn screening of this leukodystrophy. As for assays for more common diseases, it is important to achieve congruence when multiple clinical laboratories provide testing. Four clinical laboratories, one research laboratory, and a commercial vendor collaborated with the goal to achieve congruence in quantitative psychosine measurement in DBS. A set of DBS calibrators was prepared by a single vendor and used in each reference laboratory to make a standard curve of measured psychosine in DBS versus the stated calibrator psychosine level. Congruence between the participating five laboratories was achieved using the psychosine DBS calibrators. This allowed application of disease-specific reference ranges obtained by the reference laboratory with the most extensive data by the other reference laboratories. Congruence between multiple reference laboratories in the measurement of the absolute concentration of biomarkers in DBS (and by extension other samples) is possible by the use of a common set of DBS calibrators.

Development of a newborn screening tool based on bivariate normal limits: using psychosine and galactocerebrosidase determination on dried blood spots to predict Krabbe disease.

PURPOSE: Newborn screening for Krabbe disease (KD) originated in New York State in 2006 but has proven to have a high false positive rate and low positive predictive value. To improve accuracy of presymptomatic prediction, we propose a screening tool based on two biomarkers, psychosine and galactocerebrosidase enzyme activity (GalC). METHODS: We developed the tool using measures from dried blood spots of 166 normal newborns and tested it on dried blood spot measures from 15 newborns who later developed KD, 8 newborns identified as "high risk" by the New York screening protocol but were disease-free at follow-up, and 3 symptomatic children with onset before 4 years of age. The tool was developed from the (1-10-6)100% prediction region of the natural logarithms of psychosine and GalC measures, assuming bivariate normality, and their univariate normal limits. RESULTS: Krabbe disease was predicted correctly for every patient who developed symptoms in infancy or early childhood. None of the high-risk patients were incorrectly identified as having early KD. CONCLUSION: Bivariate analysis of psychosine and GalC in newborn blood spots can accurately predict early Krabbe symptoms, control false positive rates, and permit presymptomatic treatment.

Newborn screening for Krabbe disease in New York State: the first eight years' experience.

PURPOSE: Krabbe disease (KD) results from galactocerebrosidase (GALC) deficiency. Infantile KD symptoms include irritability, progressive stiffness, developmental delay, and death. The only potential treatment is hematopoietic stem cell transplantation. New York State (NYS) implemented newborn screening for KD in 2006. METHODS: Dried blood spots from newborns were assayed for GALC enzyme activity using mass spectrometry, followed by molecular analysis for those with low activity (≤12% of the daily mean). Infants with low enzyme activity and one or more mutations were referred for follow-up diagnostic testing and neurological examination. RESULTS: Of >1.9 million screened, 620 infants were subjected to molecular analysis and 348 were referred for diagnostic testing. Five had enzyme activities and mutations consistent with infantile KD and manifested clinical/neurodiagnostic abnormalities. Four underwent transplantation, two are surviving with moderate to severe handicaps, and two died from transplant-related complications. The significance of many sequence variants identified is unknown. Forty-six asymptomatic infants were found to be at moderate to high risk for disease. CONCLUSIONS: The positive predictive value of KD screening in NYS is 1.4% (5/346) considering confirmed infantile cases. The incidence of infantile KD in NYS is approximately 1 in 394,000, but it may be higher for later-onset forms.

Cost-effective and scalable DNA extraction method from dried blood spots.

BACKGROUND: Dried blood spot (DBS) samples have been widely used in newborn screening (NBS) for the early identification of disease to facilitate the presymptomatic treatment of congenital diseases in newborns. As molecular genetics knowledge and technology progresses, there is an increased demand on NBS programs for molecular testing and a need to establish reliable, low-cost methods to perform those analyses. Here we report a flexible, cost-efficient, high-throughput DNA extraction method from DBS adaptable to small- and large-scale screening settings. METHODS: Genomic DNA (g.DNA) was extracted from single 3-mm diameter DBS by the sequential use of red cell lysis, detergent-alkaline, and acid-neutralizing buffers routinely used in whole blood and plant tissue DNA extractions. We performed PCR amplification of several genomic regions using standard PCR conditions and detection methods (agarose gel, melting-curve analysis, TaqMan-based assays). Amplicons were confirmed by BigDye® Terminator cycle sequencing and compared with reference sequences. RESULTS: High-quality g.DNA was extracted from hundreds of DBS, as proven by mutation detection of several human genes on multiple platforms. Manual and automated extraction protocols were validated. Quantification of g.DNA by Oligreen® fluorescent nucleic acid stain demonstrated a normal population distribution closely corresponding with white blood cell counts detected in newborn populations. CONCLUSIONS: High-quality, amplifiable g.DNA is extractable from DBSs. Our method is adaptable, reliable, and scalable to low- and high-throughput NBS at low cost ($0.10/sample). This method is routinely used for molecular testing in the New York State NBS program.

Determination of psychosine concentration in dried blood spots from newborns that were identified via newborn screening to be at risk for Krabbe disease.

BACKGROUND: New York State has screened over 1.2 million newborns for Krabbe disease, and we identified 4 newborns with infantile Krabbe disease. In addition, 6 other newborns were identified with very low galactosylcerebrosidase (GALC) activity. Because these patients remain asymptomatic, we investigated whether psychosine levels could be a useful marker for disease. METHODS: HPLC-MS/MS methodology was used to determine the psychosine concentrations in dried blood spots (DBS) collected from the following cohorts: known Krabbe patients, screened babies that were determined to have infantile Krabbe disease, asymptomatic infants with low GALC activity, and normal controls. RESULTS: The psychosine concentrations from the known Krabbe patients ranged from 7 to 50 ng/ml. Newborns identified by screening who were confirmed with infantile Krabbe disease ranged from 23 to 73 ng/ml. Asymptomatic individuals with low GALC activity had concentrations ranging from 1.7 to 5.7 ng/ml. Concentrations in newborns with normal GALC activity were all <3 ng/ml. CONCLUSIONS: The psychosine concentrations in DBS from confirmed infantile patients are at least four times higher than the asymptomatic newborns and nearly an order of magnitude greater than normal newborns. Further studies are needed to determine if psychosine can be used as a predictor of disease status/progression in screen positive newborns.

Implementation of newborn screening for Krabbe disease: population study and cutoff determination.

OBJECTIVE: The aim of this study was to develop a newborn screening algorithm for Krabbe disease. DESIGN AND METHODS: We measured the galactocerebrosidase activity of 139,074 anonymous newborns, 56 known carriers, and 16 Krabbe patients using a tandem mass spectrometry method. The activities were converted to percentages of daily mean activity (%DMA), and the results from diseased and normal populations were used to establish cutoffs. RESULTS: The absolute activities for the newborns ranged from 0.17 to 355 micromol/L h (N=139,074) and activities for Krabbe-positive controls ranged from 0.08 to 0.48 micromol/L h (N=16, n=91 measurements) while activities for carriers ranged from 0.28 to 2.71 micromol/L h (N=56, n=72 measurements). Cutoffs were set based on results from Krabbe-positive and carrier controls and the newborn population distribution. CONCLUSIONS: The algorithm and cutoffs we propose provided 100% detection of all positive controls with 60/100,000 screen positive results predicted. In the course of this study, one anonymous newborn was predicted to have Krabbe disease based on enzyme activity and subsequent DNA analysis.