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[Editorials] Cerebrospinal Fluid Biomarkers in the Evaluation of Alzheimer Disease

[Editorials] Rapid and Effective Screening for Lysosomal Storage Disease: How Close Are We?

[Editorials] Requesting and Interpreting Urine Albumin Measurements in the Primary Health Care Setting

[Editorials] Multiple Thiopurine S-Methyltransferase Variation Detection: A Step toward Personalized Medicine

[Mini-Review] Utility of Kallikrein-Related Peptidases (KLKs) as Cancer Biomarkers

Background: The human kallikrein-related peptidase (KLK) family consists of 15 highly conserved serine proteases, which are encoded by the largest uninterrupted cluster of protease genes in the human genome. To date, several members of the family have been reported as potential cancer biomarkers. Although primarily known for their biomarker value in prostate, ovarian, and breast cancers, more recent data suggest analogous roles of KLKs in several other cancers, including gastrointestinal, head and neck, lung, and brain malignancies. Among the proposed KLK cancer biomarkers, prostate-specific antigen (also known as KLK3) is the most widely recognized member in urologic oncology. Content: Despite substantial progress in the understanding of the biomarker utility of individual KLKs, the current challenge lies in devising biomarker panels to increase the accuracy of prognosis, prediction of therapy, and diagnosis. To date, multiparametric KLK panels have been proposed for prostate, ovarian, and lung cancers. In addition to their biomarker utility, emerging evidence has revealed a number of critical functional roles for KLKs in the pathogenesis of cancer and their potential use as therapeutic targets. Summary: KLKs have biomarker utility in many cancer types but individually lack sufficient specificity or sensitivity to be used in clinical practice; however, groups of KLKs and other candidate biomarkers may offer improved performance.

[Mini-Review] High-Abundance Polypeptides of the Human Plasma Proteome Comprising the Top 4 Logs of Polypeptide Abundance

Background: Plasma contains thousands of proteins, but a small number of these proteins comprise the majority of protein molecules and mass. Content: We surveyed proteomic studies to identify candidates for high-abundance polypeptide chains. We searched the literature for information on the plasma concentrations of the most abundant components in healthy adults and for the molecular mass of the mature polypeptide chains in plasma. Because proteomic studies usually dissociate proteins into polypeptide chains or detect short peptide segments of proteins, we summarized data on individual peptide chains for proteins containing multiple subunits or polypeptides. We collected data on about 150 of the most abundant polypeptides in plasma. The abundant polypeptides span approximately the top 4 logs of concentration in plasma, from 650 to 0.06 µmol/L on a molar basis or from about 50 000 to 1 mg/L mass abundance. Conclusions: Data on the concentrations of the high-abundance peptide chains in plasma assist in understanding the composition of plasma and potential approaches for clinical laboratory or proteomic analysis of plasma proteins. Development of more extensive databases regarding the plasma concentrations of proteins in health and diseases would promote diagnostic and proteomic advances.

[Proteomics and Protein Markers] The Brain Injury Biomarker VLP-1 Is Increased in the Cerebrospinal Fluid of Alzheimer Disease Patients

background: Definitive diagnosis of Alzheimer disease (AD) can be made only by histopathological examination of brain tissue, prompting the search for premortem disease biomarkers. We sought to determine if the novel brain injury biomarker, visinin-like protein 1 (VLP-1), is altered in the CSF of AD patients compared with controls, and to compare its values to the other well-studied CSF biomarkers 42-amino acid amyloid-β peptide (Aβ1–42), total Tau (tTau), and hyperphosphorylated Tau (pTau). methods: Using ELISA, we measured concentrations of Aβ1–42, tTau, pTau, and VLP-1 in CSF samples from 33 AD patients and 24 controls. We compared the diagnostic performance of these biomarkers using ROC curves. results: CSF VLP-1 concentrations were significantly higher in AD patients [median (interquartile range) 365 (166) ng/L] compared with controls [244 (142.5) ng/L]. Although the diagnostic performance of VLP-1 alone was comparable to that of Aβ, tTau, or pTau alone, the combination of the 4 biomarkers demonstrated better performance than each individually. VLP-1 concentrations were higher in AD subjects with APOE 4/4 genotype [599 (240) ng/L] compared with 3/4 [376 (127) ng/L] and 3/3 [280 (115.5) ng/L] genotypes. Furthermore, VLP-1 values demonstrated a high degree of correlation with pTau (r = 0.809) and tTau (r = 0.635) but not Aβ1–42 (r = –0.233). VLP-1 was the only biomarker that correlated with MMSE score (r = –0.384, P = 0.030). conclusions: These results suggest that neuronal injury markers such as VLP-1 may have utility as biomarkers for AD.

