Midori Mitui

Comparison of Automated Nucleic Acid Extraction Methods with Manual Extraction

Automated nucleic acid extractors can improve workflow and decrease variability in the clinical laboratory. We evaluated Qiagen EZ1 (Valencia, CA) and bioMérieux (Durham, NC) easyMAG extractors compared with Qiagen manual extraction using targets and matrices commonly available in the clinical laboratory. Pooled samples were spiked with various organisms, serially diluted, and extracted in duplicate. The organisms/matrices were Bordetella pertussis/bronchoalveolar lavage, herpes simplex virus II/cerebrospinal fluid, coxsackievirus A9/cerebrospinal fluid, BK virus/plasma, and Mycoplasma pneumoniae/endotracheal tube samples. Extracts were amplified in duplicate using real-time PCR assays, and amplification of the target at a cycle threshold of 35 using the manual method was used for comparison. Amplification efficiency of nucleic acids extracted by automated methods was similar to that by the manual method except for a loss of efficiency for M. pneumoniae in endotracheal tube samples. The EZ1 viral kit 2.0 gave better results for coxsackievirus A9 than the EZ1 viral kit version 1.0. At the lowest limit of detection (past a cycle threshold of 35), the easyMAG was more likely to produce amplifiable nucleic acid than were either the EZ1 or manual extraction. Operational complexity, defined as the number of manipulations required to obtain an extracted sample, was the lowest for the easyMAG. The easyMAG was the most expensive of the methods, followed by the EZ1 kit and manual extraction.

A Lean Laboratory: Operational Simplicity and Cost Effectiveness of the Luminex xTAG Respiratory Viral Panel

During certain months of the year, viral respiratory infections lead to a dramatic increase in pediatric emergency room visits and hospital admissions. Rapid identification of the infectious organism results in timely treatment and reductions in hospital cost and length of stay. Before the introduction of molecular testing to the virology laboratory, diagnosis relied on the standard methods of immunofluorescence and culture. These tests can be labor-intensive and costly. Recent studies have demonstrated the higher sensitivity, faster turnaround, and broader diagnostic spectrum provided by multiplexed RT-PCR assays. Data comparing the laboratory cost and labor efficiency of the tests are lacking. To address this issue, we chose to implement the principles of operational workflow analysis using lean methodology to critically evaluate the potential advantages of a multiplexed RT-PCR assay both in terms of workflow and cost effectiveness. Our results indicated that the implementation of the Luminex xTAG Respiratory Viral Panel (RVP) resulted in a standardized workflow with decreased requirements in laboratory cost as well as improvement in efficiency. In summary, we demonstrate that, in our laboratory, the Luminex xTAG RVP is more operationally streamlined and cost-effective than standard viral direct fluorescent antibody and culture. Further studies are needed to highlight additional benefits of the test, including shortened hospital stay and improved patient outcome.

Isocitrate Dehydrogenase 1/2 Mutational Analyses and 2-Hydroxyglutarate Measurements in Wilms Tumors

L‐2‐Hydroxyglutaric aciduria (L‐2‐HGA) is an uncommon inborn error of metabolism, in which the patients are predisposed to develop brain tumors. Elevated levels of D‐2‐hydroxyglutarate have been demonstrated with malignant gliomas and myeloid leukemias associated with somatic mutations of the genes encoding NADP(+)‐dependent isocitrate dehydrogenases (IDH1 and IDH2, respectively). Recently, we noted a Wilms tumor in a child with L‐2‐HGA. Given the accumulating evidence that both enantiomers of 2‐hydroxyglutarate are associated with cellular transformation, we investigated if sporadic Wilms tumors are associated with IDH1 or IDH2 mutations or with elevated levels of 2‐hydroxyglutarate.

