bluuebunny/crossref_metadata_2025_split huggingface dataset:
Retrospective Evaluation of Patients Referred for Hemochromatosis Genetic Testing
Authors: Lanktree Matthew B. , Lanktree Bruce B. , Waye John S. , Pare Guillaume , Sadikovic Bekim , Crowther Mark A. 
Published: October 2019

Abstract
Background: The common genetic test for hemochromatosis involves the genotyping of two polymorphisms in the HFE gene (C282Y and H63D). Current guidelines suggest testing of patients with ferritin greater than 300 µg/L and transferrin saturation greater than 45%. Testing of first degree relatives of confirmed HFE positive cases is also endorsed. However, the current test has poor sensitivity and specificity for iron overload causing end organ damage. We sought to evaluate biochemical parameters of patients referred for HFEtesting and identify those who received phlebotomy.
Methods: After receiving ethics approval, electronic charts of patients referred to Hamilton Health Sciences, Hamilton, Ontario, Canada for HFE genetic testing between January 1, 2012 and December 31, 2012 were reviewed.
Results: HFE genetic testing in 642 patients yielded 100 (15.6%) positive C282Y homozygote or C282Y/H63D compound heterozygote results. In patients with biochemical markers of iron overload available (n=160), patients with a risk HFE genotype had significantly higher iron saturation, serum iron, and hemoglobin (P<0.001), but did not have higher ferritin or liver enzymes. Only fifty percent of patients referred had biochemical evidence of iron overload (transferrin saturation [TS] > 45% and ferritin > 300). Of patients who required phlebotomy, only 27 of 40 (67.5%) were HFEpositive.
Conclusion: Many factors affect the decision to phlebotomize a patient with suspected iron overload. However, it appears that most of the current HFE genetic testing being performed did not alter patient management.

Disclosures
Crowther: Asahi Kasai: Membership on an entity's Board of Directors or advisory committees; Bayer: Speakers Bureau; Celgene: Speakers Bureau; Shire: Speakers Bureau; Boehriniger Ingelheim: Consultancy; CSL Behring: Speakers Bureau; Leo Pharma: Membership on an entity's Board of Directors or advisory committees, Research Funding; Portola: Membership on an entity's Board of Directors or advisory committees; Viropharma: Membership on an entity's Board of Directors or advisory committees.
Clinical and Laboratory Characterization of the c.*233G>C Beta Globin Gene Substitution: Evidence Against a Thalassemic Phenotype
Authors: Smith Debra , Park Jason , Mitui Midori , Luu Hung , Timmons Charles 
Published: October 2019

Abstract
Purpose:
The c.*233G>C nucleotide substitution in the HBB gene was recently proposed as a novel β thalassemia allele based on genetic analysis of 11 thalassemic individuals. Based on this single report, the c.*233G>C allele has been provisionally included as a β (+) thal allele in a commonly used β globin database (HbVar).
The c.*233G>C is frequently seen in HBB gene sequencing for hemoglobinopathies. A clinical review was performed to determine the phenotype and hematologic characteristics of the c.*233G>C nucleotide substitution.
Methods:
At a tertiary-care pediatric institution, a retrospective review was performed of all HBB gene sequencing tests over a 30-month period. A chart review was performed for all cases with the c.*233G>C variant.
Results:
Of a total of 104 HBB cases, the c.*233G>C variant was identified in 18 individuals; 17 were heterozygotes and 1 was a homozygote (Patient 17) (Table 1). HBB gene sequencing was performed as part of the work-up for suspected variant hemoglobin (Hb) but can detect non-coding alterations including β thalassemia alleles as well.
Red cell indices in our series (Table 1) do not demonstrate the characteristics of heterozygous β thalassemia (microcytosis, mildly elevated RBC count, and mild anemia). MCV and RBC count data available for 9 individuals showed MCV values in the normocytic or macrocytic range and RBC counts within the normal range for all 9 patients. Of 13 patients with recorded Hb values, only one patient (Patient 14) was anemic but was found to have iron deficiency.
The patients in our series did not have elevated Hb A2, which in patients with β thalassemia is typically above the normal range (>4%); the 9 patients who could be evaluated for Hb A2 had values ranging from 2.4% to 3.7%. The remaining individuals were either too young to be evaluated for Hb A2 or possessed a variant Hb that co-eluted with HbA2.
The 13 patients in our series who were heterozygous for a Hb variant showed no excess in the variant Hb relative to Hb A. In patients with compound heterozygosity with a β thalassemia allele, the amount of the variant hemoglobin typically exceeds the amount of Hb A.

