Cell Hb Research Articles

A Low-Loss Hybrid Bypass for DC Fault Protection of Modular Multilevel Converters

Without additional circuitry, the half-bridge modular multilevel converter (HB-MMC) is endangered under dc-side faults. Typically, a bypass thyristor is augmented to each HB cell to take up fault current until ac circuit breakers interrupt the dc fault. This paper proposes a dc fault protection concept for HB-MMC stations that requires insignificant extra silicon area relative to the thyristor bypass concept. Herein, bypass thyristors of typical HB cells are rearranged such that an independent modular shadow rectifier bridge (SRB) is formed. A low-loss switch assembly is utilized to immediately isolate the MMC following fault detection and the SRB suppresses the fault current by injecting a reverse dc voltage. Among several advantages, the proposed arrangement incurs insignificant losses in steady state, and in some arrangements the MMC is capable of operating in STATCOM mode briefly after fault inception to support ac grid voltage. The proposed concept may be suitable for clearing temporary faults on overhead HVdc lines. Several structural variations will be viewed and discussed. Applicability for a two-level voltage source converter will be addressed. The concept is validated by detailed numerical simulations of a ±200-kV HB-MMC station under dc fault.

Connectivity map identifies HDAC inhibition as a treatment option of high-risk hepatoblastoma

ABSTRACTHepatoblastoma (HB) is the most common liver tumor of childhood, usually occurring in children under the age of 3 y. The prognosis of patients presenting with distant metastasis, vascular invasion and advanced tumor stages remains poor and children that do survive often face severe late effects from the aggressive chemotherapy regimen. To identify potential new therapeutics for high risk HB we used a 1,000-gene expression signature as input for a Connectivity Map (CMap) analysis, which predicted histone deacetylase (HDAC) inhibitors as a promising therapy option. Subsequent expression analysis of primary HB and HB cell lines revealed a general overexpression of HDAC1 and HDAC2, which has been suggested to be predictive for the efficacy of HDAC inhibition. Accordingly, treatment of HB cells with the HDAC inhibitors SAHA and MC1568 resulted in a potent reduction of cell viability, induction of apoptosis, reactivation of epigenetically suppressed tumor suppressor genes, and the reversion of the 16-gene HB classifier toward the more favorable expression signature. Most importantly, the combination of HDAC inhibitors and cisplatin – a major chemotherapeutic agent of HB treatment - revealed a strong synergistic effect, even at significantly reduced doses of cisplatin. Our findings suggest that HDAC inhibitors skew HB cells toward a more favorable prognostic phenotype through changes in gene expression, thus indicating a targeted molecular mechanism that seems to enhance the anti-proliferative effects of conventional chemotherapy. Thus, adding HDAC inhibitors to the treatment regimen of high risk HB could potentially improve outcomes and reduce severe late effects.

Abstract B16: NR5A2 as a potential therapeutic target for hepatoblastoma

Abstract Hepatoblastoma (HB) is the most common malignant liver tumor in very young children. Because of multidrug resistance and severe side effects of anti-HB drugs during HB chemotherapy, it is urgent and important to develop new effective therapeutic agents with less toxicity for the treatment of pediatric patients with liver cancer. Nuclear Receptor Subfamily 5 Group A Member 2 (NR5A2; LRH-1) has been linked to various types of malignancies, and it is an emerging therapeutic target for many cancers. However the role of NR5A2 in liver cancer remains elusive. In this study, we found that both mRNA and protein expression levels of NR5A2 were significantly higher in HepG2 and HuH6 human HB cell lines and mouse orthotopic HB liver than the normal liver cell and normal liver tissue. Lentiviral-Mediated Short Hairpin RNA (shRNA) knockdown of NR5A2 inhibited HepG2 and HuH6 cell proliferation and colony formation compared with control cells. Furthermore, we found that silencing of NR5A2 induced downregulation of cell cycle gene CCND1 and the regulator gene c-myc. Similar to the effects of shRNA, we found the NR5A2 antagonist treatment also inhibited the proliferation of human HB cell lines (HepG2, HuH6 and HepT1 cells), reduced colony formation and CCND1 and c-Myc expression. Finally, flow cytometry results showed that the NR5A2 antagonist treatment induced a decreased percentage of cells in S phase which resulted in a significant decrease in the rate of cell proliferation and tumorigenicity, and induced an increased number of cells in the G2/M phase, leading to HB cell cycle arrest at G2/M phase. Collectively, our results suggest that NR5A2 play an important role in HB cancer cell proliferation. Interference of NR5A2 by shRNA or the NR5A2 antagonist inhibits HB cell proliferation and colony formation by inhibiting the cell cycle process through downregulation of CCND1 and c-Myc expression. By targeting NR5A2 signaling pathway, NR5A2 antagonist has promising therapeutic potential in the treatment of HB cancer. Note: This abstract was not presented at the conference. Citation Format: Jingling Jin, Roma Patel, Nan Ge Jin, Yan Shi, Wenjing Sun, Jianhua Yang, Jed G. Nuchtern, Sanjeev A. Vasudevan. NR5A2 as a potential therapeutic target for hepatoblastoma. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr B16.

