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.) to manual titration with gel Column Agglutination Technology (CAT), as well as the reproducibility of both methods. MATERIALS AND METHODS: Ten different locations participated in this study. The equivalency study included 70 random samples at each site. The reproducibility study tested the same blinded 30-sample panel at each study site. Anti-A and anti-B IgM and IgG antibody titres were tested with both the automated and manual methods; additionally, dithiothreitol (DTT) treatment was used to inactivate IgM antibodies in the manual CAT method. RESULTS: The equivalency between CAT manual method and Galileo NEO automated titres at each site ranged from 38 to 88%; equivalency for each isotype was 66.2% for IgM, 60.6% for IgG, and 88.5% for DTT-treated IgG. The reproducibility study evaluated the titre variation of each sample obtained from the 10 sites. The average titre ranges (in doubling dilutions) for the automated and manual methods, respectively, were 2.15±1.0 and 4.03±1.8 for IgM, and 1.53±0.7 and 4.10±1.9 for IgG; for the manual DTT-treated IgG, the average titre range was 3.45±1.8 doubling dilutions. DISCUSSION: The results demonstrated that the Galileo NEO automated and manual CAT ABO titres are not equivalent. However, the study also demonstrated that titre reproducibility is enhanced with the Galileo NEO automated ABO titration assays relative to the manual CAT ABO titration method. Therefore, to improve management of patients receiving care across multiple institutions, our study supports the use of automated ABO titration.

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. We evaluated the safety of this national policy using a prospective repository of donors/recipient pairs. METHODS: We used highly sensitive nucleic acid testing (NAT) assays to test repository and follow-up samples from donors who were initially classified as negative by minipool NAT assays (6-MP), but were later found to carry occult HBV DNA. When available, we also analysed recipients' pre- and post-transfusion samples, collected in the context of a repository financed by the European Commission (the BOTIA project). RESULTS: Between 2008 and 2011 6-MP NAT assays identified 18 carriers of occult HBV infection among 12,695 donors; 28 samples from previous donations were available from 13 of these carriers. Highly sensitive HBV DNA detection methods showed that 6-MP HBV DNA screening failed to identify 14/28 (50%) viraemic donations, that were released for transfusion. HBV marker testing of such blood product recipients revealed two cases of transfusion transmitted HBV infection, documented by donor-recipient sequence identity. CONCLUSIONS: Viraemic blood donations from occult HBV infection carriers remain undetected by current minipool HBV DNA screening, and transfusion transmission of HBV continues to occur in susceptible patients. More effective individual HBV DNA screening and/or tests for antibodies to HBV core antigen should be considered to improve blood safety.

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. STUDY DESIGN AND METHODS: Plasma samples from 97 candidate donors and 80 controls were tested for malarial antibodies using a commercial enzyme immunoassay. The conserved 18S rRNA and the mitochondrial genes of Plasmodium were amplified to detect and quantify parasite genomes (copies/mL). Plasmodium species were identified with a species-specific nested polymerase chain reaction. Parasitemic samples were further tested by amplification of polymorphic repetitive regions in MSP-1 Block 2, MSP-2 Block 3, and glutamate-rich protein (GLURP) confirmed by sequencing. RESULTS: Three of 83 seropositive (3.6%) and one of 14 seronegative at-risk candidate donors carried Plasmodium genome (4 × 10(3) -8.5 × 10(4) copies/mL): two P. falciparum, one P. malariae (seronegative sample), and one coinfection with P. malariae and P. ovale. Alleles of MSP-1 (MAD20 and K1), MSP-2 (3D7 and FC27), and GLURP were amplified from Sample 261. In Sample 282 only one allele in MSP-2 (FC27) and GLURP was amplified. No alleles were detected in Samples 283 and 331. CONCLUSIONS: Immigrants from endemic countries might carry infectious Plasmodium after 2 to 5 years of continuous residence in Italy. Serologic screening may miss donors carrying P. malariae. Permanent exclusion or screening for both antibodies and genome are needed to prevent TTM.

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 (4.8-19.3). HDV-RNA was first measured by qualitative in-house nested RT-PCR and quantified by in-house real-time PCR. HDV RNA levels only appeared significantly associated to HCC (univariate analysis: OR 1.32, 95% CI 1.02-1.71; p = 0.037; multivariate analysis: OR 1.42, 95% CI 1.04-1.95; p = 0.03). In non-cirrhotics at first presentation (n = 105), HDV RNA levels were associated with progression to cirrhosis (univariate analysis: OR = 1.57, 95% CI 1.20-2.05, p<0.001; multivariate analysis: OR = 1.60, 95% CI 1.20-2.12, p = 0.007) and development of HCC (univariate analysis: OR = 1.66, 95% CI 1.04-2.65, p = 0.033; multivariate analysis: OR = 1.88, 95% CI 1.11-3.19, p = 0.019). ROC analysis showed that approximately 600,000 HDV RNA copies/mL was the optimal cut-off value in our cohort of patients for discriminating the development of cirrhosis. High levels of HDV viremia in non-cirrhotic patients are associated with a considerable likelihood of progression to cirrhosis and the development of HCC. Once cirrhosis has developed, the role of HDV replication as a predictor of a negative outcome lessens.

