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ABSTRACT: 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.
DNA was extracted from 10 whole blood and 10 FFPE specimens using six different commercial kits.
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).
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.
Clinical Chemistry and Laboratory Medicine 10/2011; 50(2):267-72. · 2.15 Impact Factor
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ABSTRACT: Enteroviruses have been implicated as a cause of low Apgar scores in conjunction with perinatal seizures and respiratory insufficiency. Using in-situ reverse transcriptase polymerase chain reaction (in-situ PCR), Nuovo et al detected enterovirus in up to 86% of placentas from perinates exhibiting these symptoms. In-situ PCR has been the only method employed to assess for the presence ofenterovirus in this specific patient population. The purpose of our study was to use PCR amplification of enterovirus from extracted RNA to confirm these observations. RNA was extracted from 26 placentas of infants with low Apgar scores, perinatal seizures, and respiratory insufficiency. Each extraction was positive for beta-actin RNA, which confirmed that the integrity of RNA was maintained in the sample. Enterovirus RNA was not detected in any of the cases. Our results indicate that enterovirus is not present in placentas from neonates with the combination of low Apgar scores, respiratory insufficiency, and seizures, as previously reported.
Pediatric and Developmental Pathology 10/2008; 12(3):177-9. · 0.99 Impact Factor
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ABSTRACT: 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.
Journal of Molecular Diagnostics 08/2008; 10(4):311-6. · 3.58 Impact Factor
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ABSTRACT: Human Epstein-Barr virus (EBV) and cytomegalovirus (CMV) can cause serious complications in immunocompromised patients. Rapid diagnosis of EBV and CMV infection is critical in the management of the disease so that anti-viral therapy can be started early. Here we describe the development of real-time PCR assays using TaqMan probes and molecular beacons and compare the performance of both assays with a well-established, validated, gel-based PCR method for the quantification of EBV and CMV in patients' samples. The TaqMan and molecular beacon assays were linear between 10 to 10(7) viral genomes/reaction. Both assays generated calibration curves with strong correlation and low intra-assay and interassay variation. Results of EBV and CMV viral load determination inpatient samples obtained by the gel-based and real-time PCR were very similar. The real-time PCR assays showed increases in viral load before clinical measures of viral disease and decreases in viral load during anti-viral therapy in two of six pediatric patients. The data indicate that these TaqMan and molecular beacon approaches are accurate, rapid, and reliable assays for the diagnosis and monitoring of EBV and CMV infections in patients.
Journal of Molecular Diagnostics 03/2003; 5(1):15-20. · 3.58 Impact Factor
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ABSTRACT: To evaluate the contribution of placental histopathology to the diagnosis of congenital syphilis.
From January 1, 1986, through December 31, 1998, all pregnant women presenting to a large, urban Dallas County labor and delivery unit with untreated syphilis at delivery and who had placental evaluation performed were identified. Women were clinically staged, and the infants were evaluated for congenital syphilis using a standard protocol. Each placenta was evaluated by two independent pathologists. Histologic characteristics of the placenta related to congenital syphilis in live-born and stillborn infants were then analyzed.
Sixty-seven women met the study criteria: 33 (49%) stillborn and 18 (27%) live-born infants with congenital syphilis, 15 (22%) uninfected live-born infants, and one uninfected stillborn fetus diagnosed by current criteria. There were no differences between the groups with regard to demographic characteristics, prenatal care, or stage of syphilis. Stillborn infants were more likely to deliver preterm (P <.001). Controlling for gestational age, histopathology revealed necrotizing funisitis, villous enlargement, and acute villitis associated with congenital syphilis. Erythroblastosis was more common in stillborn infants with congenital syphilis than all live-born infants (odds ratio 16, 95% confidence interval 1, 370). The addition of histologic evaluation to conventional diagnostic evaluations improved the detection rate for congenital syphilis from 67% to 89% in live-born infants, and 91% to 97% in stillborn infants.
Our results show that histopathologic examination of the placenta is a valuable adjunct to the contemporary diagnostic criteria used to diagnose congenital syphilis.
Obstetrics and Gynecology 07/2002; 100(1):126-33. · 4.73 Impact Factor
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ABSTRACT: Acute fatty liver of pregnancy (AFLP) is a rare and dreaded complication of pregnancy, almost exclusively seen in the third trimester. The histopathologic features of AFLP closely resemble those seen in metabolic disorders characterized by deficiency of fatty acid oxidative enzymes. Several reports have established a strong association between AFLP in the mother and fetal deficiency of the enzyme long-chain L-3-hydroxyacyl-CoA dehydrogenase (LCHAD). However, these studies have an inevitable selection bias resulting from ascertainment through an affected infant, rather than an unselected population of patients with AFLP. We retrospectively examined a series of 10 women with pregnancies complicated by AFLP to determine the prevalence of the common LCHAD mutation (G1528C) in this population. The existing LCHAD primers, which produce a 640-bp amplicon (IJlst L, Ruiter JP, Hoovers JM, Jakobs ME, Wanders RJ: J Clin Invest 98:1028-1033, 1996), were modified to make them amenable to analysis of fragmented DNA obtained from microdissected formalin-fixed material. None of the patients were found to harbor the common G1528C mutation. It is likely that AFLP arising in the context of fetal LCHAD deficiency represents only one of the possible etiologies for this uncommon disorder, and the metabolic basis of AFLP is more heterogeneous than previously believed.