Abstract B29: MDM4 is a major p53 regulator in hepatoblastoma

Abstract Purpose: Unresectable or metastatic hepatoblastoma generally confers a poor survival. MDM4 and MDM2 are the major negative regulators of p53, which has a predominantly wild type status in hepatoblastoma. Having previously shown that MDM4 has copy gain/amplification associated with the 1q32.1 amplicon in hepatoblastoma, we hypothesize that MDM4 is the predominant negative regulator of p53 function in hepatoblastoma and that blocking MDM4 will cause tumor cell death due to uninhibited p53 tumor suppressor activity. Methods: An MTT assay was used to measure NSC207895 (MDM4 inhibitor) and Nutlin-3a (MDM2 inhibitor) cytotoxicity on hepatoblastoma cell lines (HepG2, HepT1, and Huh-6) as well as a patient-derived hepatoblastoma cell line (PDCL-1). CCK-8 was used to assess effects of low dose NSC207895 on HB cell line proliferation. Soft agar was used to assess the effect of NSC207895 on hepatoblastoma colony formation. Immunoblotting and quantitative RT-PCR was used to measure expression of p53 downstream targets in hepatoblastoma after treatment with NSC207895. P53 knockdowns were created to assess the effect of NSC207895 in hepatoblastoma in the absence of p53. Cell cycle analysis was performed using BRDU. Results: Huh-6, HepG2, and HepT1 were all tested with Nutlin-3a and did not show significant cell death (IC50 >10μΜ) inhibition with NSC207895 caused significant cell death in Huh-6 (IC50=1.27μΜ), HepG2 (1.62μΜ), and HepT1 (2.05μΜ). A decrease in cell proliferation was noted with concentrations of NSC207895 as low as 0.05μΜ, 0.1μΜ and 0.3μΜ in HepG2, HepT1, and Huh-6 respectively. An increase in IC50 was seen in the p53 knockdowns compared to each respective control (HepG2: 12.6μΜ vs 3.38μΜ; HepT1: >50μΜ vs 1.45μΜ; Huh-6: 20.2μΜ vs 7.52μΜ). Increased PARP cleavage products and decreased MDM4 expression were seen on immunoblotting after treatment with NSC207895. By both immunoblotting and qPCR, the p53 downstream transcriptional targets, p21, BAX and PUMA, were increased after exposure to NSC207895 in all three cell lines. Cell cycle analysis showed an increased percentage of cells in G1/G0-phase and decreased percentage of actively replicating S-phase cells in HepG2 with NSC207895 treatment. Conclusion: Our data supports the hypothesis that in hepatoblastoma, MDM4 may be a dominant inhibitor of p53 and inhibition of MDM4 causes p53-mediated cell death and growth inhibition. Therefore, inhibition of MDM4 may be a viable drug target for treating hepatoblastoma. Citation Format: Yan Shi, Roma Patel, Emporia Hollingsworth, Stephen Sarabia, Jingling Jin, Dolores Lopez-Terrada, Sanjeev Vasudevan. MDM4 is a major p53 regulator in hepatoblastoma. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr B29.

Clinical Significance of Red Blood Cell Distribution Width and Inflammatory Factors for the Disease Activity in Rheumatoid Arthritis.

To evaluate the significance of red blood cell distribution width (RDW) and other factors for the disease activity in rheumatoid arthritis (RA). Each patient underwent clinical examination and blood sampling for assessment of serum high-sensitivity C-reactive protein (hs-CRP) levels, erythrocyte sedimentation rate (ESR), hemoglobin concentration (Hb), hematocrit (HCT), RDW, and other erythrocyte parameters (mean corpuscular volume [MCV], mean cell Hb [MCH], mean corpuscular Hb concentration [MCHC]). Rheumatoid factors (RF-IgA, -IgG, -IgM) and anti-cyclic citrullinated protein antibodies (anti-CCP) were purified from the plasma and detected by ELISA. RA patients were divided into two groups based on DAS28 scores: active RA group (DAS28 > 5.1) and inactive RA group (DAS28 2.6 - 3.2). Patient samples were within normal ranges for Hb (15.2 ± 1.3 g/L), HCT (29.9 ± 2.2%), and other red blood cell (RBC) parameters (MCV 80.3 ± 12.1 fL, MCH 26.6 ± 3.5 pg, MCHC 323 ± 25 g/L). The RDW was higher in the active RA group than in the inactive group (16.5 ± 3.2 vs. 13.9 ± 1.5%, p < 0.01). Similarly, the proportion of patients positive for RF and anti-CCP was higher in the active group than in the inactive group (RF 62 vs. 53%, CCP 83 vs. 61%). RF and anti-CCP were present at higher titers in patients with active RA. The bone erosion rate in the 110 RA patients was 67%. Patients with erosion had higher RDW than those without erosion (17.1 ± 2.2 vs. 13.9 ± 3.5%). High titers of anti-CCP and RF-IgM, -IgG, and -IgA were also associated with high bone erosion rates (67%, 77%, 67%, and 73%, respectively). Our results suggest an association between RDW and levels of inflammatory factors and autoantibodies in RA. This association may be linked to the underlying proinflammatory state and increased oxidative stress, both of which correlate with impaired erythrocyte maturation. RDW, RF, and anti-CCP are key players in the proinflammatory and proatherogenic status of RA, and they may represent useful markers to improve characterization of disease activity in RA patients, thereby helping the clinician to define more appropriate therapeutic strategies.