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. RESULTS: The primary cell populations (hcc-1, -2 and -3) and two clones generated by means of limiting dilutions from hcc-1 (clone-1/7 and -1/8) differently expressed a number of tumour-associated stem cell markers, including EpCAM, CD49f, CD44, CD133, CD56, Thy-1, ALDH and CK19, and also showed different doubling times, drug resistance and tumorigenic potential. Moreover, we found that ALDH expression, in combination with CD44 or Thy-1 negativity or CD56 positivity identified subpopulations with a higher clonogenic potential within hcc-1, hcc-2 and hcc-3 primary cell populations, respectively. Karyotyping revealed the clonal evolution of the cell populations and clones within the primary tumour. Importantly, the primary tumour cell population with the greatest tumorigenic potential and drug resistance showed more chromosomal alterations than the others and contained clones with epithelial and mesenchymal features. CONCLUSIONS: Individual HCCs can harbor different self-renewing tumorigenic cell types expressing a variety of morphological and phenotypical markers, karyotypic evolution and different gene expression profiles. This suggests that the models of hepatic carcinogenesis should take into account TPC heterogeneity due to intratumour clonal evolution.

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%). Sequencing and phylogenetic analyses described a B/F recombinant clade with anomalous breakpoints that did not allow it to be classified as CRF12_BF. Viral load was not quantified efficiently because of a mismatch in the primers and probes used by commercial assays. An assessment of the clinical management, and epidemiological, immunological, and virological characteristics of these patients, who were receiving non-nucleoside reverse transcriptase inhibitor (NNRTI)- or protease inhibitor (PI)-based regimens, showed that the immunological and virological mismatch delayed the start of treatment by a mean of 6.8 months. Therapy was started in nine patients. Both NNRTI- and PI-based regimens led to full virological suppression after a mean 36 weeks of treatment; the PI-based regimens proved to be more effective in terms of immunological recovery (1,341 vs. 544 CD4+ cells/mm(3) ). The spread of non-B subtypes is increasing throughout the world but their response to treatment is still unclear. PIs and NNRTIs are effective but further tests are needed to allow the more efficient recognition of these viral strains and establish how they should be treated.

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. During donor follow-up, HIV RNA became detectable, but the viral load was 2 to 3 log lower than measured with other NATs targeting different genome regions. Genome sequencing revealed a novel B/F recombinant with mutations affecting primers and probe annealing accounting for the poor performance of routine NAT. A total of 553 HIV-1-infected patients attending the hospital clinic were subsequently tested prospectively using the routine assay and an in-house assay specifically designed to detect the B/F strains. RESULTS: The routine assay substantially underestimated viremia (1-5 log) in 19 cases (3.5%), 11 (58%) of which were infected with the same B/F strain observed in the index donor samples. Two other non-B circulating recombinant forms of HIV-1 (A/G, B/G subtypes) were identified as poorly detected. Newly introduced NATs targeting two HIV-1 regions improved assay performance. CONCLUSION: HIV-1 increasing heterogeneity affects the efficiency of NATs and consequently the safety of the blood supply as well as diagnosis and patient management.

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. The optimized protocols included a 1:4 forward:reverse primer ratio for asymmetric PCR, and Cy5-dCTP multiple incorporation for the generation of a labeled PCR product to be hybridized to complementary probes bound to the chip surface. RESULTS: Final conditions were applied to the fully integrated LoC platform for the detection of the IVSI-110 G > A mutation in the human beta-globin (HBB) gene associated with beta-thalassemia, used as a model of genetic application, allowing for correct genotyping of 25 samples that were heterozygous, homozygous or wild-type for this mutation. CONCLUSIONS: The overall results show that the present platform is very promising for rapid identification of DNA sequence variations in an integrated, cost effective and convenient silicon chip format.

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. Electronic stringency provides quality control for the hybridization process and ensures that any bound pairs of DNA are truly complementary; the microchip can be easily customized by the end user, allowing for assembly of specific probes onto the microchip to perform individualized analyses. Assays for 10 frequent mutations in the beta-globin gene causing beta-thalassemia and sickle cell anemia are presented that can be applied, in turn, to population screening or family study and prenatal diagnosis in single cases.