[Pediatric Clinical Chemistry] Newborn Screening for Pompe Disease by Measuring Acid {alpha}-Glucosidase Activity Using Tandem Mass Spectrometry

background: Pompe disease, caused by the deficiency of acid -glucosidase (GAA), is a lysosomal storage disorder that manifests itself in its most severe form within the first months of life. Early detection by newborn screening is warranted, since prompt initiation of enzyme replacement therapy may improve morbidity and mortality. We evaluated a tandem mass spectrometry (MS/MS) method to measure GAA activity for newborn screening for Pompe disease. methods: We incubated 3.2-mm punches from dried blood spots (DBS) for 22 h with the substrate [7-benzoylamino-heptyl)-{2-[4-(3,4,5-trihydroxy-6-hydroxymethyl-tetrahydro-pyran-2-yloxy)-phenylcarbamoyl]- ethyl}-carbamic acid tert-butyl ester] and internal standard [7-d5-benzoylamino-heptyl)-[2-(4-hydroxy-phenylcarbamoyl)-ethyl]-carbamic acid tertbutyl ester]. We quantified the resulting product and internal standard using MS/MS. We assessed inter- and intrarun imprecision, carryover, stability, and correlation between enzyme activities and hematocrit and punch location and generated a Pompe disease–specific cutoff value using routine newborn screening samples. results: GAA activities in DBS from 29 known Pompe patients were <2 µmol/h/L. GAA activities in routine newborn screening samples were [mean (SD)] 14.7 (7.2) µmol/h/L (n = 10 279, median 13.3, 95% CI 14.46–14.74 µmol/h/L) and in normal adult samples 9.3 (3.3) µmol/h/L (n = 229, median 9, 95% CI 8.88–9.72 µmol/h/L). GAA activity was stable for 28 days between 37 °C and –80 °C. Carryover could not be observed, whereas intrarun and interrun imprecision were <10%. The limit of detection was 0.26 µmol/h/L and limit of quantification 0.35 µmol/h/L. conclusions: The measurement of GAA activities in dry blood spots using MS/MS is suitable for high-throughput analysis and newborn screening for Pompe disease.

[Evidence-Based Laboratory Medicine and Test Utilization] Postanalytical External Quality Assessment of Urine Albumin in Primary Health Care: An International Survey

background: Microalbuminuria (MA) is recognized as an important risk factor for cardiovascular and renal complications in diabetes. We sought to evaluate how screening for MA is conducted and how urine albumin (UA) results are interpreted in primary care internationally. methods: General practitioners (GPs) received a case history–based questionnaire depicting a male type 2 diabetes patient in whom UA testing had not been performed. Questions were related to type of urine sample used for UA testing, need for a repeat test, whether UA testing was performed in the office laboratory, and what changes in UA results were considered clinically important [critical difference (CD)]. Participants received national benchmarking feedback reports. results: We included 2078 GPs from 9 European countries. Spot urine samples were used most commonly for first time office-based testing, whereas timed collections were used to a larger extent for hospital-based repeat tests. Repeat tests were requested by 45%–77% of GPs if the first test was positive. Four different measurement units were used by 70% of participants in estimating clinically important changes in albumin values. Stated CDs varied considerably among GPs, with similar variations in each country. A median CD of 33% was considered clinically important for both improvement and deterioration in MA, corresponding to an achievable analytical imprecision of 14%, when UA is reported as an albumin/creatinine ratio. conclusions: Guidelines on diagnosing MA are followed only partially, and should be made more practicable, addressing issues such as type of samples, measurement units, and repeat tests.

