ABO antibody titres: a multisite comparative study of equivalency and reproducibility for automated solid-phase and haemagglutination titration, and manual dilution with gel column agglutination technology.

Background: ABO antibody titres are important in many clinical decisions; however, much variability is observed in titre results. For reliable and reproducible titre results, automated ABO titration methods have been developed. In this 10-site study, we evaluated the equivalency of the automated ABO titration assays on the Galileo NEO, a fully automated blood bank analyzer (Immucor, Inc.

Poor efficacy of nucleic acid testing in identifying occult HBV infection and consequences for safety of blood supply in Italy.

Background & Aims: In Italy, DNA screening of blood donations for hepatitis B virus (HBV) was introduced to prevent the transmission of window period and occult HBV infection. Anti-HBc screening is not recommended in order to avoid shortage of the blood supply. To contain costs, donor samples are generally pooled before testing.

Plasmodium genome in blood donors at risk for malaria after several years of residence in Italy.

Background: At present, the main risk of transfusion-transmitted malaria (TTM) in nonendemic countries is chronic, asymptomatic immigrants from malaria-endemic areas. Semi-immune donors may carry undetected parasitemia. This study examines Plasmodium infection in at-risk blood donors in Northern Italy.

High serum levels of HDV RNA are predictors of cirrhosis and liver cancer in patients with chronic hepatitis delta.

Chronic infection with the hepatitis delta virus (HDV) is a risk factor for cirrhosis and hepatocellular carcinoma (HCC), but little is known whether the outcome of hepatitis is predicted by serum markers of HDV and hepatitis B virus (HBV) infection. The aim of the study was to investigate these correlations in 193 patients with chronic HDV infection who had been followed up for a median of 9.5 years (4.

A cluster of patients with recombinant B/F HIV-1 infection: epidemiological, clinical, and virological aspects.

Migratory processes have caused changes in human immunodeficiency virus (HIV) epidemiology and non-B subtypes are now playing an increasing role. In a cohort of 553 HIV-infected outpatients tested to identify non-B isolates, the largest group consisted of 13 subjects with a recombinant B/F form (prevalence 2.4%).

Evidence of distinct tumour-propagating cell populations with different properties in primary human hepatocellular carcinoma.

Background And Aims: Increasing evidence that a number of malignancies are characterised by tumour cell heterogeneity has recently been published, but there is still a lack of data concerning liver cancers. The aim of this study was to investigate and characterise tumour-propagating cell (TPC) compartments within human hepatocellular carcinoma (HCC).

Methods: After long-term culture, we identified three morphologically different tumour cell populations in a single HCC specimen, and extensively characterised them by means of flow cytometry, fluorescence microscopy, karyotyping and microarray analyses, single cell cloning, and xenotransplantation in NOD/SCID/IL2Rγ/⁻ mice.

A cluster of human immunodeficiency virus Type 1 recombinant form escaping detection by commercial genomic amplification assays.

Background: Nucleic acid testing (NAT)-based methods for the detection and quantification of human immunodeficiency virus Type 1 (HIV-1) RNA are used to increase transfusion safety and to diagnose and manage HIV-1-infected patients. We describe a novel HIV-1 recombinant form associated with lack of reactivity or substantial underestimation of viral load by commercial NAT assays.

Study Design And Methods: We observed a repeat blood donor seroconverting to anti-HIV in whom HIV RNA was initially undetectable with routine NAT was observed.

Integrated PCR amplification and detection processes on a Lab-on-Chip platform: a new advanced solution for molecular diagnostics.

Background: Several microdevices have been developed to perform only a single step of a genotyping process, such as PCR or detection by probe hybridization. Here, we describe a Lab-on-Chip (LoC) platform integrating a PCR amplification microreactor with a customable microarray for the detection of sequence variations on human genomic DNA.

Methods: Preliminary work was focused on developing the single analytical steps including PCR and labeling strategies of the amplified product by conventional reference systems.

A fast microelectronic array for screening and prenatal diagnosis of beta-thalassemia.

The electronic microchip is a recently developed technology for the fast and reliable detection of known single-nucleotide polymorphisms (SNPs) in the genome. The DNA fragment to be analyzed is directed electrophoretically into the chip, and then it is hybridized with fluorescent-tagged DNA probes specific for the mutant and wild-type sequences. The presence or absence of the mutation is detected by the fluorescence signal.

Peptide-nucleic acid-mediated enriched polymerase chain reaction as a key point for non-invasive prenatal diagnosis of beta-thalassemia.

The presence of fetal DNA in maternal plasma can be exploited to develop new procedures for non-invasive prenatal diagnosis. Tests to detect 7 frequent beta-globin gene mutations in people of Mediterranean origin were applied to the analysis of maternal plasma in couples where parents carried different mutations. A mutant enrichment amplification protocol was optimized by using peptide nucleic acids (PNAs) to clamp maternal wild-type alleles.

Analysis of ferritin genes in Parkinson disease.