Abstract A13: Functional genomic analysis of central nervous system hemangioblastomas

Abstract Hemangioblastomas (HBs) are highly vascular tumors that develop in the Central Nervous System, mainly in the cerebellum, spinal cord and retina. These tumors are usually associated with Von-Hippel Lindau (VHL) disease but they can also appear sporadically. HBs often contribute to the morbidity and mortality of VHL patients due to the location of the tumor and the lack of efficient treatment. The genomic landscape of VHL-associated HBs is underexplored. The tumors develop upon bi-allelic loss of VHL but it is unclear if there are additional oncogenic drivers that influence tumor development. In addition, HBs are highly heterogeneous tumors comprised of diverse cell sub-populations; the origin of the HB tumor cell is controversial. The lack of cell lines and animal models that mimic the disease strongly impairs the research on HB biology and the search for new therapeutic strategies. In order to create an in vitro model to study HB biology, we established tumor-derived cell lines from VHL-associated HB tumors. The cell lines are formed by sub-populations that are morphologically different and express known HB markers such as Podoplanin, VEGFR2 and CXCR4. Moreover, these cells express Hypoxia Inducible Factor 1a (HIF1a), which is stabilized in VHL-deficient cells. We characterized the cell populations corresponding to surface markers expression and we analyzed the signaling pathways of isolated single HB cells derived from human tumors. In addition, we performed next-generation sequencing in HB tumor samples to identify potential oncogenic drivers that contribute to the development of HBs. We found a list of potential hits that we are now confirming with complementary techniques. These data can lead us to the identification of abnormal oncogenic pathways that could be therapeutically targeted in this disease. Finally, we are establishing a zebrafish model that mimics the HB disease. Vhl mutants develop hyper-vascularization in the brain and in the retina which is a common initiation factor in HB development. We are characterizing the vhl brain lesions using transgenic models with labeled vasculature and hematopoietic stem cell compartment. In conclusion, there are no current therapies for HBs and the lack of research models profoundly compromises the research advancement. In this work we established, for the first time, long-term cell lines that can be used to study the disease. The use of next-generation genomics allowed us to dissect the genomic landscape of this tumor type and will allow us to identify the HB cell of origin. All these new genomic data combined with novel resources, including the zebrafish model, will allow us to advance the current knowledge about HBs and may help to identify HB specific therapeutic targets. Citation Format: Ana Martins Metelo, Atanas Kamburov, James Kim, Elizabeth Lockerman, Dana Lee, Frederick Barker, Jeffrey Engelman, Anat Stemmer-Rachamimov, Gad Getz, Othon Iliopoulos. Functional genomic analysis of central nervous system hemangioblastomas. [abstract]. In: Proceedings of the AACR Special Conference: Advances in Brain Cancer Research; May 27-30, 2015; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2015;75(23 Suppl):Abstract nr A13.

SUPPLEMENTATION OF COFFEE HUSK FERMENTED WITH Pleurotus ostreatus: EFFECT ON PERFORMANCE AND BLOOD PROFILE OF GASTROINTESTINAL NEMATODES INFECTED GOAT

The aim of this study was to investigate the potency of coffee husk fermented with P.ostreatus as a natural anthelmintic supplement by measuring the performance and blood profile of goat suffered gastrointestinal nematodes infection. Eighteen local male goats of Kacang goat with body weight of 9.23 ± 1.71 kg and aged at ± 10 months were arranged into three treatments in completely randomized design. The treatments were T0 (group without chemical anthelmintic treatment and no supplementation of fermented coffee husk), T1 (without chemical anthelmintic treatment, but supplied with fermented coffee husk) and T2 (group with chemical anthelmintic and no supplementation of fermented coffee husk). All goats were offered a basal diet in the ratio of 60% natural grasses along with 40% concentrate. Fermented coffee husk was added in the diet as much as 6% from the dry matter need. The result showed that there were no significant effect to dry matter intake, daily weight gain, PCV value and eosinophil counts (P>0.05). The supplementation of fermented coffee husk (T1) enhanced Hb and red blood cell (RBC) value (P<0.05) while no significant difference to T2. There were no nematodes infection in T1 and T2 with the eggs count were zero while the animals in T0 were still infected. This result indicated that fermented coffee husk can be used as a promising natural anthelmintic supplement with the improvement of Hb value, RBC, egg counts and daily weight gain.