[Molecular Diagnostics and Genetics] Highly Multiplexed Genotyping of Thiopurine S-Methyltransferase Variants Using MALDI-TOF Mass Spectrometry: Reliable Genotyping in Different Ethnic Groups

Background: To avoid severe hematotoxicity in patients, determination of the TPMT (thiopurine S-methyltransferase) genotype before commencing thiopurine therapy has become accepted. Methods: We used MALDI-TOF mass spectrometry (MS) based on Sequenom iPLEX® technology to develop novel multiplex assays for comprehensive testing of TPMT. Two assays, a 15-plex and a 7-plex assay, consisting of multiplex PCR, shrimp alkaline phosphatase treatment, primer extension, and MALDI-TOF MS analysis, allow detection of all currently known functionally relevant 24 TPMT alleles (TPMT*2 to *18, *20 to *23). Previously identified variant DNA samples and newly constructed synthetic templates were used as quality controls. Results: Assay evaluation performed on a panel of 586 genomic DNA samples previously genotyped by other methods (denaturing HPLC, sequencing) resulted in 100% agreement. Analyses of the distribution of TPMT alleles in 116 samples from a Ghanaian population revealed a TPMT*8 allele frequency of 3.4%. In a Korean population of 118 unrelated individuals, we found a TPMT*6 allele frequency of 1.3%. Conclusions: The newly developed multiplex MALDI-TOF MS assay allows efficient genotyping for all currently known functional TPMT variants. To achieve the most accurate prediction of TPMT phenotype, molecular diagnosis of TPMT should include all these variants.

[Molecular Diagnostics and Genetics] Snapback Primer Genotyping with Saturating DNA Dye and Melting Analysis

Background: DNA hairpins have been used in molecular analysis of PCR products as self-probing amplicons. Either physical separation or fluorescent oligonucleotides with covalent modifications were previously necessary. Methods: We performed asymmetric PCR for 40–45 cycles in the presence of the saturating DNA dye, LCGreen Plus, with 1 primer including a 5' tail complementary to its extension product, but without any special covalent modifications. Samples were amplified either on a carousel LightCycler for speed or on a 96/384 block cycler for throughput. In addition to full-length amplicon duplexes, single-stranded hairpins were formed by the primer tail "snapping back" and hybridizing to its extension product. High-resolution melting was performed on a HR-1 (for capillaries) or a LightScanner (for plates). Results: PCR products amplified with a snapback primer showed both hairpin melting at lower temperature and full-length amplicon melting at higher temperature. The hairpin melting temperature was linearly related to the stem length (6–28 bp) and inversely related to the log of the loop size (17–135 bases). We easily genotyped heterozygous and homozygous variants within the stem, and 100 blinded clinical samples previously typed for F5 1691G>A (Leiden) were completely concordant by snapback genotyping. We distinguished 7 genotypes in 2 regions of CFTR exon 10 with symmetric PCR using 2 snapback primers followed by product dilution to favor intramolecular hybridization. Conclusions: Snapback primer genotyping with saturating dyes provides the specificity of a probe with only 2 primers that are free of special covalent labels in a closed-tube system.

[Molecular Diagnostics and Genetics] Genotyping {beta}-Globin Gene Mutations on Copolymer-Coated Glass Slides with the Ligation Detection Reaction

Background: Methods are needed to analyze small amounts of samples for variation in disease-causing genes. One means is to couple the sensitivity and multiplexing capability of the ligation detection reaction (LDR) with the use of simple glass slides specifically functionalized with a novel polymer coating to enhance sensitivity. Methods: We developed an array-based genotyping assay based on glass slides coated with copolymer (N,N-dimethylacrylamide, N,N-acryloyloxysuccinimide, and 3-(trimethoxysilyl)propyl methacrylate). The assay consists of an LDR with genomic DNA followed by a universal PCR (U-PCR) of genomic DNA–templated LDR product. The LDR occurs in the presence of 3 primers for each sequence variant under investigation: 2 distinguishing primers (allele specific and perfectly complementary to wild-type and mutant alleles) and 1 common locus-specific primer. The 2 allele-specific primers have different capture sequences for binding different complementary probes on a tag array. The LDR product templated from genomic DNA is made fluorescent during the U-PCR via incorporation of a Cy5-labeled universal primer into all LDR products; detection occurs on the coated glass slides. Results: The assay was designed to detect 7 prevalent mutations in the β-globin gene (HBB, hemoglobin, beta) in a multiplex format, and signals for the different alleles are detected by their fluorescence. The assay was applied to 40 genomic DNA samples from both control individuals and patients with known β-thalassemia mutations. Results show good correspondence between the patients’ genotypes as assessed by DNA sequence analysis and those generated from the LDR assays. Conclusions: The developed technology allows accurate identification of sequence variants in a simple, cost-effective way and offers good flexibility for scaling to other applications with different numbers of single-nucleotide polymorphisms or mutations to be detected.