Conclusions:
Red cell indices and Hb proportions in this series of 18 individuals with c.*233G>C substitution are not consistent with a thalassemic phenotype. Our analysis supports a hypothesis that c.*233G>C substitution is not a β (+) thalassemia allele but is rather a fairly common benign polymorphism. This information has important implications in the diagnosis and genetic counseling of individuals with the c.*233G>C substitution.
References:
1. Sirdah et al. The spectrum of β-thalassemia mutations in Gaza Strip, Palestine. Blood Cells, Molecules and Diseases. 2013; 50: 247-251.
2. HbVar: A Database of Human Hemoglobin Variants and Thalassemias. http://globin.cse.psu.edu/hbvar/menu.html.

Disclosures
No relevant conflicts of interest to declare.
Regulation of Na+/Mg2+ Exchange in Sickle Erythrocytes By Endothelin-1
Authors: Rivera Pablo A. , Inostroza Yaritza , Romero Jose R. , Rivera Alicia 
Published: October 2019

Abstract
Excess levels of endothelin-1 (ET-1), erythrocyte sickling and chronic inflammation have been proposed as important contributors to the pathophysiology of sickle cell disease (SCD). We have shown that ET-1 receptor antagonists improve hematological parameters by reducing Gardos channel activity in two transgenic mouse models of SCD while reducing oxidant stress by decreasing circulating levels of protein disulfide isomerase. Magnesium (Mg2+) deficiency, mediated in part via increased erythrocyte Na+/Mg2+ exchanger activity, has been demonstrated to contribute to erythrocyte dehydration, K+ loss and sickling in SCD. However, the relationship between ET-1 and the Na+/Mg2+ exchanger in SCD remains unclear. We measured Na+/Mg2+ exchange activity in ex vivo red cells and observed increased activity following in vitro incubation of human (2.2 ± 0.2 to 3.2 ± 0.1 mmol/1013 cell x h, P<0.03, n=5) and mouse red blood cells with ET-1 (P<0.001, n=5); events that were significantly blocked by pre-incubation of cells with 1 μM BQ788, a selective inhibitor of ET-1 type B receptors. In addition, in vitro deoxygenation of sickle red cells led to increased exchanger activity that was inhibited by impramine, a Na+/Mg2+ exchange inhibitor, and associated with reduced deoxygenation-stimulated sickle cell dehydration. These results suggest an important role for ET-1 and cellular magnesium homeostasis in sickle cell disease. To this end, we studied Na+/Mg2+ exchange activity in ex vivo erythrocytes from three transgenic sickle mouse models and observed increased activity in these cells when compared to red cells from either Hb A transgenic or C57BL/J6 wild-type mice (P<0.03, n=4). We then tested the in vivo effects of ET-1 receptor antagonists on erythrocyte Na+/Mg2+ exchange activity in the BERK mouse, a transgenic model of SCD. We blocked ET-1 receptors type A and B by in vivo treatment with BQ-788 and BQ-123 (360mg/Kg/Day) for 14 days and observed lower erythrocyte exchanger activity when compared to cells from vehicle treated BERK mice (P<0.02, n=6). Thus our results suggest that ET-1 receptor blockade represents an important therapeutic approach to control erythrocyte volume and magnesium homeostasis that may lead to improved inflammatory and vascular complications observed in SCD. Supported by NIH R01HL090632 to AR.

Disclosures
No relevant conflicts of interest to declare.
A Novel 2 Amino Acids Deletion in the Leucine Zipper Domain of C/EBPε Leads to Neutrophil-Specific Granule Deficiency (SGD)
Authors: Akagi Tadayuki , Wada Taizo , Muraoka Masahiro , Toma Tomoko , Kaji Kenzo , Agematsu Kazunaga , Koeffler H. Phillip , Yokota Takashi , Yachie Akihiro 
Published: October 2019

