Multi-Laboratory Evaluation of Prototype Dried Blood Spot Quality Control Materials for Creatine Kinase-MM Newborn Screening Assays.

Pilot studies to detect newborns with Duchenne Muscular Dystrophy (DMD) by newborn bloodspot screening (NBS) have been conducted under the New York State Newborn Screening Program (NYS) and are currently in progress as part of the Early Check Program at Research Triangle Institute (RTI) International. The Newborn Screening Quality Assurance Program (NSQAP) at the U.S. Centers for Disease Control and Prevention (CDC) produced a set of seven prototype dried blood spot (DBS) reference materials spiked with varying levels of creatine kinase MM isoform (CK-MM). These DBS were evaluated over a 3-week period by CDC, NYS, and RTI, all using the same CK-MM isoform-specific fluoroimmunoassay. Results from each laboratory were highly correlated with the relative proportion of CK-MM added to each of the six spiked pools. Based on reference ranges established by NYS and RTI for their pilot studies, these contrived DBS collectively spanned the CK-MM ranges found in typical newborns and the elevated ranges associated with DMD. This set allows quality assessment over the wide range of fluctuating CK-MM levels in typical and DMD-affected newborns.

William Harry Hannon-A Life Well Lived.

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Serum microRNA profiles among dioxin exposed veterans with monoclonal gammopathy of undetermined significance.

Previously an increased risk for monoclonal gammopathy of undetermined significance (MGUS), a precursor of multiple myeloma (MM), was reported among Vietnam veterans exposed to Agent Orange and its contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Dysregulated expression of certain microRNAs (miRNAs) was demonstrated in MGUS and MM. Given the important role of miRNAs in cellular homeostasis, the aim of this study was to determine if there was an association between serum levels of selected miRNAs and TCDD in 47 MGUS cases identified in our previous investigation using serum specimens and exposure data archived by the Air Force Health Study (AFHS). A total of 13 miRNA levels (let-7a, let-7i, miR-16, miR-20a, miR-21, miR-34a, miR-106b, miR-146a, miR-181a, miR-192, miR-205, miR-335, and miR-361) was measured in serum stored during the 2002 AFHS follow-up and the relationship to lipid-adjusted serum TCDD levels in 1987 was determined. miR-34a showed the strongest relationship with TCDD; after age-adjustment, this positive association was more pronounced. In contrast, the other 12 miRNAs displayed absolute values of age adjusted coefficient estimates below 1.16 and non-significant p-values. The observed strong positive association between high body burdens of TCDD and miR-34a, a tumor suppressor regulated by p53, in this MGUS population warrants clarification of the TCDD-miR-34a relationship and its role in the pathogenesis of MGUS and risk for MM.

MALDI-TOF-MS Assay to Detect the Hemizygous 22q11.2 Deletion in DNA from Dried Blood Spots.

BACKGROUND: A hemizygous deletion of 1.5-3 Mb in 22q11.2 causes a distinct clinical syndrome with variable congenital defects. Current diagnostic methods use fluorescent in situ hybridization (FISH) or comparative genomic hybridization by microarray to detect the deletion. Neither method is suitable for newborn screening (NBS), since they cannot be performed on dried blood spots (DBS). We developed a MALDI-TOF-MS assay that uses DBS to measure the hemizygous deletion of UFD1L, located within the 22q11.2 region. METHODS: We used DBS from 54 affected patients, previously tested by FISH or microarray, and 100 cord blood samples to evaluate the performance of the MALDI-TOF-MS assay. With a single primer pair, a 97-base oligonucleotide within UFD1L was amplified, as was a sequence on chromosome 18 that differs by 2 nucleotides. A multiplexed, single-base extension reaction created allele-specific products for MALDI-TOF-MS detection. The products were spotted onto a silicon chip, and the height of the spectral peaks identified the relative amounts of target and reference gene. RESULTS: The median ratio of the spectral peak for each UFD1L target:reference base was 0.96 and 0.99 for controls, compared with 0.35 and 0.53 for 22q11 deletion syndrome patients. There was 100% concordance between FISH/microarray and MALDI-TOF-MS in all patients with 22q11.2 deletion syndrome. CONCLUSIONS: This method can be reliably performed with DBS and is suitable for high sample throughput. This assay may be considered for use in population-based NBS for 22q11.2 deletion.

Agent Orange Exposure and Monoclonal Gammopathy of Undetermined Significance: An Operation Ranch Hand Veteran Cohort Study.