[Molecular Diagnostics and Genetics] Microfluidics Digital PCR Reveals a Higher than Expected Fraction of Fetal DNA in Maternal Plasma

Background: The precise measurement of cell-free fetal DNA in maternal plasma facilitates noninvasive prenatal diagnosis of fetal chromosomal aneuploidies and other applications. We tested the hypothesis that microfluidics digital PCR, in which individual fetal-DNA molecules are counted, could enhance the precision of measuring circulating fetal DNA. Methods: We first determined whether microfluidics digital PCR, real-time PCR, and mass spectrometry produced different estimates of male-DNA concentrations in artificial mixtures of male and female DNA. We then focused on comparing the imprecision of microfluidics digital PCR with that of a well-established nondigital PCR assay for measuring male fetal DNA in maternal plasma. Results: Of the tested platforms, microfluidics digital PCR demonstrated the least quantitative bias for measuring the fractional concentration of male DNA. This assay had a lower imprecision and higher clinical sensitivity compared with nondigital real-time PCR. With the ZFY/ZFX assay on the microfluidics digital PCR platform, the median fractional concentration of fetal DNA in maternal plasma was ≥2 times higher for all 3 trimesters of pregnancy than previously reported. Conclusions: Microfluidics digital PCR represents an improvement over previous methods for quantifying fetal DNA in maternal plasma, enabling diagnostic and research applications requiring precise quantification. This approach may also impact other diagnostic applications of plasma nucleic acids, e.g., in oncology and transplantation.

[Endocrinology and Metabolism] Serum Total Prolactin and Monomeric Prolactin Reference Intervals Determined by Precipitation with Polyethylene Glycol: Evaluation and Validation on Common ImmunoAssay Platforms

Background: Macroprolactin is an important source of immunoassay interference that commonly leads to misdiagnosis and mismanagement of hyperprolactinemic patients. We used the predominant immunoassay platforms for prolactin to assay serum samples treated with polyethylene glycol (PEG) and establish and validate reference intervals for total and monomeric prolactin. Methods: We used the Architect (Abbott), ADVIA Centaur and Immulite (Siemens Diagnostics), Access (Beckman Coulter), Elecsys (Roche Diagnostics), and AIA (Tosoh) analyzers with samples from healthy males (n = 53) and females (n = 93) to derive parametric reference intervals for total and post-PEG monomeric prolactin. Concentrations of immunoreactive prolactin isoforms in serum samples from healthy individuals were established by gel filtration chromatography (GFC). We then used samples from 22 individuals whose hyperprolactinemia was entirely attributable to macroprolactin and 32 patients with true hyperprolactinemia to compare patient classifications and prolactin concentrations measured by GFC with the newly derived post-PEG reference intervals. Results: Parametric reference intervals for post-PEG prolactin in male and female serum samples, respectively, were (in mIU/L): 61–196, 66–278 (Centaur); 63–245, 75–381 (Elecsys); 70–301, 92–469 (Access); 72–229, 79–347 (Architect); 73–247, 83–383 (AIA); and 78–263, 85–394 (Immulite). Concordance between GFC and immunoassay-specific post-PEG reference intervals was observed in 311 of 324 cases and for 31 of 32 patients with true hyperprolactinemia and 17 of 22 patients with macroprolactinemia. Results leading to misclassification occurred in a few analyzers for 5 macroprolactinemia patient samples with relatively minor increases in post-PEG prolactin (mean 61 mIU/L). Conclusions: Our validated normative reference data for sera pretreated with PEG and analyzed on the most commonly used immunoassay platforms should facilitate the more widespread introduction of macroprolactin screening by clinical laboratories.