Papillary thyroid carcinoma shows elevated levels of 2-hydroxyglutarate

Elevated levels of d-2-hydroxyglutarate (d-2-HG) occur in gliomas and myeloid leukemias associated with mutations of IDH1 and IDH2. l-2-Hydroxyglutaric aciduria, an inherited metabolic disorder, predisposes to brain tumors. Therefore, we asked whether sporadic cancers, without IDH1 or IDH2 hot-spot mutations, show elevated 2-hydroxyglutarate levels. We retrieved 15 pairs of frozen papillary thyroid carcinoma (PTC) and adjacent non-neoplastic thyroid, and 14 pairs of hyperplastic nodule (HN) and adjacent non-hyperplastic thyroid. In all lesions, exon 4 sequencing confirmed the absence of known mutations of IDH1 and IDH2. We measured 2-hydroxyglutarate by liquid chromatography-tandem mass spectrometry. Compared to normal thyroid, PTCs had significantly higher d-2-HG and l-2-hydroxyglutarate (l-2-HG) levels, and compared to HNs, PTCs had significantly higher d-2-HG levels. d-2-HG/l-2-HG levels were not significantly different between HNs and normal thyroid. Further studies should clarify if elevated 2-hydroxyglutarate in PTC may be useful as cancer biomarker and evaluate the role of 2-hydroxyglutarate in cancer biology.

A subset of cranial fasciitis is associated with dysregulation of the Wnt/β-catenin pathway

Cranial fasciitis, an unusual fibroproliferative lesion that occurs in the scalp of infants, is considered a posttraumatic reactive process similar to nodular fasciitis. Its pathobiology has not been investigated. Over the last 15 years, we diagnosed cranial fasciitis in six children; in one case, the lesion recurred after 4 years. This lesion and two others showed aberrant, diffuse nuclear reactivity for β-catenin. One of the lesions with aberrant nuclear β-catenin occurred in a child with a history of familial adenomatous polyposis (FAP) and a germline frameshift adenomatous polyposis coli (APC) mutation, c.878delG. The other APC allele in this tumor showed an acquired nonsense mutation, c.4132C → T. Both these mutations lead to translation of a truncated APC protein. The other two cases of cranial fasciitis with aberrant nuclear β-catenin occurred sporadically. One of these showed a point mutation, c.122C → T, in exon 3 of CTNNB1. This mutation causes replacement of threonine with isoleucine at codon 41, leading to loss of a phosphorylation site in the β-catenin protein. The third case with nuclear β-catenin staining was the single one that showed recurrence. This tumor did not show mutations in exon 3 of CTNNB1 or in exons 8/9/16 of APC. The results of this small study indicate a dysregulation of the Wnt/β-catenin pathway in a subset of cranial fasciitis, suggesting that this subset is pathobiologically related to desmoid fibromatoses rather than to nodular fasciitis. Occasional cases of cranial fasciitis may be associated with FAP and serve as an early indicator of this disease, information that would be important in the early diagnosis of FAP in patients without a family history of polyposis.

Evaluation of Maxwell® 16 for automated DNA extraction from whole blood and formalin-fixed paraffin embedded (FFPE) tissue.

Abstract Background: The aim of the study was to assess the performance of Promega, Maxwell® 16 for the extraction of genomic DNA from whole blood and FFPE tissue. Methods: DNA was extracted from 10 whole blood and 10 FFPE specimens using six different commercial kits. Results: For whole blood, the mean DNA concentration obtained by Maxwell® 16 was significantly greater than either easyMAG® (p<0.0001) or QIAamp® Blood DNA kit (p<0.001). For FFPE, the mean DNA concentration obtained by the AllPrep® FFPE specific DNA/RNA kit was significantly greater than either the Maxwell® 16 (p<0.0001) or the general AllPrep® DNA/RNA kit (p<0.0001). Conclusions: Comparative evaluation of the six DNA extraction kits indicated that the semi-automated Maxwell® 16 was superior for whole blood extraction while the manual AllPrep® FFPE DNA/RNA kit (Qiagen) performed better for FFPE DNA extraction in terms of quantity of DNA obtained. All six extraction methods (blood and FFPE) performed well in terms of purity. Although there were variances in the quantity of DNA obtained, there were no significant differences in the efficiency of these methods in yielding amplifiable DNA extracts, as demonstrated by β-actin for whole blood specimens. In evaluation of FFPE DNA extraction methods, the Qiagen AllPrep® FFPE DNA/RNA Mini Kit was the best for applications requiring larger amplicons, but for smaller amplicons the Maxwell was most consistent.