Haematological effects of 2-phenoxyethanol and etomidate in carp (Cyprinus carpio L.)

ObjectiveTo evaluate the effects of handling alone versus handling under anaesthesia with 2-phenoxyethanol or etomidate on haematological parameters in carp. Study designProspective, randomized, laboratory experiment. AnimalsSeventy-two juvenile carp (Cyprinus carpio) weighing 35.9 ± 10.4 g were divided into six groups of 12 fish. MethodsEither 2-phenoxyethanol or 2% etomidate were administered to induce deep anaesthesia (0.3 mL L−1 and 0.6 mL L−1, respectively) or deep sedation (0.15 mL L−1 and 0.3 mL L−1, respectively). Fish were handled with and without sedation. Blood was sampled at 1 hour and 1 week post-treatment. Phagocyte oxidative activity [nitrotetrazolium blue reduction test (NBT)] and differential erythrocyte [red blood cell (RBC)] and leukocyte (white blood cell) counts were evaluated. ResultsAt 1 hour after the induction of anaesthesia, haematocrit (Ht) and haemoglobin (Hb) were increased in fish anaesthetized with 2-phenoxyethanol, and mean corpuscular haemoglobin (MCH) was increased in fish anaesthetized with etomidate. At 1 week, an increase in RBC, erythroblastosis, erythrocyte damage, lymphopenia, neutrophilia, monocytosis and thrombocytosis occurred in both groups. Red blood parameters did not change 1 hour after handling alone, but after 1 week Ht, Hb and mean cell volume decreased, whereas MCH concentration (MCHC) and abnormal erythrocytes increased. Lymphopenia, neutrophilia, monocytosis, thrombocytosis and a decrease in NBT occurred. Fish handled under sedation showed an increase in Hb and MCHC followed by a decrease at 1 week in Ht, Hb and MCH, erythroblastosis and increased abnormal erythrocytes. Lymphopenia and neutrophilia were less pronounced than in fish handled without sedation, but a decrease in NBT was noted at 1 week post-treatment. Conclusions and clinical relevanceDeep anaesthesia with 2-phenoxyethanol or etomidate induced significant haematological alterations in juvenile carp. Deep sedation reduced the immediate immunosuppressive effects of handling but did not eliminate longterm effects. These anaesthetics should be avoided during experimental procedures involving haematological measurements. In contexts that require the short-term handling of carp, these drugs should be used with caution in view of their possible side effects.

Correlation between the Lactate Dehydrogenase Levels with Laboratory Variables in the Clinical Severity of Sickle Cell Anemia in Congolese Patients.

BackgroundSickle cell anemia is an inflammatory disease and is characterized by chronic hemolysis. We sought to evaluate the association of lactate dehydrogenase levels with specific clinical phenotypes and laboratory variables in patients with sickle cell anemia.MethodsThe present cross-sectional study was conducted in Sickle Cell Centre of Yolo in Kinshasa, the Democratic Republic of Congo. Two hundred and eleven patients with Sickle Cell Anemia in steady state were recruited. Seventy-four participants with normal Hb (Hb-AA) were selected as a control group.ResultsThe average rates of hemoglobin, hematocrit, and red blood cells tended to be significantly lower in subjects with Hb-SS (p<0.001). The average rates of white blood cells, platelets, reticulocytes and serum LDH were significantly higher in subjects with Hb-SS (p<0.001). The average rates of Hb, HbF, hematocrit and red blood cells of Hb-SS patients with asymptomatic clinical phenotype were significantly higher than those of the two other phenotypes. However, the average rates of white blood cells, platelets, reticulocytes, and LDH of Hb-SS patients with the severe clinical phenotype are higher than those of two other clinical phenotypes. Significant correlations were observed between Hb and white blood cell in severe clinical phenotype (r3 = -0.37 *) between Hb and red blood cells in the three phenotypes (r1 = 0.69 * r2 * = 0.69, r3 = 0.83 *), and finally between Hb and reticulocytes in the asymptomatic clinical phenotype and severe clinical phenotype (r1 = -0.50 * r3 = 0.45 *). A significant increase in LDH was observed in patients with leg ulcer, cholelithiasis and aseptic necrosis of the femoral head.ConclusionThe increase in serum LDH is accompanied by changes in hematological parameters. In our midst, serum LDH may be considered as an indicator of the severity of the disease.