[Endocrinology and Metabolism] Plasma Insulinlike Growth Factor 1 and Binding-Protein 3 and Risk of Myocardial Infarction in Women: A Prospective Study

Background: The aim of this study was to prospectively evaluate relationships between plasma concentrations of insulinlike growth factor 1 (IGF1) and insulinlike growth factor binding protein 3 (IGFBP3) and subsequent myocardial infarction (MI) in women. Methods: We used case-control sampling to select study participants from women who had already been selected for inclusion in the prospective Nurses’ Health Study cohort. Blood samples were collected from 32 826 women in 1989–1990. During the follow-up period from sample collection through June 1998, MI (fatal and nonfatal) was diagnosed in 245 women. Cases were matched to controls 1:2 by age, cigarette-smoking status, and month and fasting status at the time of blood collection. Conditional logistic regression was used to adjust for potential confounders (menopausal status, parental history of MI, postmenopausal hormone use, diabetes mellitus, hypertension, hypercholesterolemia, aspirin use, alcohol use, body mass index, and physical activity). Results: Multivariable adjusted analyses did not reveal a statistically significant linear relationship between IGF1 or IGFBP3 concentrations or their molar ratio and risk of MI. Women in the highest IGF1 quartile had a multivariable-adjusted rate ratio of 1.46 (95% CI 0.79, 2.72; P for trend = 0.46) for MI, compared with those in the lowest. The corresponding rate ratios (95% CI) for IGFBP3 and the IGF1:IGFBP3 mol/L ratio were 1.24 (0.71, 2.17) and 1.29 (0.70, 2.37), respectively. Conclusions: We did not observe a monotonic relationship between IGF1 or IGFBP3 and MI among predominantly postmenopausal women. Future studies are warranted to evaluate these relationships in other demographic groups including younger women.

[Automation and Analytical Techniques] Clinical Evaluation of Bionime Rightest GM310 Biosensors with a Simplified Electrode Fabrication for Alternative-Site Blood Glucose Tests

Background: Most processes for fabricating biosensors applied to screen-printed carbon electrodes (SPCEs) are complex. This study presents a novel one-step process for manufacturing electrodes for injection-molding biosensors. Methods: During the sensor-fabrication process, barrel-plated gold electrodes were inserted into an injection-molded base. The electrode directly touched the electrical contact of a meter. We analyzed technical measurements for this biosensor, including tests of the measurement range, within-run imprecision, and between-meter imprecision. In clinical trials, experienced technicians tested 3 alternative sites (fingertip, palm, and arm). The results were simultaneously compared with plasma values obtained with the hexokinase method on the Olympus AU640 instrument. Analytical results were evaluated according to International Standards Organization 15197 (ISO 15197:2003) criteria and by Clarke error grid analysis (EGA), and CVs were calculated to evaluate within-run imprecision. Results: The glucose measurement range was 0.6– 33.3 mmol/L (y = 0.96x + 0.07 mmol/L; r2 = 0.9977). The CVs in the within-run imprecision test were 1.7%–3.5%, and the overall CV was 2.1%, indicating good reproducibility of results. The Student t-tests of mean values from 5 meters revealed statistically insignificant differences (P > 0.05). In clinical trials, the agreement of the Rightest GM310 meter results with those of a laboratory method complied with ISO 15197:2003 criteria. In the EGA, 100% of the values were within the acceptable zones (A + B), and the proportion of values within zone A exceeded 95%. Conclusions: The Bionime Rightest GM310 meter applied a simplified process for biosensor fabrication and displayed acceptable performance for monitoring glucose concentrations at alternative test sites.

