Continuous ambulatory peritoneal dialysis, ascitic and pleural body fluids evaluation with the Mindray BC-6800 hematology analyzer.

BACKGROUND: Accurate evaluation of hematology analyzers is recommended before these devices can be broadly introduced for the routine testing of continuous ambulatory peritoneal dialysis (CAPD), ascitic, and pleural fluids. METHODS: We evaluated the performance of Mindray BC-6800 for white blood cell (WBC) and differential cell count in 50 CAPD, 60 ascitic and 40 pleural compared with manual microscopy. Within-run precision, limit of blank (LoB), limit of detection (LoD), limit of quantitation (LoQ), and carryover were assessed. RESULTS: The Passing-Bablok regression in all fluids showed the following equations: yWBC =1.05x+3.31 (95%CI slope 0.95 to 1.12; intercept -0.25 to 5.52); yMN =0.85x+15.63 (95%CI slope 0.72 to 1.05; intercept -24.18 to 84.47); and yPMN =1.21x+13.37 (95%CI slope 1.03 to 1.35; intercept 4.00 to 32.47) with bias 78 cells/µL. The AUC for clinical PMN cut-off was 0.88 (95%CI: 0.77 to 0.98). In ascitic, pleural, and CAPD fluids the AUC for clinical PMN cut-off were 0.88 (95%CI: 0.63 to 1.00), 0.83 (95%CI: 0.68 to 0.99), and 1.00 (95%CI: 1.00 to 1.00) respectively. CV ranged from 3%-34%. LoB of 3 cell/µL was verified. LoD and LoQ reported the same result (8 cells/µL). Carry over never exceeded 0.05%. CONCLUSION: The effectiveness of BC-6800 to categorize cells from different body fluids was not compromised by the slight positive bias observed. This conclusion is supported by the high AUC and agreement between the automated method and the reference method. The results show that BC-6800 offers rapid, accurate, and reproducible results for clinical management of CAPD, ascitic, and pleural fluids.

A novel NUP98/RARG gene fusion in acute myeloid leukemia resembling acute promyelocytic leukemia.

Chromosomal translocations in hematological malignancies often result in novel fusion chimeric genes. We report a case of acute myeloid leukemia with a clonal translocation t(11;12)(p15;q13) displaying morphologic and immunophenotypic features resembling the classical hypergranular subtype of acute promyelocytic leukemia. The gene fused to NUP98 (nucleoporin 98) was detected by comparative genomic hybridization array as the retinoid acid receptor gamma gene (RARG). The involvement of RARG in a chimeric fusion transcript has not been reported previously in human leukemia.

Adaptive response to starvation in the fish pathogen Flavobacterium columnare: cell viability and ultrastructural changes.

BACKGROUND: The ecology of columnaris disease, caused by Flavobacterium columnare, is poorly understood despite the economic losses that this disease inflicts on aquaculture farms worldwide. Currently, the natural reservoir for this pathogen is unknown but limited data have shown its ability to survive in water for extended periods of time. The objective of this study was to describe the ultrastructural changes that F. columnare cells undergo under starvation conditions. Four genetically distinct strains of this pathogen were monitored for 14 days in media without nutrients. Culturability and cell viability was assessed throughout the study. In addition, cell morphology and ultrastructure was analyzed using light microscopy, scanning electron microscopy, and transmission electron microscopy. Revival of starved cells under different nutrient conditions and the virulence potential of the starved cells were also investigated. RESULTS: Starvation induced unique and consistent morphological changes in all strains studied. Cells maintained their length and did not transition into a shortened, coccus shape as observed in many other Gram negative bacteria. Flavobacterium columnare cells modified their shape by morphing into coiled forms that comprised more than 80% of all the cells after 2 weeks of starvation. Coiled cells remained culturable as determined by using a dilution to extinction strategy. Statistically significant differences in cell viability were found between strains although all were able to survive in absence of nutrients for at least 14 days. In later stages of starvation, an extracellular matrix was observed covering the coiled cells. A difference in growth curves between fresh and starved cultures was evident when cultures were 3-months old but not when cultures were starved for only 1 month. Revival of starved cultures under different nutrients revealed that cells return back to their original elongated rod shape upon encountering nutrients. Challenge experiments shown that starved cells were avirulent for a fish host model. CONCLUSIONS: Specific morphological and ultrastructural changes allowed F. columnare cells to remain viable under adverse conditions. Those changes were reversed by the addition of nutrients. This bacterium can survive in water without nutrients for extended periods of time although long-term starvation appears to decrease cell fitness and resulted in loss of virulence.