Elevated Steady State WBC and Platelet Counts Are Associated with Frequent Emergency Room Use in Adults with Sickle Cell Disease

Patients with sickle cell disease (SS/Sβ0) often utilize the emergency department (ED) for treatment of painful vaso-occlusive crisis and other sequalae of their disease. However there is significant variation in use, with a minority of patients making up the majority of visits. We studied whether objective steady state laboratory parameters might be associated with frequent ED use and whether hydroxyurea use modified this relationship.Methods: We identified all patients with sickle cell disesase seen at our medical center in 2012. Patients were identified as having sickle cell disease if hemoglobin (Hb) electrophoresis demonstrated sickle hemoglobin (HbS), fetal hemoglobin (HbF) and HbA2 but no Hemoglobin A, C, or other detectable hemoglobinopathies. ED, clinic, and inpatient admissions over the entire year were calculated and ED use was categorized as either 0-1, 2-5, or >6 visits a year. Steady state laboratory tests were defined as those not within a day of an ED visit or a week of a hospital admission. All 2012 steady state parameters retrieved for a given laboratory test for a given patient were averaged. “Active” laboratory tests, defined as those within one day of an ED visit, were averaged separately. HbF and weight were not separated by activity. Data were analyzed for normality; parametric values were assessed by mean and SD, non-parametric values were assessed as medians and interquartile ranges. Parametric and non-parametric bivariate tests of association were used as appropriate.Results: 432 adult sickle cell patients were identified, ages 18-87yrs; 54% were female. 181 patients had 0-1 ED visits within the year, 143 had 2-5 visits in the year and 96 had >6 visits for a total of 2259 visits. Patients who had >6 visits accounted for 1750 (77%) of the total visits for the year. When steady state labs were examined, high WBC and platelet counts were most strongly associated with frequent admissions. Steady state WBC of >12.0 x10^9/L were significantly more likely to have >6 visits/year (OR 2.6; 95% CI: 1.6-4.2, p=.0004). Platelet counts of >420 x10^9/L were also strongly associated with >6 ED visits (OR 2.6, 95% CI:1.5-4.4, p=.0007). LDH and AST were also shown to correlate significantly with ED (p=0.02 and 0.005 respectively) use while Hb and albumin were negatively associated (p<0.001 and 0.02 respectively). Hydroxyurea scripts were associated with increased ED visits (p<0.001); 38.1% of the population had been given a script for hydroxyurea within the year; patients with frequent ED use were 2.8x more likely to have been given a script for hydroxyurea within the year. These patients had higher MCV and Hb levels and lower, but still elevated, WBC and platelet counts per ED visit cohort stratification, a suggestion that hydroxyurea was being taken, but despite this, had similar significant associations with ED visits as the non-hydroxyurea group. MCV, MCHC, absolute reticulocyte count, weight, and %HbF did not demonstrate an association with ED visit frequency. When “active” parameters were examined, leukocytosis was present in all categories and WBC count could no longer predict a predisposed cohort; Hb level, LDH and AST were also no longer significant. Albumin and platelet count remained associated with ED visits. When older patients >40 yrs (n=124) were compared with <40yrs (n=308 ), high WBC and platelet counts remained significantly associated with high (>6) ED visits in both cohorts.Conclusions: Our data suggest that baseline WBC and platelet count are strongly associated with frequent ED utilization and may be better predictors than Hb, HbF, and other red cell or hemolytic parameters. Hydroxyurea use was not associated with fewer ED visits but those patients with frequent ED use still have relatively higher WBC and platelet counts than those with less frequent ED use, suggesting that hydroxyurea under-dosing may be an important issue. To what extent objective steady state parameters can identify a population that needs more aggressive baseline care and to what extent maximization of therapy can decrease frequent ED visits needs further study. DisclosuresNo relevant conflicts of interest to declare.

Abstract 3086: The genetic landscape of the childhood liver cancer hepatoblastoma

Abstract Hepatoblastoma (HB) is the most common malignant liver tumor in children with a histology that resembles various stages of the developing liver. To explore the genetic origin of this cancer, we performed whole-exome sequencing of 8 tumors and matched germline tissue. In average, we detected only three sequence variants per tumor, which is the lowest mutation rate ever reported for any cancer. Mutations of ß-catenin (CTNNB1) displayed the only recurrent event occurring in all tumors, thereby underscoring the significance of activated Wnt signaling in the genesis of HB. Gene ontology analysis revealed a predominance of candidate genes involved in the regulation of gene expression and transcription, including the nuclear factor (erythroid-derived 2)-like 2 (NFE2L2). Targeted resequencing of a cohort of 50 HB cases and cell lines revealed NFE2L2 mutations in 10% of all tumors. Interestingly, all mutations were located in or adjacent to the DLG and ETGE motifs of the NFE2L2 protein that are known to be recognized from the KEAP1/CUL3 complex for proteasomal degradation. In line with this, cells transfected with mutant NFE2L2 were insensitive to KEAP1-mediated downregulation of NFE2L2 signaling. Clinically, overexpression of the NFE2L2 target gene NQO1 in tumor tissues was significantly associated with high tumor stage, metastasis, and poor outcome. These data suggest that activation of NFE2L2-KEAP1 signaling through mutations in NFE2L2 is a prominent characteristic in pediatric liver tumors, thereby advocating inhibition of aberrant NFE2L2 activity as a therapeutic option, especially in high-risk patients with advanced disease. Citation Format: Roland Kappler, Melanie Eichenmüller, Franziska Trippel, Tim M. Strom, Dietrich von Schweinitz. The genetic landscape of the childhood liver cancer hepatoblastoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3086. doi:10.1158/1538-7445.AM2014-3086