Pediatric Research 06/2002; 51(5):658-61. · 2.70 Impact Factor
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ABSTRACT: Interleukin-6 (IL6) and suppurating placental inflammation are markers of neonatal sepsis. The purpose of this study was to define a relationship between IL6 and acute chorioamnionitis and funisitis of the placenta, and to compare IL6 levels in term and preterm neonates. Umbilical venous IL6 was measured in 137 term and 110 preterm neonates. Acute chorioamnionitis was graded as none, mild, moderate, severe, and necrotizing. Funisitis was graded as none, 1 vessel, 2 vessels, 3 vessels, or necrotizing. A 2-way analysis of variance with interaction was used to compare the IL6 levels. There was a stepwise progression of IL6 levels with increasing severity of acute chorioamnionitis and funisitis. Term neonates showed an IL6 elevation with mild acute chorioamnionitis and single-vessel vasculitis, which increased progressively until the inflammation became severe. In contrast, IL6 levels in preterm neonates did not increase significantly until severe acute chorioamnionitis or 3-vessel vasculitis was seen. Statistically significant differences in IL6 levels were seen in term versus preterm infants when the acute chorioamnionitis was mild or moderate or when the funisitis involved either 1 or 2 vessels (P < 0.05). The difference may be related to the relative immaturity of the preterm immune system, as has been demonstrated in vivo and in vitro. However, differences in management could be confounding factors. In conclusion, umbilical venous IL6 levels correlate with the severity of acute placental inflammation, with greater IL6 elevations in term infants compared to preterm infants until the inflammation becomes severe.
Human Pathlogy 03/2002; 33(3):335-40. · 2.88 Impact Factor
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ABSTRACT: To assess whether the semiquantitative peripheral blood Epstein-Barr virus (EBV) polymerase chain reaction (PCR) test correlates
with post-transplant lymphoproliferative disorder (LPD), we compiled the results of the test done over a 3-year period ending
July 1997. Six hundred seventy-six tests were done on 185 patients. Four hundred-thirty tests (63%) were negative, 167 (25%)
were weak positive, 67 (10%) were moderate positive, and 12 (2%) were strong positive. Twelve of the patients developed a
lymphoproliferative disorder (LPD) during this time. The EBV PCR tests proximate to the diagnosis of LPD in the 12 patients
with EBV-positive LPD were 6 strong positive, 5 moderate positive, 1 weak positive. No patient with LPD had a negative result
at diagnosis. Stated another way, 6/12 (50%) of strong-positive PCR tests, 5/67 (7%) moderate-positive tests, and 1/167 (.6%)
of weak-positive tests correlated with LPD. Serologic evaluation for EBV done on 7 patients at the time of LPD showed low
serologic responses in 5 of the 7 patients. The EBV PCR temporally associated with the serology indicated moderate to large
viral burdens. In each patient evaluated serially, the EBV PCR test rose before the diagnosis of LPD and fell with treatment
for the disorder. In conclusion, the EBV PCR test may be used as an adjunct to the diagnosis of patients with LPD and may
be used to monitor response to therapy for the disorder.
Pediatric and Developmental Pathology 10/1998; 1(6):480-486. · 0.99 Impact Factor
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ABSTRACT: Chronic villitis is a histologic diagnosis that may be either associated with infection, or termed villitis of undetermined etiology (VUE). The lymphocytic infiltrate in VUE has been reported to consist of maternal lymphocytes, but the origin of the lymphocytic infiltrate in infectious villitis has not been identified. The purpose of our study was to compare the maternal vs. fetal origin of the infiltrating lymphocytes in VUE and syphilitic villitis, and to expand the immunophenotypic data provided by previous studies. Paraffin-embedded placentas from four males with VUE and two males with syphilitic villitis were subjected to fluorescence in situ hybridization (FISH) for the X and Y chromosomes. Serial sections were stained with antibodies to CD3, CD4, CD8, CD68, HLA-DR, and CD20. Quantitation of the relative number of cells marking with each antibody was done for four villi in each slide. CD3 lymphocytes predominated in both VUE and syphilitic villitis, with slightly more CD8 cells compared to CD4 cells. CD68 and HLA-DR positive cells were as frequent as CD3 cells, and B-lymphocytes were rare. Maternal cells were the predominant intravillous population in both VUE and syphilitic villitis, and neutrophils in syphilitic villitis were also maternal. These data indicate that the immune response in both syphilitic villitis and VUE is similar, raising the possibility of a similar immunopathogenetic pathway.
Pediatric and Developmental Pathology 7(5):453-8; discussion 421. · 0.99 Impact Factor
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ABSTRACT: Three children, two with liver transplants and one with acquired human immunodeficiency virus (HIV) infection, presented with hepatitis accompanied by elevated gamma glutamyl transpeptidase. Biopsies revealed cholangiohepatitis caused by adenovirus infection. There was a progressive loss of interlobular bile ducts in two of the patients. In one patient, infection of the biliary tree was marked by a necrotizing cholangitis, with adenoviral inclusions noted in the biliary epithelium. In each patient, there was evidence of adenovirus gastrointestinal infection. This is the first report of adenoviral infection of the biliary tree in humans. It is hypothesized that adenovirus cholangiohepatitis occurs as a result of ascending infection from the gastrointestinal tract to the biliary tree.
Pediatric and Developmental Pathology 6(2):156-9. · 0.99 Impact Factor