Fragment length analysis screening for detection of CEBPA mutations in intermediate-risk karyotype acute myeloid leukemia.

During last years, molecular markers have been increased as prognostic factors routinely screened in acute myeloid leukemia (AML). Recently, an increasing interest has been reported in introducing to clinical practice screening for mutations in the CCAAT/enhancer-binding protein α (CEBPA) gene in AML, as it seems to be a good prognostic factor. However, there is no reliable established method for assessing CEBPA mutations during the diagnostic work-up of AMLs. We describe here a straightforward and reliable fragment analysis method based in PCR capillary electrophoresis (PCR-CE) for screening of CEBPA mutations; moreover, we present the results obtained in 151 intermediate-risk karyotype AML patients (aged 16-80 years). The method gave a specificity of 100% and sensitivity of 93% with a lower detection limit of 1-5% for CEBPA mutations. The series found 19 mutations and four polymorphisms in 12 patients, seven of whom (58%) presented two mutations. The overall frequency of CEBPA mutations in AML was 8% (n = 12). CEBPA mutations showed no coincidence with FLT3-ITD or NPM1 mutations. CEBPA mutation predicted better disease-free survival in the group of patients without FLT3-ITD, NPM, or both genes mutated (HR 3.6, IC 95%; 1.0-13.2, p = 0.05) and better overall survival in patients younger than 65 of this group without molecular markers (HR 4.0, IC 95%; 1.0-17.4, p = 0.05). In conclusion, the fragment analysis method based in PCR-CE is a rapid, specific, and sensitive method for CEBPA mutation screening and our results confirm that CEBPA mutations can identify a subgroup of patients with favorable prognosis in AML with intermediate-risk karyotype.

Analysis of SNP rs16754 of WT1 gene in a series of de novo acute myeloid leukemia patients.

The single nucleotide polymorphism (SNP) rs16754 of the WT1 gene has been previously described as a possible prognostic marker in normal karyotype acute myeloid leukemia (AML) patients. Nevertheless, the findings in this field are not always reproducible in different series. One hundred and seventy-five adult de novo AML patients were screened with two different methods for the detection of SNP rs16754: high-resolution melting (HRM) and FRET hybridization probes. Direct sequencing was used to validate both techniques. The SNP was detected in 52 out of 175 patients (30 %), both by HRM and hybridization probes. Direct sequencing confirmed that every positive sample in the screening methods had a variation in the DNA sequence. Patients with the wild-type genotype (WT1(AA)) for the SNP rs16754 were significantly younger than those with the heterozygous WT1(AG) genotype. No other difference was observed for baseline characteristic or outcome between patients with or without the SNP. Both techniques are equally reliable and reproducible as screening methods for the detection of the SNP rs16754, allowing for the selection of those samples that will need to be sequenced. We were unable to confirm the suggested favorable outcome of SNP rs16754 in de novo AML.

Prognostic value of FLT3 mutations in patients with acute promyelocytic leukemia treated with all-trans retinoic acid and anthracycline monochemotherapy.