IMPORTANCE: Multiple myeloma has been classified as exhibiting "limited or suggestive evidence" of an association with exposure to herbicides in Vietnam War veterans. Occupational studies have shown that other pesticides (ie, insecticides, herbicides, fungicides) are associated with excess risk of multiple myeloma and its precursor state, monoclonal gammopathy of undetermined significance (MGUS); however, to our knowledge, no studies have uncovered such an association in Vietnam War veterans. OBJECTIVE: To examine the relationship between MGUS and exposure to Agent Orange, including its contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), in Vietnam War veterans. DESIGN, SETTING, AND PARTICIPANTS: This was a prospective cohort study conducted in 2013 to 2014, testing for MGUS in serum specimens collected and stored in 2002 by the Air Force Health Study (AFHS). The relevant exposure data collected by the AFHS was also used. We tested all specimens in 2013 without knowledge of the exposure status. The AFHS included former US Air Force personnel who participated in Operation Ranch Hand (Ranch Hand veterans) and other US Air Force personnel who had similar duties in Southeast Asia during the same time period (1962 to 1971) but were not involved in herbicide spray missions (comparison veterans). Agent Orange was used by the US Air Force personnel who conducted aerial spray missions of herbicides (Operation Ranch Hand) in Vietnam from 1962 to 1971. We included 479 Ranch Hand veterans and 479 comparison veterans who participated in the 2002 follow-up examination of AFHS. EXPOSURES: Agent Orange and TCDD. Serum TCDD levels were measured in 1987, 1992, 1997, and 2002. MAIN OUTCOMES AND MEASURES: Risk of MGUS measured by prevalence, odds ratios (ORs), and 95% CIs. RESULTS: The 479 Ranch Hand veterans and 479 comparison veterans had similar demographic and lifestyle characteristics and medical histories. The crude prevalence of overall MGUS was 7.1% (34 of 479) in Ranch Hand veterans and 3.1% (15 of 479) in comparison veterans. This translated into a 2.4-fold increased risk for MGUS in Ranch Hand veterans than comparison veterans after adjusting for age, race, BMI in 2002, and the change in BMI between 2002 and the time of blood draw for TCDD measurement (adjusted OR, 2.37; 95% CI, 1.27-4.44; P=.007). CONCLUSIONS AND RELEVANCE: Operation Ranch Hand veterans have a significantly increased risk of MGUS, supporting an association between Agent Orange exposure and multiple myeloma.

Newborn blood spot screening test using multiplexed real-time PCR to simultaneously screen for spinal muscular atrophy and severe combined immunodeficiency.

BACKGROUND: Spinal muscular atrophy (SMA) is a motor neuron disorder caused by the absence of a functional survival of motor neuron 1, telomeric (SMN1) gene. Type I SMA, a lethal disease of infancy, accounts for the majority of cases. Newborn blood spot screening (NBS) to detect severe combined immunodeficiency (SCID) has been implemented in public health laboratories in the last 5 years. SCID detection is based on real-time PCR assays to measure T-cell receptor excision circles (TREC), a byproduct of T-cell development. We modified a multiplexed real-time PCR TREC assay to simultaneously determine the presence or absence of the SMN1 gene from a dried blood spot (DBS) punch in a single reaction well. METHOD: An SMN1 assay using a locked nucleic acid probe was initially developed with cell culture and umbilical cord blood (UCB) DNA extracts, and then integrated into the TREC assay. DBS punches were placed in 96-well arrays, washed, and amplified directly using reagents specific for TREC, a reference gene [ribonuclease P/MRP 30kDa subunit (RPP30)], and the SMN1 gene. The assay was tested on DBS made from UCB units and from peripheral blood samples of SMA-affected individuals and their family members. RESULTS: DBS made from SMA-affected individuals showed no SMN1-specific amplification, whereas DBS made from all unaffected carriers and UCB showed SMN1 amplification above a well-defined threshold. TREC and RPP30 content in all DBS were within the age-adjusted expected range. CONCLUSIONS: SMA caused by the absence of SMN1 can be detected from the same DBS punch used to screen newborns for SCID.

Comparative evaluation of newborn bloodspot specimen cards by experienced laboratory personnel and by an optical scanning instrument.