Multi-Institutional FASTQ File Exchange as a Means of Proficiency Testing for Next-Generation Sequencing Bioinformatics and Variant Interpretation

Next-generation sequencing is becoming increasingly common in clinical laboratories worldwide and is revolutionizing clinical molecular testing. However, the large amounts of raw data produced by next-generation sequencing assays and the need for complex bioinformatics analyses present unique challenges. Proficiency testing in clinical laboratories has traditionally been designed to evaluate assays in their entirety; however, it can be alternatively applied to separate assay components. We developed and implemented a multi-institutional proficiency testing approach to directly assess custom bioinformatics and variant interpretation processes. Six clinical laboratories, all of which use the same commercial library preparation kit for next-generation sequencing analysis of tumor specimens, each submitted raw data (FASTQ files) from four samples. These 24 file sets were then deidentified and redistributed to five of the institutions for analysis and interpretation according to their clinically validated approach. Among the laboratories, there was a high rate of concordance in the calling of single-nucleotide variants, in particular those we considered clinically significant (100% concordance). However, there was significant discordance in the calling of clinically significant insertions/deletions, with only two of seven being called by all participating laboratories. Missed calls were addressed by each laboratory to improve their bioinformatics processes. Thus, through our alternative proficiency testing approach, we identified the bioinformatic detection of insertions/deletions as an area of particular concern for clinical laboratories performing next-generation sequencing testing.

Novel Helicobacter pylori Sequencing Test Identifies High Rate of Clarithromycin Resistance

Objectives: Eradication therapy selection for Helicobacter pylori gastritis requires knowledge of the local resistance rate to clarithromycin. There is minimal population-based or regional data in the United States on pediatric clarithromycin resistance. Although commercial methods such as fluorescence in situ hybridization and DNA probe assays are available in Europe for the evaluation of H pylori 23S rRNA mutations associated with resistance, clinical testing for 23S rRNA in the United States is not widely available. This study examined a single pediatric institutions clarithromycin resistance rate by a DNA polymerase chain reaction/sequencing assay applied to archived gastric biopsy specimens. Methods: From the period 2010 to 2012, 38 H pylori–infected gastric biopsies were examined from archived formalin-fixed paraffin-embedded (FFPE) material. The 23S rRNA gene of H pylori was polymerase chain reaction amplified and sequenced for the identification of point mutations that are associated with clarithromycin therapeutic resistance. Results: By 23S rRNA gene sequencing, 50% (n = 19) of the specimens contained H pylori with mutations significant for clarithromycin resistance. Conclusions: This study is consistent with other pediatric reports suggesting significant H pylori clarithromycin resistance in the United States. Furthermore, the method used in this study can be used by hospital-based clinical laboratories to assess local clarithromycin resistance from archived biopsy material.

Development and validation of a quantitative real time PCR assay for BK virus.

Several studies have shown that BK viral load in plasma and urine are reliable markers for the detection of BK virus associated nephropathy (BKVAN) in renal transplant patients. We developed a quantitative real time PCR assay based on TaqMan technology for the measurement of BK viral load in plasma and urine. Considering the high similarity of the nucleotide sequence of the BK virus (BKV) with the JC virus (JCV), we designed this assay to specifically amplify BKV. We determined the viral DNA recovery rate on manual (QIAGENs QIAamp DNA Blood Mini Kit) and automated (BioMerieuxs NucliSENS EasyMAG) extraction methods. The comparison showed a higher viral DNA recovery rate on the automated extraction (61-76% in plasma and 52-65% in urine) as compared to the manual method (49-52% in plasma and 33-56% in urine). Quantitation of the viral load was performed using an external standard curve that was constructed with serial dilution of a plasmid containing the full length of the BKV genome. Commercially available quantitative BKV standards showed good correlation with the plasmid standard. The reproducibility of the assay was determined based on the Ct values of the amplified products as well as in BK copies per milliliter of sample. This assay is linear over a 7 log range (10 to 1 × 10(7) copies per reaction), no cross-reactivity was detected with the closest-related polyomavirus JCV, as well as other viruses that may be found in immunocompromised patients, and human genomic DNA. The limit of detection of the assay is 300 copies per milliliter in both plasma and urine and the limit of quantitation is 1000 copies per milliliter using the NATtrol BK Virus Linearity Panel (ZeptoMetrix). This real time PCR assay provides a reliable and sensitive method for the quantitation of BKV in plasma and urine samples.

Long-range PCR based sequencing of the highly homologous genes, SFTPA1 and SFTPA2

In humans, Surfactant Protein A exists as two highly homologous genetic isoforms, SFTPA1 and SFTPA2. Mutations in these two genes are associated with idiopathic pulmonary fibrosis (IPF) and lung cancer. We have developed a Sanger DNA sequencing assay which utilizes long-range PCR to detect mutations in these two genes.