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. By this approach, 41 prenatal diagnoses were performed by microelectronic microchip analysis, with total concordance of results obtained on fetal DNA extracted from chorionic villi. Among these, 27/28 were also confirmed by direct sequencing and 4 by pyrosequencing.

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. RESULTS: In the H-ferritin gene, we found one new and rather common intronic polymorphism and the K54R substitution in two controls. The L-ferritin gene showed a very common L55L polymorphism and four other types of DNA variations, three of which were in the patient cohort. A mutation of the conserved His133 to Pro was found in a PD patient and in his daughter. The patient did not show signs of neuroferritinopathy, but the mutation was associated with low L-ferritin levels and with mild chronic anemia. CONCLUSIONS: The results support the hypothesis that DNA variations in the ferritin genes are not a common cause for PD.

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). These DNA analogues bind wild-type DNA, thus interfering with its amplification while still allowing the mutant DNA to become detectable. We have synthesized different PNAs, which are highly effective in clamping wild-type DNA in the beta-globin gene region, where four beta-thalassemia mutations are located (IVSI.110, CD39, IVSI.1, IVSI.6) plus HbS. The fluorescence microchip readout allows us to monitor the extent of wild-type allele inhibition, thus facilitating the assessment of the optimal PNA concentration.

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. Thus, we analyzed 24 mutations spanning over a DNA stretch of 48 nucleotides, including four deletions 2-29 nucleotides long and 20 substitutions, seven of which were conservative transversions. With this unique experimental model we developed a microchip diagnostic platform for identifying known molecular defects in the L-ferritin IRE structure with a microelectronic array approach, which we optimized after studying the effects of various parameters. The system enables electronic deposition of biotinylated amplicons to selected pads. Under optimized conditions, no cross-hybridization was found, even for mutations that affected the same or adjacent nucleotide positions. The same cartridge could be serially hybridized with all the 24 reporter probe sets, which allowed correct genotyping right up until the end of the analysis. Extensive validation on 200 samples in a blinded fashion gave total concordance of results. This pilot study represents a first step toward developing a diagnostic microchip for large-scale analyses for epidemiological studies and screening of mutations associated with iron disorders.

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. The DNA at each pad is then hybridized with specific oligonucleotide probes, complementary to normal or mutant sequences, that labeled with Cy3 or Cy5 dyes, respectively. The array is imaged, and fluorescence signals are scanned, monitored, and quantified by highly developed, digital image-processing procedures. The experimental steps to be performed for the development and execution of a microchip assay are described. Attention is focused on the fundamental aspects of probe design, and guidelines and useful suggestions are given. Protocols for sample preparation, addressing, reporting, and data analysis are also detailed.

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. This review describes the application of microelectronics to all the processes involved in a genetic test, particularly to sample preparation, DNA amplification and sequence variation detection.

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. The biotinylated amplicon was electronically addressed on the chip to selected pads, where it remained embedded through interaction with streptavidin in the permeation layer. The DNA at each test site was then hybridized to a mixture of fluorescently labeled wild-type or mutant probes. RESULTS: Assays conditions were established based on the analysis of 700 DNA samples from compound heterozygotes or homozygotes for the nine mutations. The assays were blindly validated on 250 DNA samples previously genotyped by other methods, with complete concordance of results. Alternative multiplexed formats were explored: the combination of multiplex PCR with multiple addressing and/or hybridization allowed analysis of all nine mutations in the same sample on one test site of the chip. CONCLUSIONS: The open flexible platform can be designed by the user according to the local prevalence of mutations in each geographic area and can be rapidly extended to include the remaining mutations causing beta-thalassemia in other regions of the world.

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. L-ferritin 5'UTR has been extensively studied and up to 21 different mutations have been identified. Only one mutation has been reported for H-ferritin 5'UTR; this mutation modified IRE structure and was apparently associated with high serum ferritin levels and iron overload. DESIGN AND METHODS: To identify other mutations in H ferritin 5'UTR we developed a fast DNA scanning method based on denaturing high performance liquid chromatography (HPLC). Five artificial DNA mutants were produced in order to validate the analytical conditions of the system for the identification of all mutations by single runs at 68 degrees C. The system was used to screen 660 DNA samples from subjects with high serum ferritin levels. RESULTS: Two abnormal patterns were identified carrying the mutations C20G and G34T. Structural data and the analysis of ferritin levels in red blood cells suggest that these mutations do not affect the functionality of the IRE. INTERPRETATION AND CONCLUSIONS: This large and first population analysis indicates that mutations in the H-ferritin 5'UTR are rare and do not seem to contribute to hyperferritinemia or iron overload.