A major factor in determining the suitability of a dried blood spot (DBS) specimen is the subjective nature of evaluation by laboratory personnel. Using newborn screening DBS specimen cards as they were submitted to a public health NBS program, we conducted a systematic pilot study of DBS evaluation by multiple experienced laboratory personnel (ELP) and by an automated optical scanning instrument (OSI) (CardScan (tm), BSD Robotics). OSI confirmed the satisfactory status of all newborn DBS specimen cards that passed initial review by the first ELP. Among the questionable cards selected for further review, 58% passed multiple ELP consensus assessment, and 62% passed OSI evaluation. The overall agreement between ELP and OSI was 86%. Among questionable specimen cards, ELP and OSI were more strongly correlated when multiple ELP assessment was unanimous. We conclude that subjective assessment by ELP is essential and that OSI evaluation is a useful adjunct when ELP assessment does not reach consensus. OSI further allows the selection of optimal locations for punching DBS from unsatisfactory or questionable specimens, optimizing the quality of interim analyses that may be conducted while repeat specimens are being collected. Instrument evaluation of specimen cards would also be valuable as an independent reference method for training laboratory and specimen collection personnel. OSI technology merits further studies to confirm and extend our findings.

Monoclonal B-cell lymphocytosis in healthy blood donors: an unexpectedly common finding.

Circulating monoclonal B cells may be detected in healthy adults, a condition called monoclonal B-cell lymphocytosis (MBL). MBL has also been identified in donated blood, but no systematic study of blood donors has been reported. Using sensitive and specific laboratory methods, we detected MBL in 149 (7.1%; 95% confidence interval, 6.0% to 8.3%) of 2098 unique donors ages 45 years or older in a Midwestern US regional blood center between 2010 and 2011. Most of the 149 donors had low-count MBL, including 99 chronic lymphocytic leukemia-like (66.4%), 22 atypical (14.8%), and 19 CD5(-) (12.8%) immunophenotypes. However, 5 donors (3.4%) had B-cell clonal counts above 500 cells per µL, including 3 with 1693 to 2887 cells per µL; the clone accounted for nearly all their circulating B cells. Four donors (2.7%) had 2 distinct MBL clones. Of 51 MBL samples in which immunoglobulin heavy chain (IGH)V-D-J genotypes could be determined, 71% and 29% used IGHV3- and IGHV4-family genes, respectively. Sequencing revealed 82% with somatic hypermutation, whereas 18% had >98% germ-line identity, including 5 with entirely germ-line sequences. In conclusion, MBL prevalence is much higher in blood donors than previously reported, and although uncommon, the presence of high-count MBL warrants further investigations to define the biological fate of the transfused cells in recipients.

Prevalence of monoclonal B-cell lymphocytosis: a systematic review.

BACKGROUND: Individuals with monoclonal B-cell lymphocytosis (MBL) have been identified in clinic outpatients, in unaffected relatives of patients with chronic lymphocytic leukemia (CLL), and in general populations. MBL and its relationship with CLL have been actively investigated over the last decade. This report systematically reviews the prevalence of MBL in the context of the populations studied and the evolution of laboratory methods used to define MBL. METHODS: To identify published studies that have assessed the prevalence of MBL, we systematically searched the MEDLINE databases and consulted with members of the International MBL Study Group. We reviewed the 10 articles that were identified by this process. We abstracted information on study populations, laboratory tests, criteria for designating MBL, and the reported frequencies. RESULTS: Three of the ten studies were published in 2009, three between 2007 and 2008, and four between 2002 and 2004. Reported prevalences varied widely, ranging from 0.12 to 18.2%. This variability was clearly associated with both the laboratory methods and the populations studied. MBL was more common among older individuals and kindred of persons with CLL. The most common MBL subtype was CLL-like MBL. CONCLUSIONS: Large population-based studies of MBL that employ standardized laboratory methods with a consensus case definition are needed to assess prevalence and establish risk factors. These studies should include prospective follow-up of MBL cases to determine the relationship between MBL and CLL. Data from original studies should be reported in sufficient detail to allow future synthesis of information from multiple studies, such as meta-analysis.

Commentary: Comparison of current flow cytometry methods for monoclonal B cell lymphocytosis detection.