Screening of Thalassaemia Carriers and Its Limitations

The α and β-thalassaemias (thal) are common genetic disorders of globin chain synthesis where the carriers have deficiency of α or β globin chain respectively. In resource limited settings, the naked eye single tube red cell osmotic fragility (NESTROFT) has been used for screening of thalassaemia. It has low specificity compared with its sensitivity. Screening of carriers encompasses automated/semi-automated techniques and manual methods for presumptive diagnosis of thalassaemia. The current strategy in screening involves a step by step process simplified into the following acronym, BHES. B, refers to blood counts and a film; H, haemoglobin (Hb) subtyping; E, Hb electrophoresis; and S, stability (H-inclusion and Sickle cell test). Blood counts and red blood cell indices are now available by automated instrumentation where a cut-off value of mean corpuscular Hb (MCH)<27 pg necessitates further investigation for thalassaemia or iron deficiency. The distribution of normal Hb subtypes (HbA, HbA2 and HbF) is done commonly by high performance liquid chromatography (HPLC) or capillary zone electrophoresis (CZE). The thalassaemia trait phenotype is indicated by a near normal Hb, microcytic and hypochromic red cell indices. In classical β-thalassaemia trait, the cut-off value of HbA2 value is 4% and above. However, milder mutations can result in HbA2 values between 3.2 to 3.9%. In alpha thalassaemia trait the HbA2 falls within the normal range. In a carrier of α0-thalassaemia, occasional or few H-inclusions suggests its presence. However, absence of H-inclusions does not rule out its presence. Recently, it has been shown that the current screening method of thalassaemia carriers has its limitations. Mild mutations may show normal or near normal red cell indices and be missed in the screening process. Carriers with these mutations can cause thalassaemia intermedia. In addition there are mutations that have more profound effects resulting in more severe outcome but have carriers with normal red cell indices. In α-thalassaemia intermedia, HbH disease, those with nondeletional mutations are clinically more severe. These non-deletion mutations include Hb Constant Spring, Hb Pakse and Hb Adana. Carriers with these non-deletional α-thal mutations have normal red cell indices. Cases of hydrops foetalis have been reported with a homozygous state of Hb Adana and also in α0 with Hb Adana. Coinheritance of α or β-thal also effects the red cell indices making them near normal. Thalassaemia intermedia and major have serious implications and prevention requires strategies to detect couples at risk. Mutational spectrum of the globin genes is updated continuously with improved diagnostic technology. Clinical programs preventing thalassaemia are presently based upon prospective screening and prenatal diagnosis. In order for prevention to be effective, there is a need to be aware that mutations can exist in thalassaemia carriers where the red cell indices are normal.

Upregulation of a potential prognostic biomarker, miR-155, enhances cell proliferation in patients with oral squamous cell carcinoma

We sought to investigate the role and diagnostic value of microRNA 155 (miR-155) in OSCC patients. Using real-time quantitative polymerase chain reaction analysis, miR-155 expression levels were assessed in OSCC cell lines and a cancerous HB cell line. The correlation between miR-155 expression level and clinical parameters was analyzed in 46 patients with OSCC. In addition, the effects of miR-155 on OSCC cell proliferation were evaluated by modulating its expression using an miR-155 mimic and antisense miR-155. Significant upregulation of miR-155 was found in OSCC cell lines and in tissues of patients with OSCC. The receiver operator characteristic analysis indicated fair-to-good predictability. Overexpression of miR-155 correlated with the histologic grade (P = .033), and the upregulation of miR-155 enhanced OSCC cell proliferation. In OSSC, upregulation of miR-155 correlated with the histologic grade and can be used as a potential prognostic biomarker.

Evaluation of clinical and laboratory correlates of sickle leg ulcers

The exact mechanism for the occurrence of sickle leg ulcers (SLUs) has not been fully explained, although, popular opinion supports a multifactorial etio-pathogenetic process. Leg ulceration in sickle cell is a chronic and debilitating condition which is difficult to treat and may worsen the psychosocial impact of this illness. This study aims to evaluate the laboratory and clinical correlates of SLUs. One hundred sixty-seven patients who had been diagnosed with sickle cell anemia (homozygous S) had their steady-state hemoglobin concentration (Hb), hematocrit, white cell count, platelet count, serum bilirubin, and aspartate transaminase (AST) as well as frequency of crisis per annum evaluated with respect to their relationship to the occurrence of leg ulcers. They were aged 6-53 years (mean age 24.3 years), and prevalence of leg ulcer was found to be 2.75 per 1000 (2.54 per 1000 in females and 2.83 per 1000 in males). The independent sample t-test showed a significant difference in the serum AST levels in those with SLU (p = 0.029), though a positive correlation did not exist. Other predictors of disease severity found to have positive relationship with each other were the AST and total serum bilirubin 0.207 (p = 0.012); Hb and age 0.130 (p = 0.035); Hb and white cell count -0.159 (p = 0.010), white cell count and age -0.113 (p = 0.018). SLUs do not occur in patients with severe disease in sickle cell. The clinical and laboratory indicators of severe sickle cell disease do not correlate positively with the occurrence of SLU. Serum AST may have a relationship with leg ulceration in these patients. Environmental factors most likely play a major part in the etiopathogenesis of leg ulcer and this may require further studies in different sociocultural settings.