[Cancer Diagnostics] Prognostic Value of Mature MicroRNA-21 and MicroRNA-205 Overexpression in Non-Small Cell Lung Cancer by Quantitative Real-Time RT-PCR

Background: microRNA (miRNA) expression profiles are being intensively investigated for their involvement in carcinogenesis. We evaluated the prognostic value of mature microRNA-21 (miR-21) and mature microRNA-205 (miR-205) overexpression in non–small cell lung cancer (NSCLC). Patients and methods: We studied 48 pairs of NSCLC fresh frozen tissue specimens collected at time of surgery and before chemotherapy. Highly specific amplification and quantification of mature miR-21 and mature miR-205 was achieved using looped real time RT-PCR. Results: miRNA expression, determined by real time RT-PCR, was defined by Ct measurements. We detected overexpression of mature miR-21 in 25 (52.0%) of the 48 NSCLC paired specimens and overexpression of miR-205 in 31 (64.6%). Overexpression was assessed after comparison of miRNA expression in NSCLC tissues and in their corresponding noncancerous tissues with respect to U6 expression. During the follow-up period, 29 of 48 (60.4%) patients relapsed, and 23 of 48 died (47.9%). Mature miR-21 was upregulated in 16 of 29 (55.2%) patients who relapsed and 15 of 23 (65.2%) patients who died. Mature miR-205 was overexpressed in 19 of 29 patients who relapsed (65.5%) and 15 of 23 patients who died (65.2%). Mature miR-21 overexpression correlated with overall survival (OS) of the patients (P = 0.027), whereas overexpression of mature miR-205 did not. Conclusions: Our results suggest that overexpression of mature miR-21 is an independent negative prognostic factor for OS in NSCLC patients.

[Cancer Diagnostics] Intraplatform Reproducibility and Technical Precision of Gene Expression Profiling in 4 Laboratories Investigating 160 Leukemia Samples: The DACH Study

Background: Gene expression profiling has the potential to offer consistent, objective diagnostic test results once a standardized protocol has been established. We investigated the robustness, precision, and reproducibility of microarray technology. Methods: One hundred sixty individual patient samples representing 11 subtypes of acute and chronic leukemias, myelodysplastic syndromes, and nonleukemia as a control group were centrally collected and diagnosed as part of the daily routine in the Munich Leukemia Laboratory. The custom AmpliChip Leukemia research microarray was used for technical analyses of quadruplicate mononuclear cell lysates in 4 different laboratories in Germany (D), Austria (A), and Switzerland (CH) (the DACH study). Results: Total-RNA preparations were successfully performed in 637 (99.5%) of 640 cases. Mean differences between pairs of laboratories in the total-RNA yield from the same sample ranged from 0.02 µg to 1.03 µg. Further processing produced 622 successful in vitro transcription reactions (97.6%); the mean differences between laboratories in the cRNA yield from the same sample ranged from 0.40 µg to 6.18 µg. After hybridization to microarrays, a mean of 47.6%, 46.5%, 46.2%, and 46.4% of probe sets were detected as present for the 4 laboratories, with mean signal-intensity scaling factors of 3.1, 3.7, 4.0, and 4.2, respectively. In unsupervised hierarchical cluster and principal component analyses, replicates from the same patient always clustered closely together, with no indications of any association between gene expression profiles due to different operators or laboratories. Conclusions: Microarray analysis can be performed with high interlaboratory reproducibility and with comparable quality and high technical precision across laboratories.

[Cancer Diagnostics] Analysis of MicroRNAs in Pancreatic Fine-Needle Aspirates Can Classify Benign and Malignant Tissues

Background: MicroRNAs (miRNAs) are RNA molecules that are involved in the regulation of many cellular processes, including those related to human cancers. The aim of this study was to determine, as a proof of principle, whether specific candidate miRNAs could be detected in fine-needle aspirate (FNA) biopsies of pancreatic ductal adenocarcinoma (PDAC) and could accurately differentiate malignant from benign pancreatic tissues. Methods: We used TaqMan® assays to quantify miRNA levels in FNA samples collected in RNARetain (n = 16) and compared the results with a training set consisting of frozen macrodissected pancreatic samples (n = 20). Results: Quantitative reverse-transcription PCR analysis confirmed that miRNA levels are affected in PDAC FNAs and correlate well with the changes observed in the training set of frozen pancreatic samples. Analysis of the amounts produced for a few specific miRNAs enabled identification of PDAC samples. The combination of miR-196a and miR-217 biomarkers further improved the ability to distinguish between healthy tissue, PDAC, and chronic pancreatitis in the training set (P = 8.2 x 10–10), as well as segregate PDAC FNA samples from other FNA samples (P = 1.1 x 10–5). Furthermore, we showed that miR-196a production is likely specific to PDAC cells and that its incidence paralleled the progression of PDAC. Conclusions: To the best of our knowledge, this study is the first to evaluate the diagnostic potential of miRNAs in a clinical setting and has shown that miRNA analysis of pancreatic FNA biopsy samples can aid in the pathologic evaluation of suspicious cases and may provide a new strategy for improving the diagnosis of pancreatic diseases.