Abstract
Neutrophil-specific granule deficiency (SGD) is a rare congenital disorder. Neutrophils of SGD patients show abnormal bilobed nuclei and lack secondary and tertiary granule gene expression. Since neutrophils of SGD patients are deficient for bactericidal activity, the patients display immunodeficiency and suffer from frequent and severe infections. The transcription factor CCAAT/enhancer binding protein epsilon (C/EBPε) is one of the major regulators of granulopoiesis and is known to be the responsible gene for the disease. At least two homozygous germline mutations (5bp deletion and A-nucleotide insertion) of C/EBPε, which are functionally defective, have been reported.
Here, we report a novel in-frame deletion of the C/EBPεgene in a 55 years-old female patient with a life-long history of recalcitrant skin infections with ulcer and scar formation. Peripheral blood smear showed characteristic changes, including reduction of cytoplasmic granules and increased bilobed nuclei of the neutrophils, monocytosis, absence of eosinophils and increased basophils. Flow cytometric examination revealed significant reduction of CD16 and abnormal expression of CD14 on the neutrophil surface.
Sequencing of the C/EBPε gene revealed a homozygous deletion of 6 base-pairs (arginine and serine residues deletion; ΔRS) within the leucine zipper domain. Activation of luciferase driven by G-CSF receptor promoter was not induced by ΔRS; and endogenous expression of granule genes, including B9, NGAL and lactoferrin, was induced by wild-type C/EBPε but not by ΔRS in NIH3T3 cells, suggesting that transcriptional activity of ΔRS was diminished. GFP-tagged ΔRS, as well as wild-type C/EBPε, were localized to the nucleus in NIH3T3 cells, and DNA binding activity of ΔRS, which was determined by biotin-labeled DNA pulldown assay, was intact. Meanwhile, ΔRS together with Gata1 and PU.1 were not able to induce expression of eosinophil major basic protein (MBP) in NIH3T3 cells. Of note, wild-type C/EBPε associated with Gata1; however, ΔRS did not interact with Gata1, indicating that the ΔRS likely impairs protein-protein interaction of other transcription factors resulting in loss of transcriptional activation. These results further support the importance of the leucine zipper domain of C/EBPε for its essential function and indicate multiple molecular mechanisms leading to SGD.

Disclosures
No relevant conflicts of interest to declare.
Contribution of Red Blood Cells and Clot Structure to Thrombosis
Authors: Ariens Robert A S 
Published: October 2019

Abstract
The blood clot or thrombus is composed of red blood cells, platelets and white cells that interact with the fibrin meshwork produced by the coagulation system. The red blood cells form polyhedrocytes that seal the clot, platelets provide forces for clot contraction, and white cells contribute neutrophil extracellular traps, cytokines and complement activation. The fibrin network contributes clot elasticity and provides the proteinaceous backbone structure that stabilises the clot. Previous studies from our laboratory and others have shown that dense fibrin networks demonstrating small pores and increased resistance to fibrinolysis associate with thrombosis. However, the mechanisms underpinning this have not been fully understood. Recent studies using atomic force microscopy from our laboratory have shown that fibrinogen interacts with red cells with comparable affinity as that with platelets. A patient with mutations in the β3 integrin subunit showed no binding between red cells and fibrinogen, demonstrating that a β3-related integrin receptor is involved in the interaction. Mutations in the fibrinogen α-chain integrin binding sites (D97E and D574E) reduced frequency of red cell interactions with fibrinogen. Interestingly, a naturally occuring splice variant of the fibrinogen γ-chain that reduces binding to the platelets, fibrinogen γ', increased binding interactions between fibrinogen and red blood cells. Fibrinogen γ' is a naturally occurring splice variant of fibrinogen, in which the C-terminal AGDV residues of the more common γA-chain (85%) are replaced with a negatively charched VRPEHPAETEYDSLYPEDDL sequence of the γ' chain (15%). Fibrinogen γ' induced clustering of fibrin fibres into tightly interknit nuclei of fibrin fibres, interspersed by large pores that extend over more than 50 μm within the fibrin network structure. The effects of fibrinogen γ' on fibrin clot structure was independent of thrombin and FXIII as demonstrated using snake venom enzyme. Previously we showed impaired fibrin protofibril formation with fibrinogen γ' using atomic force microscopy. Using turbidimetric analysis of fibrin intrafibrillar structure, we show that fibrinogen γ' reduces protofibil packing per fibrin fiber. Furthermore, we find that reduced protofibril packing diminishes fibrin stiffness as analysed with magnetic tweezers both in purified systems as well as in plasma at (patho)physiological fibrinogen γ' levels that range from 3-40%, and in whole blood as analysed with thromboelastography. In conclusion, our data show that red blood cells and fibrinogen γ' play major roles in the regulation of clot structure and stability, and that these effects on clot structure are major determinants of the functional properties of the blood clot. Modulating fibrin clot structure and its interactions with blood cells may represent major new targets for the treatment of thrombosis.