Comparison of whole blood collection and double-unit erythrocytapheresis in preoperative autologous blood donation

We compared preoperative autologous blood donation (PABD) using serial manual whole blood (WB) and PABD using a single session, double-unit erythrocytapheresis in terms of the hemodynamic recovery and clinical outcomes. This study included 56 donors in the WB PABD group and 117 donors in the double-unit erythrocytapheresis PABD group. All subjects were men with body weight >70 kg, Hb level >13.3g/dL, Hct >40%, and who were scheduled for oral and maxillofacial surgery. Three cycles of manual WB collection for PABD or a single session, double-unit erythrocytapheresis using the Alyx was performed. There were no significant differences in donor demographic variables including age, height, weight, Hb, Hct, or red cell mass between the 2 groups. The double-unit erythrocytapheresis was completed earlier than the last manual WB PABD (at 15.3 ± 4.7 days and 6.5 ± 3.2 days before surgery, p<0.001). Hct values before surgery were higher in the double-unit erythrocytapheresis PABD group than in the manual WB PABD group (39.7 ± 3.2 vs. 38.6 ± 2.7, p=0.024). ΔHct and %ΔHct before the first PABD and before surgery were lower in the double-unit erythrocytapheresis PABD group than in the manual WB PABD group (-5.6 ± 2.8 vs. -6.8 ± 2.7, p=0.010 and -12.3 ± 5.9 vs. -14.8 ± 5.6, p=0.008, respectively). The incidence of additional allogeneic blood transfusions during or after surgery and the post-operative Hb and Hct values were similar in the 2 groups. The length of hospital stay after surgery was significantly longer in the manual WB PABD group than in the double-unit erythrocytapheresis group (6.1 ± 2.5 vs. 5.4 ± 1.9, p=0.043). Of the 33 donors in the double-unit erythrocytapheresis PABD group, 7 (21.2%) reported discomforts related to the procedure, and 6 graded the discomforts (hypocalcemia, perioral tingling sense, paresthesia, dizziness, stuffiness, pain on the intravenous site, and muscle tension) as mild. The single session, double-unit erythrocytapheresis prolonged the time interval between PABD and surgery and led to better hemodynamic recovery than the serial manual WB PABD, and hypocalcemic symptoms were mild.

Fetal Red Blood Cell Parameters in Thalassemia and Hemoglobinopathies

Introduction: With the lack of fetal blood specimens in routine practice, little is known about red blood cell (RBC) parameters of fetuses with various thalassemia syndromes. This study aimed to describe these in various forms of thalassemia. Materials and Methods: The study was performed on 93 fetal blood specimens obtained from pregnant women by cordocentesis during 18-24 weeks of gestation. RBC parameters were recorded on automated analyzer. Hemoglobin (Hb) and DNA analyses were performed for definite genotyping. Results: No significant difference in RBC parameters was observed between non-thalassemic fetuses and those with β-thalassemia trait, Hb E trait, homozygous Hb E and β-thalassemia/Hb E disease. However, in those with α⁰-thalassemia trait and double heterozygous α⁰-thalassemia/Hb E, slight reduction in mean corpuscular volume (MCV) was noted. Fetuses with the Hb H disease showed significant reductions in Hb, MCV and mean corpuscular Hb (MCH). Marked reductions in Hb, hematocrit, MCH and mean cell Hb concentration and increased RBC distribution width with numerous nucleated RBC were clearly observed in Hb Bart's hydrops fetalis. Conclusion: Simple analysis of fetal RBC parameters is useful for making presumptive prenatal diagnosis of α-thalassemia syndromes including Hb H disease and Hb Bart's hydrops fetalis which can then be confirmed by Hb and DNA analyses.