BACKGROUND: Fms-like tyrosine kinase-3 (FLT3) gene mutations are frequent in acute promyelocytic leukemia but their prognostic value is not well established. DESIGN AND METHODS: We evaluated FLT3-internal tandem duplication and FLT3-D835 mutations in patients treated with all-trans retinoic acid and anthracycline-based chemotherapy enrolled in two subsequent trials of the Programa de Estudio y Tratamiento de las Hemopatías Malignas (PETHEMA) and Hemato-Oncologie voor Volwassenen Nederland (HOVON) groups between 1996 and 2005. RESULTS: FLT3-internal tandem duplication and FLT3-D835 mutation status was available for 306 (41%) and 213 (29%) patients, respectively. Sixty-eight (22%) and 20 (9%) patients had internal tandem duplication and D835 mutations, respectively. Internal tandem duplication was correlated with higher white blood cell and blast counts, lactate dehydrogenase, relapse-risk score, fever, hemorrhage, coagulopathy, BCR3 isoform, M3 variant subtype, and expression of CD2, CD34, human leukocyte antigen-DR, and CD11b surface antigens. The FLT3-D835 mutation was not significantly associated with any clinical or biological characteristic. Univariate analysis showed higher relapse and lower survival rates in patients with a FLT3-internal tandem duplication, while no impact was observed in relation to FLT3-D835. The prognostic value of the FLT3-internal tandem duplication was not retained in the multivariate analysis. CONCLUSIONS: FLT3-internal tandem duplication mutations are associated with several hematologic features in acute promyelocytic leukemia, in particular with high white blood cell counts, but we were unable to demonstrate an independent prognostic value in patients with acute promyelocytic leukemia treated with all-trans retinoic acid and anthracycline-based regimens.

Adhesion dynamics of Flavobacterium columnare to channel catfish Ictalurus punctatus and zebrafish Danio rerio after immersion challenge.

The adhesion dynamics of Flavobacterium columnare to fish tissues were evaluated in vivo by immersion challenge followed by bacterial plate count and confirmatory observations of gill-adhered bacterial cells using scanning electron microscopy. Adhesion of F. columnare genomovar I (ARS-1) and II (BGFS-27) strains to skin and gill of channel catfish Ictalurus punctactus and gill of zebrafish Danio rerio was compared. At 0.5 h post-challenge, both strains adhered to gill of channel catfish at comparable levels (10(6) colony forming units [CFU] g(-1)), but significant differences in adhesion were found later in the time course. Channel catfish was able to effectively reduce ARS-1 cells on gill, whereas BGFS-27 persisted in gill beyond the first 24 h post-challenge. No significant difference was found between both strains when adhered to skin, but adhered cell numbers were lower (10(3) CFU g(-1)) than those found in gill and were not detectable at 6 h post-challenge. Adhesion of BGFS-27 cells to gill of zebrafish also occurred at high numbers (> 10(6) CFU g(-1)), while only < 10(2) CFU g(-1) of ARS-1 cells were detected in this fish. The results of the present study show that particular strains of F. columnare exhibit different levels of specificity to their fish hosts and that adhesion to fish tissues is not sufficient to cause columnaris disease.

High intragenomic heterogeneity of 16S rRNA genes in a subset of Vibrio vulnificus strains from the western Mediterranean coast.

Heterogeneity among ribosomal operons in Vibrio vulnificus is purported as a probabilistic indicator of strain virulence and classifies V. vulnificus strains as 16S rRNA genes type A and B. In this study, 16S rRNA genes typing of V. vulnificus strains isolated from the Valencia city coast, in the western Mediterranean, showed that 24 out of 30 isolates were type A, one was type B and five could not be typed. Single strand conformation polymorphism (SSCP) analysis of this gene region revealed complex patterns indicative of intragenomic ribosomal operon sequence heterogeneity. The 16S rRNA genes of three untypeable isolates C27, C30, and C34, along with type A (ATCC 27562) and B (C7184) reference strains, were amplified, cloned and sequenced. The number of unique 16S rRNA gene sequences was 4, 3, and 4 for the environmental isolates. The type strain of the species (ATCC 27562) presented only two 16S rRNA gene types, while the reference isolate C7184 of clinical origin had only one 16S rRNA gene type. Sequences differed from five to 35 bp (99.6% to 97.6% sequence similarity). Areas of variability concentrated in helices 10, 18, and 37 and included variants with short intervening sequences in helix 10. Most of the substitutions showed compensatory mutations suggesting ancient sequence divergence generated by lateral gene transfer.

Minimal residual disease detection in acute myeloid leukemia by mutant nucleophosmin (NPM1): comparison with WT1 gene expression.