Disclosures
No relevant conflicts of interest to declare.
Megaloblastic Anemia from Methophenobarbital
Authors: CALVERT R. J. , HURWORTH E. , MACBEAN A. L. 
Published: October 2019

Abstract
A 39-year-old spinster presented with a severe megaloblastic anemia after treatment of her epilepsy for eight years with the barbiturate, methophenobarbital, taken regularly and alone in therapeutic dose. This drug appears to have caused the anemia, which may have been conditioned by a folic-acid-poor diet. Hematologic remission followed the administration of folic acid, which she has since continued to take along with her previous dose of methophenobarbital. Disordered pigment metabolism, including methemalbuminemia, is reported and discussed.
Glucose-6-Phosphate Dehydrogenase Deficiency in Saudi Arabia: A Survey
Authors: GELPI A. P. 
Published: October 2019

Abstract
A survey of red cell G-6-PD deficiency has been conducted among 1296 Saudi subjects. Thirteen per cent of randomly selected male subjects and 2.4 per cent of the females tested were found to be G-6-PD deficient. In this study, the screening test employed did not detect female (partially G-6-PD deficient) heterozygotes. The deficiency appeared to be localized to the oasis of the Eastern province, and in village studies a very high incidence was found in male children from the Qatif and Al-Hasa oases. The geographic limits of G-6-PD deficiency correspond precisely to the areas known to be hyperendemic for malaria in previous years. However, the population group affected represents a distinct minority in terms of religious and cultural tradition, and anthropometric type. In Saudi Arabia, falciparum malaria does not appear to be the only significant factor determining marked regional differences in the incidence of G-6-PD deficiency: this genetic marker is essentially confined to the Shiite muslim population. The Sunni population, regardless of its proximity to areas of endemic falciparum malaria, has a low incidence of G-6-PD deficiency.
The Normal Development of the Lymphoid Mass in the Golden Hamster and its Relationship to the Effects of Thymectomy
Authors: ADNER MARVIN M. , SHERMAN JOSEPH D. , DAMESHEK WILLIAM 
Published: October 2019

Abstract
A study of lymphoid organ sections and imprints of normal hamsters from fetal to adult state revealed a sequential pattern of organ structure and growth. The thymus developed an adult structure by 1 week, maximum size at 6 weeks. The mesenteric lymph node had an adult structure by 4 weeks, the spleen by 6 weeks, with an accelerated growth of both organs between 6 to 8 weeks. The growth and development of the peripheral lymphoid organs appears to be under thymic control until the attainment of a "critical peripheral lymphoid mass." This appears capable of functioning independently of thymic control. Thymectomy is effective in producing lymphoid atrophy with resultant immunosuppression and wasting disease only if performed prior to the attainment of the "critical peripheral lymphoid mass."
Effects of Endotoxin and Nitrogen Mustard on Leukocyte Kinetics
Authors: HERION JOHN C. , WALKER RICHARD I. , HERRING WILLIAM B. , PALMER JEFFRESS G. 
Published: October 2019

Abstract
In rabbits given a single large dose of HN2 circulating granulocytes are maintained at a normal concentration until 50-52 hours; thereafter the onset of a rapid fall in concentration coincides with maximum marrow hypocellularity. Recovery from granulocytopenia, however, does not begin until after 90 hours when considerable recovery of marrow cellularity is evident.
Bacterial endotoxin in differing doses given at various times up to 24 hours after HN2 produces the same changes in circulating granulocytes as in normal animals. The rapid decline in circulating granulocytes, heralding exhaustion of the pool of postmitotic granulocytes, occurs earlier in HN2-treated animals given bacterial endotoxin; they also recover sooner than controls from HN2-induced granulocytopenia. Endotoxin given at 51 hours after HN2 when the postmitctic pool is exhausted, increases the rate of fall in the concentration of circulating granulocytes producing an earlier appearance of maximum granulocytopenia, but recovery by these animals from HN2-induced granulocytopenia does not occur sooner than in controls. Thus the time of recovery from HN2-induced granulocytopenia appears to be related to the time of depletion of marrow stores of postmitotic cells.
Rabbits made tolerant to endotoxin and given HN2 show an earlier onset of the rapid fall in circulating granulocytes and an earlier recovery from granulocytopenia. Maximum marrow hypocellularity appears sooner after HN2 in tolerant animals, but marrow cellularity is always greater than in nontolerant rabbits. The increased cellularity of the marrow of tolerant animals may represent an increase in the size of the mitotic compartment of cells since the postmitotic compartment seems smaller than in normal animals.
The granulocytopenia produced by endotoxin results from an increased removal of granulocytes from the circulation and significant numbers of these cells do not appear to recirculate. Granulocytosis following the injection of endotoxin results from movement of cells out of the marrow and occurs only if there is a store of postmitotic granulocytes. Release of cells from the marrow following this stimulus, however, appears to be related to cell maturity; only the more mature ones enter the circulation.
Movement of mature cells out of the marrow, depleting the reservoir of these elements, may provide the stimulus for the increased cell production that appears in normal animals given endotoxin.
The effect of chemotherapy on the kinetics and proliferative capacity of normal and tumorous tissues in vivo
Authors: Rosenoff SH , Bull JM , Young RC 
Published: October 2019