Monoclonal B cell lymphocytosis (MBL) is now recognized as the B-lymphocyte analogue of a monoclonal gammopathy of unknown significance. MBL can be the precursor of chronic lymphocytic leukemia or associated with non-Hodgkin's lymphoma. It may be associated with an autoimmune abnormality or be related to aging (immunosenescence). The combination of available new fluorochrome-conjugated monoclonal antibody reagents, multilaser instrumentation, and improved software tools have led to a new level of multicolor analysis of MBL. Presently, several centers, including the University of Salamanca (Spain), Duke University (Durham, NC), Mayo Clinic (Rochester, MN), and the National Cancer Institute (Bethesda, MD) in conjunction with the Genetics and Epidemiology of Familial chronic lymphocytic leukemia Consortium, the Food and Drug Administration (Bethesda, MD), and the Centers for Disease Control and Prevention/Agency for Toxic Substances and Disease Registry (Atlanta, GA) in collaboration with Saint Luke's Hospital (Kansas City, MO), the Università Vita-Salute San Raffaele in Milan (Italy), and Leeds Teaching Hospital (UK) are all actively conducting studies on MBL. This commentary is an updated summary of the current methods used in these centers. It is important to note the diversity of use in reagents, instruments, and methods of analysis. Despite this diversity, there is a consensus in what constitutes the diagnosis of MBL and its subtypes. There is also an emerging consensus on what the next investigative steps should be.

Long-term follow-up of monoclonal B-cell lymphocytosis detected in environmental health studies.

BACKGROUND: Four individuals in whom Monoclonal B cell Lymphocytosis (MBL) had been previously detected were evaluated for the fourth time after 15-18 years since initial testing. All four were environmental health study participants without hematologic malignancies who had elevated absolute B cell counts at initial testing. METHODS: The current laboratory evaluation included complete blood counts, lymphocyte immunophenotypes, immunoglobulin heavy-chain variable (IGHV) gene mutation status, and serum tests for monoclonal immunoglobulins and free light chains. Results from this evaluation were compared with those from the three previous evaluations. Clinical status was assessed by reviewing medical records. RESULTS: B-cell clones with phenotypic characteristics of the original MBL clone were detected in three of the four individuals. Since the last evaluation in 2003, one participant who had a clinical diagnosis of Waldenstrom's Macroglobulinemia had developed a diffuse large cell lymphoma and was treated. Another participant continued to show a decline in lymphocyte and B cell counts, reaching clinical lymphocytopenia and B cell lymphopenia. The MBL clone was still detectable. The remaining two participants had stable blood counts and MBL phenotypes. Neither had been diagnosed with a hematologic malignancy. However, molecular analysis revealed clonal changes in both: one showed a marked decline in the percentage of somatically-mutated B cells, and the other showed a clonal transition from IGHV3-13 to IGHV4-34. CONCLUSIONS: A diversity of clonal evolution was observed in these MBL cases. These observations suggest that long-term follow-up studies using standardized MBL subcategories are essential to understanding B-cell pathobiology and optimizing clinical management.

Proficiency testing of human leukocyte antigen-DR and human leukocyte antigen-DQ genetic risk assessment for type 1 diabetes using dried blood spots.

BACKGROUND: The plurality of genetic risk for developing type 1 diabetes mellitus (T1DM) lies within the genes that code for the human leukocyte antigens (HLAs). Many T1DM studies use HLA genetic risk assessment to identify higher risk individuals, and they often conduct these tests on dried blood spots (DBSs) like those used for newborn bloodspot screening. One such study is The Environmental Determinants of Diabetes in the Young (TEDDY), a long-term prospective study of environmental risk factors. To provide quality assurance for T1DM studies that employ HLA genetic risk assessment, the Centers for Disease Control and Prevention (CDC) conducts both a voluntary quarterly proficiency testing (VQPT) program available to any laboratory and a mandatory annual proficiency testing (PT) challenge for TEDDY laboratories. METHODS: Whole blood and DBS samples with a wide range of validated HLA-DR and HLA-DQ genotypes were sent to the participating laboratories. Results were evaluated on the basis of both the reported haplotypes and the HLA genetic risk assessment. RESULTS: Of the reported results from 24 panels sent out over six years in the VQPT, 94.7% (857/905) were correctly identified with respect to the relevant HLA-DR or HLA-DQ alleles, and 96.4% (241/250) were correctly categorized for risk assessment. Significant improvement was seen over the duration of this program, usually reaching 100% correct categorization during the last three years. Of 1154 reported results in four TEDDY PT challenges, 1153 (99.9%) were correctly identified for TEDDY eligibility. CONCLUSIONS: The different analytical methods used by T1DM research centers all provided accurate (>99%) results for genetic risk assessment. The two CDC PT programs documented the validity of the various approaches to screening and contributed to overall quality assurance.