A Study of Inflammation-Based Predictors of Tumor Response to Neoadjuvant Chemoradiotherapy for Locally Advanced Rectal Cancer

Background: The ability of pretreatment laboratory markers of acute-phase inflammatory reactions like serum albumin level (SAL), hemoglobin (Hb), and absolute blood cell counts to predict complete pathological response (CPR) to neoadjuvant chemoradiotherapy (NACRT) in patients with locally advanced rectal cancer (LARC) has not yet been fully studied. Methods: We retrospectively examined the relation between SAL, Hb and absolute blood cell counts, and CPR rates in 140 LARC patients treated with NACRT. Results: Univariate analysis showed a significantly higher probability of CPR to NACRT in patients with clinical stage (CS) III LARC who had SAL >3.5 mg/dl (OR = 2.39; p = 0.04) and a neutrophil-to-lymphocyte ratio (NLR) value <5 (OR = 2.86; p = 0.03). The relation of CPR with SAL (OR = 2.11; p = 0.048) and NLR (OR = 2.54; p = 0.04) was confirmed by multivariate analysis in the same subset of patients. None of the parameters studied predicted CPR in patients with CS II disease. Patients who achieved CPR to NACRT had a higher probability of 5-year overall survival (HR 0.48; p = 0.01) and 5-year disease-free survival (HR 0.33; p = 0.003). Conclusions: Our data indicate that SAL >3.5 mg/dl and NLR <5 may be positively related to CPR after NACRT in patients with CS III LARC. Hypoalbuminemia and a high NLR may be considered an indication for a more aggressive approach to NACRT and postoperative adjuvant chemotherapy in this subset of patients. This hypothesis requires confirmation in a randomized study.

Red cell indices in classification and treatment of anemias

Measurements of red cell volume, hemoglobin (Hb) concentration and Hb content continue to play a crucial role in the differential diagnosis of anemias 80 years after the publication of Wintrobe's seminal work. Modern hematology analyzers provide additional data on the heterogeneity of these parameters (distribution width) and quantify similar parameters of reticulocytes as well. Red cell and reticulocyte cellular indices are widely used in the diagnosis and monitoring of hematological diseases. Quantification of hypochromic cells is valuable in the differential diagnosis of thalassemia trait and iron deficiency, and in monitoring therapeutic response to erythropoietic stimulating agents, while hyperchromic cells are an essential diagnostic component for hereditary spherocytosis and may correlate with hemolytic parameters in sickle cell disease. Values for these parameters however depend on the technology used. Red cell clearance is associated with a reduction in both Hb content and cell volume: normal cells are likely to be removed by the time they reach a volume of 72 fl. Reticulocyte parameters such as Hb content (CHr or ret-He) or maturity index (RMI) have shown value in a variety of hematological conditions. New findings from genetic association studies have identified several potential novel genes affecting red cell indices, which are not mediated by changes in iron availability. Red cell indices continue to provide an essential support to the diagnosis and monitoring of hematological diseases.

Low iron storage in children with tilt positive neurally mediated syncope

The mechanisms under neurally mediated syncope (NMS) are not fully understood. This study aimed to assess the level of storage iron in children with different hemodynamic patterns in head-up tilt test. Altogether 210 children (11.31±2.49 years) with syncope or pre-syncope treated between May 2008 and September 2010 were studied prospectively. Following history taking and physical examination, their levels of hemoglobin (Hb), hematocrit (Hct) and serum ferritin were measured. In the 210 children, 162 (77.1%) had NMS and 48 (22.9%) had syncope due to other causes. In the 162 children with NMS, 98 children were subjected to positive tilt test. The level of serum ferritin was significantly lower in the 98 children with NMS (P<0.001). The comparison of levels of Hb, Hct and mean cell volume (MCV) displayed no significant difference between the two groups. Reduced iron storage (serum ferritin <25 ng/mL) was found to be more prevalent in children with NMS (63% vs. 20%, P<0.001). Prevalence of iron deficiency was also significantly higher in children with NMS than in children with syncope due to other causes (27% vs. 6%, P=0.003). In head-up tilt test positive children with NMS, the level of serum ferritin should be evaluated. Low storage iron may be one of the underlying mechanisms of NMS.

Correlation of haemoglobin and red cell indices with serum ferritin in Indian women in second and third trimester of pregnancy

BackgroundIron deficiency anaemia (IDA) is the most common cause of anaemia in pregnancy in Indians and is associated with increased risk of low birth-weight infants. Studies from developed countries recommend iron supplementation based on serum ferritin levels. However, screening by serum ferritin is not feasible in all cases in India. This study was undertaken to document haematological profile of pregnant Indian women. MethodsWe studied the correlation between second and third trimester ferritin concentration and haemoglobin (Hb) and red cell indices in 100 consecutive ANC cases to select the best haematologic characteristic to identify women who needed iron therapy. Hb and red cell indices, RBC count, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, red cell distribution width were analysed and PBS studied to subtype anaemia if present. ResultsProportion of iron deficiency anaemia in pregnancy was 34% and significant correlation was found between serum ferritin and RDW-CV% and TRBC. No correlation was found between ferritin levels and Hb, MCV, MCH and MCHC. Serum ferritin levels were <12 ng/mL in 30 out of 52 non-anaemic cases suggesting prevalence of sub-clinical iron deficiency in 58% cases. None of the red cell indices correlated with ferritin level in this group. Only TRBC showed some correlation with ferritin (r = −0.090, p > 0.05). ConclusionAll pregnant women in India should continue to get iron supplements unlike what is recommended in the developed countries where iron supplementation is based on serum ferritin levels.