[Brief Communications] Multiplex Enzyme Assay Screening of Dried Blood Spots for Lysosomal Storage Disorders by Using Tandem Mass Spectrometry

Background: Reports of the use of multiplex enzyme assay screening for Pompe disease, Fabry disease, Gaucher disease, Niemann-Pick disease types A and B, and Krabbe disease have engendered interest in the use of this assay in newborn screening. We modified the assay for high-throughput use in screening laboratories. Methods: We optimized enzyme reaction conditions and procedures for the assay, including the concentrations of substrate (S) and internal standard (IS), assay cocktail compositions, sample clean-up procedures, and mass spectrometer operation. The S and IS for each enzyme were premixed and bottled at an optimized molar ratio to simplify assay cocktail preparation. Using the new S:IS ratio, we validated the modified assay according to CLSI guidelines. Stability of the S, IS, and assay cocktails were investigated. Dried blood spots from 149 healthy adults, 100 newborns, and 60 patients with a lysosomal storage disorder (LSD) were tested using the modified assay. Results: In our study, the median enzyme activity measured in adults was generally increased 2–3–fold compared to the original method, results indicating higher precision. In the multiplex format, each of the 5 modified enzyme assays enabled unambiguous differentiation between samples from healthy individuals (adults and newborns) and the corresponding disease-specific samples. Conclusions: The modified multiplex enzyme assay with premixed S and IS is appropriate for use in high-throughput screening laboratories.

[Brief Communications] Isopropanol Protein Precipitation for the Analysis of Plasma Free Metanephrines by Liquid Chromatography-Tandem Mass Spectrometry

Background: High-performance liquid chromatography–tandem mass spectrometric (LC-MS/MS)1 analysis of plasma free metanephrines is the most diagnostically sensitive and specific screening test for the diagnosis of pheochromocytoma. We sought to develop an in-house method for this expensive test Methods: We used off-line isopropanol protein precipitation of plasma to remove interfering substances before LC-MS/MS analysis. We compared the extraction efficiency and limits of quantification of protein precipitation to those of previously reported solid-phase techniques. Results: The new method had limits of quantification of 0.09 nmol/L and 0.17 nmol/L for metanephrine and normetanephrine, respectively. Method comparison with a previously described solid-phase extraction method revealed Deming regression slopes of 0.904 and 0.994, intercepts of 0.007 and 0.023, and SEs of the residuals (Sy|x) of 0.071 and 0.284 for metanephrine and normetanephrine, respectively. Extraction efficiency of isopropanol protein precipitation was 66% for metanephrine and 35% for normetanephrine, results that were superior to the efficiencies of 4% and 1% for our adapted solid-phase extraction method. No ion suppression was observed at the retention times for metanephrine and normetanephrine. Conclusions: Isopropanol protein precipitation is a novel and effective off-line sample preparation method for metanephrines that offers a less expensive alternative to on-line solid-phase extraction for low-volume testing and requires a sample volume of only 200 µL. The mass spectrometric analysis time is equivalent to that of solid-phase techniques.

[Clinical Case Study] A Girl with Severe Hand Swelling and Abdominal Cramps

[Clinical Case Study] Commentary

[Letters to the Editor] Calibration of Fractionated Metanephrines in Urine: Still an Issue?

[Letters to the Editor] Maple Syrup Urine Disease: Newborn Screening Fails to Discriminate between Classic and Variant Forms

[Letters to the Editor] Association of the FABP2 T54 Variant with Plasma Triglycerides and Insulin Resistance in a Multiethnic Population

[Letters to the Editor] Quantification of Urinary Light Chains

[Letters to the Editor] Precision of High-Throughput Single-Nucleotide Polymorphism Genotyping with Fingernail DNA: Comparison with Blood DNA