BACKGROUND: Molecular analysis of minimal residual disease is only applicable in acute myeloblastic leukemia (AML) patients with genetic markers (20-30%). This study analyzes the feasibility of the real-time quantitative polymerase chain reaction (RQ-PCR) assay to detect mutant nucleophosmin (NPM1) during follow-up in AML patients. Moreover, we compare the NPM1 results with those of WT1 expression to MRD assessment. METHODS: The study includes 97 samples from 24 AML patients with type A NPM1 mutation at diagnosis. MRD was evaluated simultaneous by RQ-PCR assay to detect NPM1-mutated and WT1 expression. RESULTS: The expression levels of WT1 and NPM1 in 93 paired samples showed a strong positive correlation (r=0.81; p<0.0001). However, the kinetics of disappearance were different, WT1 decreased rapidly after induction but maintained these residual levels after treatment in patients in complete remission, whereas NPM1 experienced a mild reduction after induction but was undetectable in long survivor patients. CONCLUSIONS: This study shows the feasibility of the RQ-PCR assay to monitor MRD in AML patients carrying NPM1 mutations and its advantage over RQ-PCR assay for WT1. Owing to NPM1-mutated is specific of leukemic cells and shows higher levels at presentation.

IgG subclasses quantitation: Analytical performance of The Binding Site SPAPLUS® human assay and comparison with Siemens BNII® assay.

OBJECTIVES: Accurate evaluation of analyzers is highly recommended before these devices are broadly introduced for routine testing. Concerning quantification of IgG subclasses (IgGSc), standardization has not yet been reached and thus different assays might lead to different results. Here we report the analytical performances of The Binding Site (TBS) SPAPLUS® human IgGSc assay and the concordance with the Siemens BNII® human IgGSc assay. DESIGN AND METHODS: We evaluated precision, LoB, LoD and linearity of TBS SPAPLUS® human IgGSc immunoassay. Quantitation of IgGSc in 53 patients' serum samples was performed in parallel on both analyzers. Results from both assays were compared. RESULTS: Analytical performances of the TBS SPAPLUS® human IgGSc assay are acceptable for routine clinical use. According to the method comparison study, TBS assay measures lower values than Siemens assay for IgG1 and IgG4, whereas for IgG2 and IgG3 TBS provides greater values. All assays present a proportional bias, greater in the case of IgG3 and IgG4 assays. Individual subclass agreement, based on the classification of samples within three categories (low, normal and high) according to assay-specific reference intervals, range from 75% (IgG1) to 92% (IgG2). However, total classification agreement over all four subclasses only account for 55% of samples. CONCLUSION: Results obtained from both assays are not interchangeable. Standardization of IgGSc assay and review of the reference ranges must be accomplished in order to achieve a higher degree of agreement between different methods.

Molecular typing of isolates of the fish pathogen, Flavobacterium columnare, by single-strand conformation polymorphism analysis.

Flavobacterium columnare intraspecies diversity was revealed by analyzing the 16S rRNA gene and the 16S-23S internal spacer region (ISR). Standard restriction fragment length polymorphism (RFLP) of these sequences was compared with single-strand conformation polymorphism (SSCP). Diversity indexes showed that both 16S-SSCP and ISR-SSCP improved resolution (D>or=0.9) when compared with standard RFLP. ISR-SSCP offered a simpler banding pattern than 16S-SSCP while providing high discrimination between isolates. SSCP analysis of rRNA genes proved to be a simple, rapid, and cost-effective method for routine fingerprinting of F. columnare.

Host-specific association between Flavobacterium columnare genomovars and fish species.

A total of 90 Flavobacterium columnare isolates were recovered from predominant wild fish species in the Mobile River, Alabama, USA. Isolates were identified and confirmed by fatty acid methyl ester analysis and specific PCR amplification. Genomovar ascription was performed using 16S-restriction fragment length polymorphism (RFLP) analysis. The majority of genomovar I isolates were recovered from threadfin shad while genomovar II isolates came from catfish (including channel and blue catfish). Additional genotyping methods, including multilocus sequence analysis (MLSA), internal spacer region-single strand conformation polymorphism analysis (ISR-SSCP) and amplified fragment length polymorphism (AFLP), confirmed a clear division of the isolates into two groups that matched genomovar ascription. Fingerprinting methods revealed a higher genetic diversity within genomovar II isolates. Our data confirmed the coexistence of F. columnare genomovars I and II in a natural environment. A statistically significant association between genomovar I and threadfin shad was demonstrated while genomovar II strains were mainly recovered from catfish species.

Plant somatic hybrid cytoplasmic DNA characterization by single-strand conformation polymorphism.