Abstract
The proliferative state of a given tissue is a major determinant of its sensitivity to both phase-specific and cycle-specific chemotherapeutic agents. To study the extent of injury induced by antitumor agents to normal and tumorous tissues, a technique for following DNA synthesis as reflected in the incorporation of tritiated thymidine (3H-TdR) into DNA was compared to the conventional radioautographic technique of the labeling index (LI) and to the functional kinetic technique of granulocyte colony formation in vitro. Alterations in DNA synthesis induced by a single dose of cyclophosphamide in normal and tumorous tissues in vivo paralleled in many respects the changes seen when the more time-consuming techniques of the LI or granulocyte colony formation were employed. However, the recovery of granulocyte colony formation after cyclophosphamide therapy laged behind the recovery of DNA synthesis in the bone marrow, obscuring a kinetic event of potential therapeutic significance. The determination of DNA synthesis simultaneously in normal and tumorous tissues in vivo was easy to perform and supplied therapeutically pertinent results comparatively quickly.
Effect of bleeding on hematopoiesis following irradiation and marrow transplantation
Authors: Boggs SS , Boggs DR 
Published: October 2019

Abstract
In previous studies, bleeding after irradiation did not affect the rate of regeneration of endogenous spleen colony-forming cells, but induced an early (4–6 days after irradiation) appearance of erythrocytic colonies which differentiated and disappeared by days 7–8. This “abortive” wave was associated with a similarly abortive wave of splenic 59Fe uptake. The present experiments were done to determine whether or not an abortive wave of erythropoiesis could be induced in the transplanted, exogenous stem cell system. Lethally irradiated mice were given normal bone marrow cells and one-half of the group were bled of about one-third their blood volume within 4 hr of irradiation. Groups were killed on days 3–10 after irradiation. Seventeen to twenty hours prior to killing, 59Fe was injected. Hematocrits, spleen weights, colony numbers, and per cent 59Fe uptake were determined. Hematocrits of bled mice averaged about 70% of those of cell-injected controls. Spleen weights, colony counts, and per cent 59Fe uptake per spleen began to increase about 1 day earlier in bled mice (days 4–5 as compared to days 5–6), and rates of increase were the same as those of controls. However, no abortive wave of erythropoiesis was detected. A large cell dose resulted in earlier increases in all parameters than a small dose. Thus, bleeding after injection of cells produced results similar to those obtained by increasing the cell dose. The inability of bleeding to induce an early abortive wave of erythropoiesis in transplanted as compared to endogenous colony-forming systems may reflect differences in the cell cycling characteristics of these systems.
Further studies of canine von Willebrand's disease
Authors: Dodds WJ 
Published: October 2019

Abstract
Additional characterization of von Willebrand's disease (VWD) in a family of German shepherd dogs is presented. Genetic studies of three generations of affected dogs indicate that about 50% of the progeny are affected if one parent has VWD and about 60% if both parents have the defect. Some of these progeny manifested an incomplete form of VWD, suggesting autosomal dominant inheritance with variable expressivity. The disease become progressively less severe with advancing age and repeated pregnancies. Ristocetin-induced platelet aggregation was significantly reduced in VWD dogs as compared with normal, thrombopathic, and hemophilic carrier dogs. Immunodiffusion and electroimmunodiffusion studies with rabbit anticanine factor VII showed the level of factor VII-related antigen to be low in VWD dogs but present in increased amounts in hemophilic dogs. VWD affected dogs had markedly delayed hemostatic plug formation, but their plugs appeared normal by light and electron microscopy. Their platelet nucleotides, ATP/ADP ration, and platelet protein content were normal. Platelet and fibrinogen survival times with [75Se] selenomethionine were also normal, although platelets from VWD dogs incorporated more radioactivity than did those from normal dogs or from dogs with incomplete VWD.