Prevalence of congenital hypothyroidism--current trends and future directions: workshop summary.

In response to published newborn-screening data that have shown an increase in the incidence (birth prevalence) rate of primary congenital hypothyroidism (CH) in the United States, a workshop was held in Atlanta, Georgia, on February 27 and 28, 2008, to examine this issue. Topics of the meeting included pathophysiology, medical management, and follow-up of CH; transient hypothyroidism (etiology, clinical implications, management, and changes in prevalence); risk factors for CH; laboratory approaches to newborn screening for CH; state-specific evaluations of trends in incidence rates of CH; and concluding discussions on future directions to resolve outstanding issues. Through presentations and discussion, gaps in knowledge were identified, such as the lack of consistent definitions for CH and transient hypothyroidism and the effects of preventable risk factors on incidence rates of CH. One outcome of the meeting was a series of accompanying articles that examined (1) trends in the incidence rates of CH in individual states and nationally, (2) effects of newborn-screening practices on CH-incidence rates, (3) the contribution of transient hypothyroidism to CH-incidence rates, and (4) future research directions. In this summary, we briefly touch on the topics of these articles and examine highlights of other presentations from the workshop that illuminated the secular trends in reported CH-incidence rates in the United States.

Newborn screening for X-linked adrenoleukodystrophy (X-ALD): validation of a combined liquid chromatography-tandem mass spectrometric (LC-MS/MS) method.

Newborn screening for X-linked adrenoleukodystrophy (X-ALD) has until now been limited in implementation because of the lack of an accepted standard methodology. We have previously reported a technique using LC-MS/MS analysis that could provide the basis for screening of newborns for X-ALD. The target analyte diagnostic for X-ALD and other peroxisomal disorders of peroxisomal beta-oxidation is 1-hexacosanoyl-2-lyso-sn-3-glycero-phosphorylcholine (26:0-lyso-PC). We report here the validation of the analytical method using an authentic standard of the target compound. The method possesses sensitivity of <1.0fmole injected on column with a correlation coefficient (R(2)) of 0.9987. A tetradeuterated analog of 26:0-lyso-PC served as the internal standard. The sensitivity of this clinical method was confirmed using 17 newborn samples of individuals with peroxisomal disorders retrieved from state newborn screening programs. These samples were run masked with over 1000 newborn samples. All affected individuals were identified with one exception. One sample which was retrieved as an affected did not have the biochemical or genetic abnormality of X-ALD and thus is considered an error in sample identity. These studies clearly show that the method is highly sensitive and accurate in identifying individuals with a defect in peroxisomal beta-oxidation such as X-ALD.

Development and evaluation of quality control dried blood spot materials in newborn screening for lysosomal storage disorders.

BACKGROUND: Lysosomal storage disorders (LSDs) comprise more than 40 genetic diseases that result in the accumulation of products that would normally be degraded by lysosomal enzymes. A tandem mass spectrometry (MS/MS)-based method is available for newborn screening for 5 LSDs, and many laboratories are initiating pilot studies to evaluate the incorporation of this method into their screening panels. We developed and evaluated dried blood spot (DBS) QC materials for LSDs and used the MS/MS method to investigate their suitability for LSD QC monitoring. METHODS: We incubated 3.2-mm punches from DBS controls for 20-24 h with assay cocktails containing substrate and internal standard. Using MS/MS, we quantified the resulting product and internal standard. Samples were run in triplicate for 3 consecutive days, and results were reported as product-to-internal standard ratios and enzyme activity units (micromol/L/h). RESULTS: Enzyme activity interday imprecision (CV) for the high, medium, and low series were 3.4%-14.3% for galactocerebroside alpha-galactosidase, 6.8%-24.6% for acid alpha-galactosidase A, 7.36%-22.1% for acid sphingomyelinase, 6.2%-26.2% for acid alpha-glucocerebrosidase, and 7.0%-24.8% for lysosomal acid alpha-glucosidase (n = 9). In addition, DBS stored at -20 degrees and 4 degrees C showed minimal enzyme activity loss over a 187-d period. DBS stored at 37 degrees and 45 degrees C had lower activity values over the 187-day evaluation time. CONCLUSIONS: Suitable QC materials for newborn screening of LSDs were developed for laboratories performing DBS LSD screening. Good material linearity was observed, with goodness-of-fit values of 0.953 and higher. The QC materials may be used by screening laboratories that perform LSD analysis by MS and/or more conventional fluorescence-based screening methods.