Unlike maternal inheritance in sexual hybridization, plant somatic hybridization allows transfer, mixing and recombination of cytoplasmic genomes. In addition to the use of somatic hybridization in plant breeding programs, application of this unique tool should lead to a better understanding of the roles played by the chloroplastic and mitochondrial genomes in determining agronomically important traits. The nucleotide sequences of cytoplasmic genomes are much more conserved than those of nuclear genomes. Cytoplasmic DNA composition in somatic hybrids is commonly elucidated either by length polymorphism analysis of restricted genome regions amplified with universal primers (PCR-RF) or by hybridization of total DNA using universal cytoplasmic probes. In this study, we demonstrate that single-stranded conformational polymorphism (SSCP) analysis is a powerful, quick and easy alternative method for cytoplasmic DNA characterization of somatic hybrids, especially for mitochondrial DNA. The technique allows detection of polymorphisms based on both size and sequence of amplified targets. Twenty-two species of the subfamily Aurantioideae were analyzed with eight universal primers (four from chloroplastic and four from mitochondrial regions). Differences in chloroplastic DNA composition were scored in 98% of all possible two-parent combinations, and different mitochondrial DNA profiles were found in 87% of them. Analysis by SSCP was also successfully used to characterize somatic hybrids and cybrids obtained by fusion of Citrus sinensis (L.) Osb. and C. excelsa Wester protoplasts.

Study of the S427G polymorphism and of MYBL2 variants in patients with acute myeloid leukemia.

Dysregulation of MYBL2 has been associated to tumorigenesis and the S427G polymorphism could induce partial inactivation of MYBL2, associating it with cancer risk. It has previously been shown that MYBL2 was over-expressed in some acute myeloid leukemias (AML), portending poor prognosis. However, to date no studies have investigated the S427G or other genetic variants of MYBL2 in AML. This study analyzed the S427G in 197 AML patients and 179 controls and screened the MYBL2 sequence in patients. In contrast to other studies in solid tumors, the S427G was not associated with the incidence of AML. This study detected four unannotated genetic alterations, of which the Q67X could be involved in MYBL2 dysfunction. Eight polymorphisms were identified, among which the rs73116571, located in a splicing region, was associated with higher incidence in AML and weaker MYBL2 expression, suggesting pre-disposition to AML. Additional functional studies should be performed to verify these genetic variations as possible targets in AML.

Influence of inflammatory and lipidic parameters on red blood cell distribution width in a healthy population.

Red blood cell distribution width (RDW) is a routine red blood cell count parameter which has been shown to be associated with inflammatory parameters. Recently, some authors proposed that RDW seems to be a marker of an adverse lipidic profile. In order to clarify whether RDW is related to inflammation, plasma lipids, or both, we determined anthropometric, hematimetric, inflammatory and lipidic parameters in 1111 healthy subjects. RDW correlated directly with age, body mass index (BMI), inflammatory parameters (plasma viscosity, erythrocyte sedimentation rate (ESR), fibrinogen, leukocyte and neutrophil count), and inversely with iron and hematimetric parameters (P <  0.05). When subjects were divided according to gender, RDW correlated inversely with triglycerides only in women (P <  0.05). When subjects were classified into RDW-quartiles, increased RDW values were accompanied by decreased serum iron levels and hematimetric indices (P <  0.01), whereas age and inflammatory markers increased according to RDW-quartiles (P <  0.001 and P <  0.05, respectively). However, plasma lipids did not change with increasing RDW-quartiles (P >  0.05). In the linear regression analysis, age, hemoglobin, MCV (beta coefficient: 0.202, -0.234, -0.316, P <  0.001) and fibrinogen (beta coefficient: 0.059, P = 0.048) were the only independent predictors of RDW. The present study indicates that RDW is associated with inflammatory markers and hematimetric indices, but not with plasma lipid levels in a healthy population.

Single-nucleotide polymorphism array-based karyotyping of acute promyelocytic leukemia.