Background: Genes that regulate iron metabolism may be involved in increasing brain iron content in Parkinson disease (PD). The ferritin L-chain is one of these genes, but the rare insertional mutations that cause neuroferritinopathy with basal ganglia degeneration have not yet been identified in PD.

Methods: We used denaturing HPLC (DHPLC) to investigate 124 PD patients and 180 controls for variations in the coding and in the 5' untranslated regions of the H- and L-ferritin genes.

Different approaches for noninvasive prenatal diagnosis of genetic diseases based on PNA-mediated enriched PCR.

The aim of this work was to develop advanced and accessible protocols for noninvasive prenatal diagnosis of genetic diseases. We are evaluating different technologies for mutation detection, based on fluorescent probe hybridization of the amplified product and pyrosequencing, a technique that relies on the incorporation of nucleotides in a primer-directed polymerase extension reaction. In a previous investigation, we have already proven that these approaches are sufficiently sensitive to detect a few copies of a minority-mutated allele in the presence of an excess of wild-type DNA, In this work, in order to further enhance the sensitivity, we have employed a mutant enrichment amplification strategy based on the use of peptide nucleic acids (PNAs).

Microelectronic DNA chip for hereditary hyperferritinemia cataract syndrome, a model for large-scale analysis of disorders of iron metabolism.

Hereditary hyperferritinemia cataract syndrome (HHCS) is caused by mutations in the regulatory iron responsive element (IRE) in the 5'UTR of the L-ferritin transcript that reduce binding affinity to the iron regulatory proteins (IRPs) and lead to a constitutive upregulation of the protein in tissue and serum. Twenty-nine mutations have been reported within the L-ferritin (FTL) IRE sequence, 21 of which were available to us. In addition, we included in this study three new mutations.

Single-nucleotide polymorphism and mutation identification by the nanogen microelectronic chip technology.

The present chapter describes a microarray technology developed by Nanogen Inc., for the identification of DNA variations based on the use of microelectronics. The NMW 1000 NanoChip Molecular Biology Workstation allows the active deposition and concentration of charged biotinylated molecules on designated test sites.

Molecular diagnostics by microelectronic microchips.

Molecular diagnostics is being revolutionized by the development of highly advanced technologies for DNA and RNA testing. One of the most important challenges is the integration of microelectronics to microchip-based nucleic acid technologies. The specific characteristics of these microsystems make the miniaturization and automation of any step of a molecular diagnostic procedure possible.

Beta-thalassemia microelectronic chip: a fast and accurate method for mutation detection.

Background: beta-Thalassemia is one of the most common genetic diseases in humans. We developed an automated electronic microchip for fast and reliable detection of the nine most frequent mutations accounting for >95% of the beta-thalassemia alleles in the Mediterranean area.

Methods: We developed a microchip-based assay to identify the nine most frequent mutations (cd39C>T, IVS1-110G>A, IVS1-1G>A, IVS1-6T>C, IVS2-745C>G, cd6delA, -87C>G, IVS2-1G>A, and cd8delAA) by use of the Nanogen Workstation.

Identification of two novel mutations in the 5'-untranslated region of H-ferritin using denaturing high performance liquid chromatography scanning.

Background And Objectives: Hereditary hyperferritinemia cataract syndrome is caused by mutations of the iron responsive elements (IREs) of L-ferritin mRNA. These alter the IRE structure and determine L-ferritin upregulation. IREs are located in 5'untranslated regions (5'UTR) of ferritin mRNAs.

Scanning mutations of the 5'UTR regulatory sequence of L-ferritin by denaturing high-performance liquid chromatography: identification of new mutations.

Hereditary hyperferritinaemia cataract syndrome is an autosomal dominant disorder caused by heterogeneous mutations of the iron regulatory element (IRE) in the ferritin l-chain mRNA. The mutations are rare and fast DNA scanning would facilitate diagnosis. The aim of the study was to compare the analytical performances of two fast DNA scanning techniques: denaturing high-performance liquid chromatography (DHPLC) and double-gradient denaturing gradient gel electrophoresis (DG-DGGE).

Analysis of clinically relevant single-nucleotide polymorphisms by use of microelectronic array technology.

Background: Microelectronic DNA chip devices represent an emerging technology for genotyping. We developed methods for detection of single-nucleotide polymorphisms (SNPs) in clinically relevant genes.

Methods: Primer pairs, with one containing a 5'-biotin group, were used to PCR-amplify the region encompassing the SNP to be interrogated.

A novel deletion of the L-ferritin iron-responsive element responsible for severe hereditary hyperferritinaemia-cataract syndrome.

In the last few years, mutations that cause disease through increased efficiency of mRNA translation have been discovered. Hereditary hyperferritinaemia-cataract syndrome (HHCS) arises from various point mutations or deletions within the iron-responsive element (IRE) in the 5'-UTR of the L-ferritin mRNA. Each unique mutation confers a characteristic degree of hyperferritinaemia and severity of cataract in affected individuals.