Acute promyelocytic leukemia (APL) is characterized by the t(15;17)(q22;q21), but additional chromosomal abnormalities (ACA) and other rearrangements can contribute in the development of the whole leukemic phenotype. We hypothesized that some ACA not detected by conventional techniques may be informative of the onset of APL. We performed the high-resolution SNP array (SNP-A) 6.0 (Affymetrix) in 48 patients diagnosed with APL on matched diagnosis and remission sample. Forty-six abnormalities were found as an acquired event in 23 patients (48%): 22 duplications, 23 deletions and 1 Copy-Neutral Loss of Heterozygocity (CN-LOH), being a duplication of 8(q24) (23%) and a deletion of 7(q33-qter) (6%) the most frequent copy-number abnormalities (CNA). Four patients (8%) showed CNAs adjacent to the breakpoints of the translocation. We compared our results with other APL series and found that, except for dup(8q24) and del(7q33-qter), ACA were infrequent (≤3%) but most of them recurrent (70%). Interestingly, having CNA or FLT3 mutation were mutually exclusive events. Neither the number of CNA, nor any specific CNA was associated significantly with prognosis. This study has delineated recurrent abnormalities in addition to t(15;17) that may act as secondary events and could explain leukemogenesis in up to 40% of APL cases with no ACA by conventional cytogenetics.

New attenuated vaccine against columnaris disease in fish: choosing the right parental strain is critical for vaccine efficacy.

Flavobacterium columnare, the causative agent of columnaris disease, is a highly diverse species comprised by three genomovars. Genomovar II strains are more virulent toward catfishes than genomovar I isolates. The objective of this study was to compare the vaccine efficacy of avirulent mutants derived from genomovars I and II using a rifampicin-resistance strategy. First, we compared the efficacy of 13 genomovar II mutants in channel catfish (Ictalurus punctatus) fingerlings and identified mutant 17-23 as the best vaccine candidate based on their relative percent survival (RPS) against a highly virulent genomovar II strain (BGFS-27). In the second experiment, we vaccinated zebrafish (Danio rerio) with two genomovar II mutants (17-23 and 16-534) and FCRR (genomovar I mutant) followed by exposure to BGFS-27 strain. RPS values were 28.4, 20.3 and 8.1% for 17-23, 16-534, and FCRR, respectively. For experiments 3 and 4, we tested both 17-23 and FCRR in channel catfish fry and Nile tilapia (Oreochromis niloticus). In both experiments, vaccinated fish were divided in two groups and each challenged with either a genomovar I (ARS-1) or a II (BGFS-27) strain. Channel catfish fry vaccinated with 17-23 and FCRR followed by challenge with BGFS-27 resulted in RPS values of 37.0% and 4.4%. When fish were challenged with ARS-1, RPS values were 90.9% and 72.7% for fish vaccinated with 17-23 and FCRR, respectively. Nile tilapia vaccinated with 17-23 and FCRR followed by challenged with BGFS-27 had RPS values of 82.1% and 16.1%, respectively. When fish were challenged with strain ARS-1, RPS values were 86.9% and 75.5%. Overall, our results demonstrated that vaccination with genomovar II mutant 17-23 confers better protection in channel catfish and Nile tilapia than FCRR against columnaris disease caused by genomovar II. Both mutants were equally protective against columnaris caused by genomovar I showing that 17-23 mutant cross-protected against both genomovars.

The deletion of exons 3-5 of BRCA1 is the first founder rearrangement identified in breast and/or ovarian cancer Spanish families.

We recently described a novel g.8097_22733del14637 deletion encompassing exons 3-5 in BRCA1 gene. This rearrangement was detected in 3 of 15 (20 %) breast and/or ovarian cancer families of Eastern Spain. This finding made us suspect that the newly identified deletion could be a founder mutation. To confirm this hypothesis we studied 18 subjects belonging to the three families under study, 11 deletion carriers and 7 non-carriers. We performed a haplotype analysis using two BRCA1 intragenic microsatellite markers and two markers surrounding the BRCA1 locus. The segregation analysis showed one common particular haplotype established by D17S1325, D17S1323, D17S855 and D17S1320 markers detected in the deletion carriers but absent in the non-carriers. Our study sustain that the deletion of exons 3-5 of BRCA1, g.8097_22733del14637, identified in families of southeastern of the Valencian Community is the first founder rearrangement until now reported in Spanish population, confirming the hypothesis that this mutation could have Iberian ancestry.